The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching

Every source the paper cites for this claim was checked and supports it.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 and the RMS2-Dependent Feedback Signal

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§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 Is a Target in the SL Signaling Pathway

Every source the paper cites for this claim was checked and supports it.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 Acts Downstream of the SL Pathways to Control Axillary Bud Outgrowth The fact that the Psbrc1 mutant did not respond to SL application suggests that PsBRC1 may be involved in the SL signaling pathway to repress axillary bud outgrowth.In support of BRC1 acting in the SL pathway, we showed that PsBRC1 expression in axillary buds was rapidly enhanced by SL treatment.PsBRC1 transcript levels were very low in axillary buds of SLdeficient (rms1) or SL-response (rms4) mutants in comparison with wild-type buds but were rapidly up-regulated by SL application, particularly in rms1, in which it was already very low, and in the wild type, rms2, and Psbrc1.As expected for a SL-response mutant, PsBRC1 transcript levels remained very low after SL treatment in buds of the rms4 response mutant.Although RMS4 has not been proven to act in the SL pathway, it has all the features expected of a protein involved in SL signaling (branching phenotype of rms4, F-box protein, nonresponse to GR24 application of the rms4 mutant, strong feedback up-regulating RMS1 expression in the rms4 mutant).As such, the RMS4-dependent response of PsBRC1 to SL is consistent with PsBRC1 acting in the SL signaling pathway for branching inhibition.The expression of PsBRC1 in the Psbrc1 mutant was similar to its expression in the wild type, and the increase of PsBRC1 expression after GR24 application was not affected in the Psbrc1 mutant, despite the fact that the branching of this mutant was not repressed by GR24 application.These data indicate that PsBRC1 transcript levels do not simply correlate negatively with the activity of axillary buds and suggest a direct effect of the PsBRC1 transcription factor in the repression of axillary bud outgrowth.In rice, the homolog of the maize TB1 gene, FC1, was found not to be transcriptionally up-regulated by SL (Minakuchi et al., 2010).In Arabidopsis, expression of AtBRC1 was strongly down-regulated in the max1 to max4 mutants (Aguilar-Martı ´nez et al., 2007), suggesting that SL up-regulates transcript levels of AtBRC1 in Arabidopsis, as we observed for PsBRC1.In contrast, the other homolog, AtBRC2, is not down-regulated in the max1 and max2 mutants; FC1 and AtBRC2 appear to be similarly unresponsive to SL, while AtBRC1 and PsBRC1 are both transcriptionally up-regulated by SL. To confirm that PsBRC1 and RMS1 act in the same pathway, rms1 Psbrc1 double mutant plants were produced and their phenotype analyzed alongside the single mutant plants in a comparable genetic background.In Arabidopsis and rice, the corresponding double mutants have the same level of branching as single mutants (Aguilar-Martı ´nez et al., 2007), suggesting that the FC1/AtBRC1 TCP transcription factors act in the SL pathway.In pea, surprisingly, total branch length was strongly enhanced in the rms1 Psbrc1 double mutant plants, mainly because of strong development of the cotyledonary branches.When branching was quantified according to the position along the stem, branching of rms1 Psbrc1 was similar to branching in rms1 at upper nodes (node 3 and above) but transgressive at basal nodes (cotyledonary node and node 1).These results suggest a possible specific regulation of branching at basal nodes in pea.Unlike in Arabidopsis, these buds are differentiated very early in the embryo at the axils of particular leaves (cotyledons and scale leaves) and their development generally occurs below the soil surface.In pea, the RMS6 gene has been shown to control bud outgrowth only at these nodes (Rameau et al., 2002).Future studies will have to decipher how branching is regulated at these basal nodes and how RMS6, SL, and PsBRC1 interact to have a better understanding of the phenotype of the rms1 Psbrc1 double mutant.

Every source the paper cites for this claim was checked and supports it.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > CK Regulates Bud Outgrowth via PsBRC1 and Sustained Bud Growth Independently of PsBRC1

Every source the paper cites for this claim was checked and supports it.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 and the RMS2-Dependent Feedback Signal

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > The Pea Psbrc1 Mutant Phenotype Shows That PsBRC1 Inhibits Bud Outgrowth

The paper states this without a source or data behind it here, so there is nothing to check it against. The quote shows it is in the paper.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > The Pea Psbrc1 Mutant Phenotype Shows That PsBRC1 Inhibits Bud Outgrowth

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 Is a Target in the SL Signaling Pathway

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > The Pea Psbrc1 Mutant Phenotype Shows That PsBRC1 Inhibits Bud Outgrowth

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > The Pea Psbrc1 Mutant Phenotype Shows That PsBRC1 Inhibits Bud Outgrowth

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 Is a Target in the SL Signaling Pathway

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

• AssumptionRests on an assumption: The T195I mutation in PsBRC1 causes loss of PsBRC1 function.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 Is a Target in the SL Signaling Pathway

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > The Pea Psbrc1 Mutant Phenotype Shows That PsBRC1 Inhibits Bud Outgrowth

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > The Pea Psbrc1 Mutant Phenotype Shows That PsBRC1 Inhibits Bud Outgrowth

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 Is a Target in the SL Signaling Pathway

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > The Pea Psbrc1 Mutant Phenotype Shows That PsBRC1 Inhibits Bud Outgrowth

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > The Pea Psbrc1 Mutant Phenotype Shows That PsBRC1 Inhibits Bud Outgrowth

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > The Pea Psbrc1 Mutant Phenotype Shows That PsBRC1 Inhibits Bud Outgrowth

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > The Psbrc1 Cam Mutant Responds to Exogenous CK Application and to Decapitation To test whether PsBRC1 is needed for axillary bud growth, CK was applied to bud 4 of wild-type Te ´re `se and Came ´or and rms4 and Psbrc1 Cam mutants and buds were measured 5 and 7 d after application (Fig. 4B).For all lines, axillary buds did not grow much without CK treatment, as in this experiment, to have a better comparison between wild-type and mutants, the primary bud at upper nodes in mutants was removed prior to the treatment, as were the basal lateral branches for all genotypes.All genotypes responded to BAP application by displaying a strong increase in bud/branch length compared with the mock-treated buds, particularly for rms4 and Psbrc1 Cam mutants.Another way to test the response to CK is decapitation, which, by depleting the source of auxin, has been shown to induce a rapid and massive decrease of RMS1 SL biosynthesis gene expression, together with rapid increases in CK level in xylem sap and in transcript levels of CK biosynthesis genes in nodal tissue (Bangerth, 1994;Foo et al., 2005;Tanaka et al., 2006;Foo et al., 2007).Decapitation of rms1 and Psbrc1 Cam mutants and their corresponding wild types resulted in increased branch lengths in all genotypes (Fig. 4C).Because the Psbrc1 Cam mutant has been shown to be insensitive to GR24 application, its response to decapitation suggests that this response may be largely due to CK level variation occurring after decapitation or to CK-independent effects of auxin depletion.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 Is a Target in the SL Signaling Pathway

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 and the RMS2-Dependent Feedback Signal

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 and the RMS2-Dependent Feedback Signal

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > CK Regulates PsBRC1 at the Transcriptional Level Independently of SL

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 Is a Target in the SL Signaling Pathway

The paper states this without a source or data behind it here, so there is nothing to check it against. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 Is a Target in the SL Signaling Pathway

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 Is a Target in the SL Signaling Pathway

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 Is a Target in the SL Signaling Pathway

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 and the RMS2-Dependent Feedback Signal

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 Is a Target in the SL Signaling Pathway

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 and the RMS2-Dependent Feedback Signal

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 and the RMS2-Dependent Feedback Signal

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > The Psbrc1 Cam Mutant Responds to Exogenous CK Application and to Decapitation To test whether PsBRC1 is needed for axillary bud growth, CK was applied to bud 4 of wild-type Te ´re `se and Came ´or and rms4 and Psbrc1 Cam mutants and buds were measured 5 and 7 d after application (Fig. 4B).For all lines, axillary buds did not grow much without CK treatment, as in this experiment, to have a better comparison between wild-type and mutants, the primary bud at upper nodes in mutants was removed prior to the treatment, as were the basal lateral branches for all genotypes.All genotypes responded to BAP application by displaying a strong increase in bud/branch length compared with the mock-treated buds, particularly for rms4 and Psbrc1 Cam mutants.Another way to test the response to CK is decapitation, which, by depleting the source of auxin, has been shown to induce a rapid and massive decrease of RMS1 SL biosynthesis gene expression, together with rapid increases in CK level in xylem sap and in transcript levels of CK biosynthesis genes in nodal tissue (Bangerth, 1994;Foo et al., 2005;Tanaka et al., 2006;Foo et al., 2007).Decapitation of rms1 and Psbrc1 Cam mutants and their corresponding wild types resulted in increased branch lengths in all genotypes (Fig. 4C).Because the Psbrc1 Cam mutant has been shown to be insensitive to GR24 application, its response to decapitation suggests that this response may be largely due to CK level variation occurring after decapitation or to CK-independent effects of auxin depletion.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 Is a Target in the SL Signaling Pathway

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 Is a Target in the SL Signaling Pathway

The paper states this without a source or data behind it here, so there is nothing to check it against. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 Acts Downstream of the SL Pathways to Control Axillary Bud Outgrowth The fact that the Psbrc1 mutant did not respond to SL application suggests that PsBRC1 may be involved in the SL signaling pathway to repress axillary bud outgrowth.In support of BRC1 acting in the SL pathway, we showed that PsBRC1 expression in axillary buds was rapidly enhanced by SL treatment.PsBRC1 transcript levels were very low in axillary buds of SLdeficient (rms1) or SL-response (rms4) mutants in comparison with wild-type buds but were rapidly up-regulated by SL application, particularly in rms1, in which it was already very low, and in the wild type, rms2, and Psbrc1.As expected for a SL-response mutant, PsBRC1 transcript levels remained very low after SL treatment in buds of the rms4 response mutant.Although RMS4 has not been proven to act in the SL pathway, it has all the features expected of a protein involved in SL signaling (branching phenotype of rms4, F-box protein, nonresponse to GR24 application of the rms4 mutant, strong feedback up-regulating RMS1 expression in the rms4 mutant).As such, the RMS4-dependent response of PsBRC1 to SL is consistent with PsBRC1 acting in the SL signaling pathway for branching inhibition.The expression of PsBRC1 in the Psbrc1 mutant was similar to its expression in the wild type, and the increase of PsBRC1 expression after GR24 application was not affected in the Psbrc1 mutant, despite the fact that the branching of this mutant was not repressed by GR24 application.These data indicate that PsBRC1 transcript levels do not simply correlate negatively with the activity of axillary buds and suggest a direct effect of the PsBRC1 transcription factor in the repression of axillary bud outgrowth.In rice, the homolog of the maize TB1 gene, FC1, was found not to be transcriptionally up-regulated by SL (Minakuchi et al., 2010).In Arabidopsis, expression of AtBRC1 was strongly down-regulated in the max1 to max4 mutants (Aguilar-Martı ´nez et al., 2007), suggesting that SL up-regulates transcript levels of AtBRC1 in Arabidopsis, as we observed for PsBRC1.In contrast, the other homolog, AtBRC2, is not down-regulated in the max1 and max2 mutants; FC1 and AtBRC2 appear to be similarly unresponsive to SL, while AtBRC1 and PsBRC1 are both transcriptionally up-regulated by SL. To confirm that PsBRC1 and RMS1 act in the same pathway, rms1 Psbrc1 double mutant plants were produced and their phenotype analyzed alongside the single mutant plants in a comparable genetic background.In Arabidopsis and rice, the corresponding double mutants have the same level of branching as single mutants (Aguilar-Martı ´nez et al., 2007), suggesting that the FC1/AtBRC1 TCP transcription factors act in the SL pathway.In pea, surprisingly, total branch length was strongly enhanced in the rms1 Psbrc1 double mutant plants, mainly because of strong development of the cotyledonary branches.When branching was quantified according to the position along the stem, branching of rms1 Psbrc1 was similar to branching in rms1 at upper nodes (node 3 and above) but transgressive at basal nodes (cotyledonary node and node 1).These results suggest a possible specific regulation of branching at basal nodes in pea.Unlike in Arabidopsis, these buds are differentiated very early in the embryo at the axils of particular leaves (cotyledons and scale leaves) and their development generally occurs below the soil surface.In pea, the RMS6 gene has been shown to control bud outgrowth only at these nodes (Rameau et al., 2002).Future studies will have to decipher how branching is regulated at these basal nodes and how RMS6, SL, and PsBRC1 interact to have a better understanding of the phenotype of the rms1 Psbrc1 double mutant.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 and the RMS2-Dependent Feedback Signal

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > CK Regulates Bud Outgrowth via PsBRC1 and Sustained Bud Growth Independently of PsBRC1

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 Acts Downstream of the SL Pathways to Control Axillary Bud Outgrowth The fact that the Psbrc1 mutant did not respond to SL application suggests that PsBRC1 may be involved in the SL signaling pathway to repress axillary bud outgrowth.In support of BRC1 acting in the SL pathway, we showed that PsBRC1 expression in axillary buds was rapidly enhanced by SL treatment.PsBRC1 transcript levels were very low in axillary buds of SLdeficient (rms1) or SL-response (rms4) mutants in comparison with wild-type buds but were rapidly up-regulated by SL application, particularly in rms1, in which it was already very low, and in the wild type, rms2, and Psbrc1.As expected for a SL-response mutant, PsBRC1 transcript levels remained very low after SL treatment in buds of the rms4 response mutant.Although RMS4 has not been proven to act in the SL pathway, it has all the features expected of a protein involved in SL signaling (branching phenotype of rms4, F-box protein, nonresponse to GR24 application of the rms4 mutant, strong feedback up-regulating RMS1 expression in the rms4 mutant).As such, the RMS4-dependent response of PsBRC1 to SL is consistent with PsBRC1 acting in the SL signaling pathway for branching inhibition.The expression of PsBRC1 in the Psbrc1 mutant was similar to its expression in the wild type, and the increase of PsBRC1 expression after GR24 application was not affected in the Psbrc1 mutant, despite the fact that the branching of this mutant was not repressed by GR24 application.These data indicate that PsBRC1 transcript levels do not simply correlate negatively with the activity of axillary buds and suggest a direct effect of the PsBRC1 transcription factor in the repression of axillary bud outgrowth.In rice, the homolog of the maize TB1 gene, FC1, was found not to be transcriptionally up-regulated by SL (Minakuchi et al., 2010).In Arabidopsis, expression of AtBRC1 was strongly down-regulated in the max1 to max4 mutants (Aguilar-Martı ´nez et al., 2007), suggesting that SL up-regulates transcript levels of AtBRC1 in Arabidopsis, as we observed for PsBRC1.In contrast, the other homolog, AtBRC2, is not down-regulated in the max1 and max2 mutants; FC1 and AtBRC2 appear to be similarly unresponsive to SL, while AtBRC1 and PsBRC1 are both transcriptionally up-regulated by SL. To confirm that PsBRC1 and RMS1 act in the same pathway, rms1 Psbrc1 double mutant plants were produced and their phenotype analyzed alongside the single mutant plants in a comparable genetic background.In Arabidopsis and rice, the corresponding double mutants have the same level of branching as single mutants (Aguilar-Martı ´nez et al., 2007), suggesting that the FC1/AtBRC1 TCP transcription factors act in the SL pathway.In pea, surprisingly, total branch length was strongly enhanced in the rms1 Psbrc1 double mutant plants, mainly because of strong development of the cotyledonary branches.When branching was quantified according to the position along the stem, branching of rms1 Psbrc1 was similar to branching in rms1 at upper nodes (node 3 and above) but transgressive at basal nodes (cotyledonary node and node 1).These results suggest a possible specific regulation of branching at basal nodes in pea.Unlike in Arabidopsis, these buds are differentiated very early in the embryo at the axils of particular leaves (cotyledons and scale leaves) and their development generally occurs below the soil surface.In pea, the RMS6 gene has been shown to control bud outgrowth only at these nodes (Rameau et al., 2002).Future studies will have to decipher how branching is regulated at these basal nodes and how RMS6, SL, and PsBRC1 interact to have a better understanding of the phenotype of the rms1 Psbrc1 double mutant.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 Acts Downstream of the SL Pathways to Control Axillary Bud Outgrowth The fact that the Psbrc1 mutant did not respond to SL application suggests that PsBRC1 may be involved in the SL signaling pathway to repress axillary bud outgrowth.In support of BRC1 acting in the SL pathway, we showed that PsBRC1 expression in axillary buds was rapidly enhanced by SL treatment.PsBRC1 transcript levels were very low in axillary buds of SLdeficient (rms1) or SL-response (rms4) mutants in comparison with wild-type buds but were rapidly up-regulated by SL application, particularly in rms1, in which it was already very low, and in the wild type, rms2, and Psbrc1.As expected for a SL-response mutant, PsBRC1 transcript levels remained very low after SL treatment in buds of the rms4 response mutant.Although RMS4 has not been proven to act in the SL pathway, it has all the features expected of a protein involved in SL signaling (branching phenotype of rms4, F-box protein, nonresponse to GR24 application of the rms4 mutant, strong feedback up-regulating RMS1 expression in the rms4 mutant).As such, the RMS4-dependent response of PsBRC1 to SL is consistent with PsBRC1 acting in the SL signaling pathway for branching inhibition.The expression of PsBRC1 in the Psbrc1 mutant was similar to its expression in the wild type, and the increase of PsBRC1 expression after GR24 application was not affected in the Psbrc1 mutant, despite the fact that the branching of this mutant was not repressed by GR24 application.These data indicate that PsBRC1 transcript levels do not simply correlate negatively with the activity of axillary buds and suggest a direct effect of the PsBRC1 transcription factor in the repression of axillary bud outgrowth.In rice, the homolog of the maize TB1 gene, FC1, was found not to be transcriptionally up-regulated by SL (Minakuchi et al., 2010).In Arabidopsis, expression of AtBRC1 was strongly down-regulated in the max1 to max4 mutants (Aguilar-Martı ´nez et al., 2007), suggesting that SL up-regulates transcript levels of AtBRC1 in Arabidopsis, as we observed for PsBRC1.In contrast, the other homolog, AtBRC2, is not down-regulated in the max1 and max2 mutants; FC1 and AtBRC2 appear to be similarly unresponsive to SL, while AtBRC1 and PsBRC1 are both transcriptionally up-regulated by SL. To confirm that PsBRC1 and RMS1 act in the same pathway, rms1 Psbrc1 double mutant plants were produced and their phenotype analyzed alongside the single mutant plants in a comparable genetic background.In Arabidopsis and rice, the corresponding double mutants have the same level of branching as single mutants (Aguilar-Martı ´nez et al., 2007), suggesting that the FC1/AtBRC1 TCP transcription factors act in the SL pathway.In pea, surprisingly, total branch length was strongly enhanced in the rms1 Psbrc1 double mutant plants, mainly because of strong development of the cotyledonary branches.When branching was quantified according to the position along the stem, branching of rms1 Psbrc1 was similar to branching in rms1 at upper nodes (node 3 and above) but transgressive at basal nodes (cotyledonary node and node 1).These results suggest a possible specific regulation of branching at basal nodes in pea.Unlike in Arabidopsis, these buds are differentiated very early in the embryo at the axils of particular leaves (cotyledons and scale leaves) and their development generally occurs below the soil surface.In pea, the RMS6 gene has been shown to control bud outgrowth only at these nodes (Rameau et al., 2002).Future studies will have to decipher how branching is regulated at these basal nodes and how RMS6, SL, and PsBRC1 interact to have a better understanding of the phenotype of the rms1 Psbrc1 double mutant.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > CK Regulates Bud Outgrowth via PsBRC1 and Sustained Bud Growth Independently of PsBRC1

The paper states this without a source or data behind it here, so there is nothing to check it against. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > The RMS2-Dependent Feedback Signal Originates between RMS4 and PsBRC1 The existence of a mobile graft-transmissible auxinindependent shoot-to-root feedback signal that up-regulates RMS1 and RMS5 transcription and downregulates X-CK export from roots was previously proposed (Beveridge et al., 2009;Dun et al., 2009).Because the rms2 mutant showed low RMS1 expression and high X-CK content in the xylem sap, in contrast to the other rms mutants (Table I), it was suggested that the shoot-to-root feedback signal was RMS2 dependent (Beveridge et al., 2000).Experiments with grafted plants bearing two shoots of different genotypes and different phenotypes indicated that the feedback signal was more likely generated by the branching shoot to suppress X-CK and up-regulate RMS1 expression, even in the presence of a nonbranching wild-type shoot.Moreover, experiments with mutants lacking axillary meristems in an rms4 background demonstrated that branching per se was not the cause of the feedback (Foo et al., 2001(Foo et al., , 2007)).It was instead suggested to be regulated by the absence of perception of SL (Foo et al., 2005;Dun et al., 2009), as various mutants unable to produce (rms1 and rms5) or to respond to (rms3 and rms4) SL have reduced export of X-CK from the roots (Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007) and increased expression of SL biosynthesis genes (Foo et al., 2005;Johnson et al., 2006;Arite et al., 2007;Hayward et al., 2009).Here, we showed that Psbrc1 Cam lacks the feedback upregulation of RMS1 gene expression and that the substantial down-regulation of X-CK content in SL biosynthesis and signaling mutants was not found in Psbrc1 Cam plants (Table I).Instead, X-CK was either nearly normal in Psbrc1 Cam or substantially increased in Psbrc1 Te mutant plants, with the response appearing to depend on genetic background in our experiments. Similarly, in rice, D10 expression was not increased in the fc1 mutant, whereas it was highly expressed in other SL-related mutants (Arite et al., 2007;Minakuchi et al., 2010).As discussed above, our results suggest that PsBRC1 acts downstream of RMS4 in the SL signaling pathway.The absence of feedback in Psbrc1 could suggest that the SL-mediated feedback regulation is generated between the action of RMS4 and PsBRC1.This is further supported by the feedback down-regulation of X-CK in Psbrc1 rms1 double and rms1 single mutants but not in Psbrc1 Te single mutants. Advances in plant hormone signaling have highlighted the role of the ubiquitin-proteasome pathway and targeted protein turnover (Santner and Estelle, 2009).Because the MAX2/RMS4 gene encodes an F-box protein (Stirnberg et al., 2002;Johnson et al., 2006), it is likely that the SL signaling pathway involves similarly targeted protein degradation.In the GA signaling pathway, DELLA proteins are repressors that act directly downstream of the GA receptor.Microarray analysis has identified early GA-and DELLA-responsive genes (Zentella et al., 2007).Among the GA-repressed and DELLA-induced targets are genes encoding GA biosynthetic enzymes, indicating the direct involvement of DELLA proteins in feedback (Domagalska and Leyser, 2011).PsBRC1 integrates the SL and CK pathways to control bud outgrowth.CK also increases bud growth via a PsBRC1-independent pathway.Auxin maintains RMS1 transcript levels, and hence SL synthesis, and down-regulates CK levels.The RMS2-dependent feedback, which upregulates SL biosynthesis and down-regulates xylem CK, is activated when there is a lack of SL signaling via RMS4 and may be independent of PsBRC1.SLs reduce PIN accumulation to the plasma membrane via RMS4 but independently of PsBRC1 and, by reducing the effectiveness of the canalization feedback loop, enhance the competition between active buds. regulation.Proteins targeted for degradation in the SL signaling pathway, still to be identified, and possibly acting as repressors of PsBRC1 may similarly regulate the feedback signal in pea controlling SL biosynthesis and X-CK.

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§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > SL Sampling and Analysis Pea plants were germinated in vermiculite for 6 d and then transferred to aerated hydroponic complete nutrient solution culture, with 12 plants in 6 L of solution in a growth cabinet set at 23°C, 55% relative humidity during the day and 15°C, 65% relative humidity at night, with a 16-h photoperiod and light intensity of 300 mmol m 22 s 21 provided by cool-white fluorescent lamps supplemented with tungsten lamps.At 19 d after germination, the solution was replaced with water, then 24 h later, batches of 12 plants were transferred to 900 mL of water into which exudate was collected for 24 h.Deuteriumlabeled SL internal standards (20 ng each of d 1 -orobanchol, d 1 -orobanchyl acetate, d 1 -epiorobanchyl acetate, d 1 -fabacyl acetate, d 6 -5-deoxystrigol, and d 6 -epi-5-deoxystrigol; all generous gifts of Koichi Yoneyama) were added to each sample.SLs were extracted with 0.6 volume of ethyl acetate, followed by back-extraction with 0.1 M KH 2 PO 4 .The ethyl acetate fraction was dried with anhydrous MgSO 4 , filtered, and evaporated to dryness at 35°C.Samples were redissolved in dry acetone, transferred to autosampler vials, redried, dissolved in acetonitrile:water (30:70, v/v), and filtered (0.45 mm) prior to analysis by liquid chromatography-mass spectrometry multiple reaction monitoring (MRM) in positive ion electrospray mode using the Agilent 1100 LC System and an Applied Biosystems Sciex QTrap mass spectrometer.The column was Phenomenex 3 mm C18 Luna 100 3 2 mm, heated to 40°C with a flow rate of 200 mL min 21 .The initial mobile phase was 45.5% acetonitrile in 0.1% aqueous formic acid.After 1 min, a linear gradient to 77% acetonitrile over 19 min was applied, then increased to 95% acetonitrile for 3 min.Appropriate MRM transitions were monitored for each labeled standard and corresponding unlabeled SL.For the quantitations reported here, the transitions were mass-to-charge ratio 406 to 232 for d 1 -fabacyl acetate and 405 to 231 for fabacyl acetate.Two biological replicates representing pools of 12 plants were analyzed for each genotype.SL content was calculated from MRM peak areas by the stable isotope ratio method.CYC/ TB1 CIN Class II LjCYC3 LjCYC1 LjCYC2 AmCYC AtTCP1 AtBRC2 OsTB1 ZmTB1 SbTB1 AtBRC1 PsBRC1 LjCYC5 OsPCF6 AtTCP24 AtTCP17 AtTCP4 AtTCP10 OsPCF5 AmCIN AtTCP16 87 100 35 33 99 94 59 98 89 77 87 84 82 62 64 39 78 Class I Figure S1.Phylogenetic tree of representative TCP proteins from Arabidopsis thaliana (At), PsBRC1 from pea and members of other plant species, Antirrhinum majus (Am), Lotus japonicus (Lj), rice (Os), maize (Zm), Sorghum bicolor (Sb).Evolutionary relationships were analyzed using a selection of predicted amino acid sequences of the TCP domains aligned with the program CLUSTALX ( Thompson et al., 1997).The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987) and the optimal tree was generated.Phylogenetic analyses were conducted in MEGA4 (Tamura et al., 2007).Arabidopsis and rice TCP genes named after (Martin-Trillo and Cubas, 2010); LjCYC1, LjCYC2, LjCYC3, and LjCYC5 (GenBank accession nos DQ202475, DQ202476, DQ202477 and DQ202478).0 20 40 60 80 Internode length between nodes 1 to 6 (mm) Psbrc1 Té Caméor (WT) Psbrc1 Cam Térèse (WT) rms1 C D Psbrc1 Cam Caméor WT Branch length at each node (mm). Figure: 1 Figure 1 . Figure 1.Structure of the PsBRC1 gene and phenotype of the corresponding mutant.A, Gene structure of PsBRC1 and locations of mutations.Bases are numbered from the start codon.The TCP domain (from bp 441 to 630) is shown in red, and the corresponding protein sequence is indicated below.Point mutations are indicated by triangles (black and red for the one studied here), boxes correspond to exons, and the blue area corresponds to the TILLed sequence.B, Comparison of a wild-type (WT) Te ´re `se plant (left) with rms4 (middle left), wild-type Came ´or (middle right), and Psbrc1 Cam (right).C, Branch length at each node of wild-type Came ´or, Psbrc1 Cam , wildtype Te ´re `se, rms1, and Psbrc1 Te .Data are means 6 SE (n = 12).D, Means of the number of cotyledonary branches per individual observed in a segregating F2 population of 103 plants between M3T-884 (rms1) and a Psbrc1 F2 plant (Psbrc1 Cam 3 Te ´re `se).n = the number of plants observed per genotypic class; ncot = the number of plants with at least one cotyledonary branch.Data are means 6 SE (n = 12). Figure: 2 Figure 2 . Figure 2. A, Effects of GR24 application on bud growth.Bud length at node 3 of wild-type Te ´re `se (black bars), rms1 (white bars), wild-type Came ´or (gray bars), and Psbrc1 Cam (hatched bars) was measured 10 d after treatment was applied to buds of stage 5 intact plants with solution containing 0 or 500 nM GR24.Data are means 6 SE (n = 12).B, SL levels in root exudates of wild-type and Psbrc1 plants.Exudates were collected into water for 24 h from 20-d-old hydroponically grown plants, and SLs were quantified by LC-MS using MRM transitions at mass-to-charge ratio 405 to 231 for fabacyl acetate and 406 to 232 for the d 1 -fabacyl acetate internal standard.Data are means 6 SE, based on analyses of two independent pools of 12 plants for each genotype. Figure: 3 Figure 3 . Figure 3. Effects of GR24 on PsBRC1 transcript levels.PsBRC1 transcript levels were determined relative to EF1a in axillary buds at node 4 after GR24 application (white bars) or mock treatment (black bars).RNA was extracted from dissected buds from pools of 30 plants at the six-node stage and quantified by real-time PCR.The data are representative of two to three independent experiments.A, Six and 24 h after GR24 application in wild-type Te ´re `se, rms1, rms2, and rms4 plants.The branching phenotype at node 4 after GR24 application is given below for each genotype.B, Six and 48 h after GR24 application in wild-type Te ´re `se, rms1, rms4, and Psbrc1 Te ´plants.Data are means 6 SE (n = 3). Figure: 4 Figure 4 . Figure 4. Effects of BAP on PsBRC1 transcript levels and on bud growth.A, PsBRC1 transcript levels were determined relative to EF1a in axillary buds at node 4 after BAP (50 mM) application in wild-type Came ´or, Psbrc1 Cam , wild-type Te ´re `se, rms1, and rms4.RNA was extracted from the dissected buds of 30 plants at the six-node stage and quantified by real-time PCR.The data are representative of three independent experiments.B, Effects of BAP (50 mM) treatment on bud growth at node 4 in wild-type Te ´re `se, rms4, wild-type Came ´or, and Psbrc1 Cam .Measurements were done 5 d after treatment.Data are means 6 SE (n = 12).C, Effects of decapitation above node 5 on total branch length at nodes 1 to 5. Intact and decapitated plants of Psbrc1 Cam , wild-type Came ´or, rms1, and wild-type Te ´re `se were used.Data are means 6 SE (n = 8).

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > The Pea Psbrc1 Mutant Phenotype Shows That PsBRC1 Inhibits Bud Outgrowth

• AssumptionRests on an assumption: The T195I mutation in PsBRC1 causes loss of PsBRC1 function.

None of the sources cited for this claim have been checked yet.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 and the RMS2-Dependent Feedback Signal

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 and the RMS2-Dependent Feedback Signal

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 and the RMS2-Dependent Feedback Signal

None of the sources cited for this claim have been checked yet.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 and the RMS2-Dependent Feedback Signal

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 and the RMS2-Dependent Feedback Signal

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 and the RMS2-Dependent Feedback Signal

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > CK Regulates PsBRC1 at the Transcriptional Level Independently of SL

None of the sources cited for this claim have been checked yet.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 and the RMS2-Dependent Feedback Signal

None of the sources cited for this claim have been checked yet.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > The RMS2-Dependent Feedback Signal Originates between RMS4 and PsBRC1 The existence of a mobile graft-transmissible auxinindependent shoot-to-root feedback signal that up-regulates RMS1 and RMS5 transcription and downregulates X-CK export from roots was previously proposed (Beveridge et al., 2009;Dun et al., 2009).Because the rms2 mutant showed low RMS1 expression and high X-CK content in the xylem sap, in contrast to the other rms mutants (Table I), it was suggested that the shoot-to-root feedback signal was RMS2 dependent (Beveridge et al., 2000).Experiments with grafted plants bearing two shoots of different genotypes and different phenotypes indicated that the feedback signal was more likely generated by the branching shoot to suppress X-CK and up-regulate RMS1 expression, even in the presence of a nonbranching wild-type shoot.Moreover, experiments with mutants lacking axillary meristems in an rms4 background demonstrated that branching per se was not the cause of the feedback (Foo et al., 2001(Foo et al., , 2007)).It was instead suggested to be regulated by the absence of perception of SL (Foo et al., 2005;Dun et al., 2009), as various mutants unable to produce (rms1 and rms5) or to respond to (rms3 and rms4) SL have reduced export of X-CK from the roots (Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007) and increased expression of SL biosynthesis genes (Foo et al., 2005;Johnson et al., 2006;Arite et al., 2007;Hayward et al., 2009).Here, we showed that Psbrc1 Cam lacks the feedback upregulation of RMS1 gene expression and that the substantial down-regulation of X-CK content in SL biosynthesis and signaling mutants was not found in Psbrc1 Cam plants (Table I).Instead, X-CK was either nearly normal in Psbrc1 Cam or substantially increased in Psbrc1 Te mutant plants, with the response appearing to depend on genetic background in our experiments. Similarly, in rice, D10 expression was not increased in the fc1 mutant, whereas it was highly expressed in other SL-related mutants (Arite et al., 2007;Minakuchi et al., 2010).As discussed above, our results suggest that PsBRC1 acts downstream of RMS4 in the SL signaling pathway.The absence of feedback in Psbrc1 could suggest that the SL-mediated feedback regulation is generated between the action of RMS4 and PsBRC1.This is further supported by the feedback down-regulation of X-CK in Psbrc1 rms1 double and rms1 single mutants but not in Psbrc1 Te single mutants. Advances in plant hormone signaling have highlighted the role of the ubiquitin-proteasome pathway and targeted protein turnover (Santner and Estelle, 2009).Because the MAX2/RMS4 gene encodes an F-box protein (Stirnberg et al., 2002;Johnson et al., 2006), it is likely that the SL signaling pathway involves similarly targeted protein degradation.In the GA signaling pathway, DELLA proteins are repressors that act directly downstream of the GA receptor.Microarray analysis has identified early GA-and DELLA-responsive genes (Zentella et al., 2007).Among the GA-repressed and DELLA-induced targets are genes encoding GA biosynthetic enzymes, indicating the direct involvement of DELLA proteins in feedback (Domagalska and Leyser, 2011).PsBRC1 integrates the SL and CK pathways to control bud outgrowth.CK also increases bud growth via a PsBRC1-independent pathway.Auxin maintains RMS1 transcript levels, and hence SL synthesis, and down-regulates CK levels.The RMS2-dependent feedback, which upregulates SL biosynthesis and down-regulates xylem CK, is activated when there is a lack of SL signaling via RMS4 and may be independent of PsBRC1.SLs reduce PIN accumulation to the plasma membrane via RMS4 but independently of PsBRC1 and, by reducing the effectiveness of the canalization feedback loop, enhance the competition between active buds. regulation.Proteins targeted for degradation in the SL signaling pathway, still to be identified, and possibly acting as repressors of PsBRC1 may similarly regulate the feedback signal in pea controlling SL biosynthesis and X-CK.

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 Acts Downstream of the SL Pathways to Control Axillary Bud Outgrowth The fact that the Psbrc1 mutant did not respond to SL application suggests that PsBRC1 may be involved in the SL signaling pathway to repress axillary bud outgrowth.In support of BRC1 acting in the SL pathway, we showed that PsBRC1 expression in axillary buds was rapidly enhanced by SL treatment.PsBRC1 transcript levels were very low in axillary buds of SLdeficient (rms1) or SL-response (rms4) mutants in comparison with wild-type buds but were rapidly up-regulated by SL application, particularly in rms1, in which it was already very low, and in the wild type, rms2, and Psbrc1.As expected for a SL-response mutant, PsBRC1 transcript levels remained very low after SL treatment in buds of the rms4 response mutant.Although RMS4 has not been proven to act in the SL pathway, it has all the features expected of a protein involved in SL signaling (branching phenotype of rms4, F-box protein, nonresponse to GR24 application of the rms4 mutant, strong feedback up-regulating RMS1 expression in the rms4 mutant).As such, the RMS4-dependent response of PsBRC1 to SL is consistent with PsBRC1 acting in the SL signaling pathway for branching inhibition.The expression of PsBRC1 in the Psbrc1 mutant was similar to its expression in the wild type, and the increase of PsBRC1 expression after GR24 application was not affected in the Psbrc1 mutant, despite the fact that the branching of this mutant was not repressed by GR24 application.These data indicate that PsBRC1 transcript levels do not simply correlate negatively with the activity of axillary buds and suggest a direct effect of the PsBRC1 transcription factor in the repression of axillary bud outgrowth.In rice, the homolog of the maize TB1 gene, FC1, was found not to be transcriptionally up-regulated by SL (Minakuchi et al., 2010).In Arabidopsis, expression of AtBRC1 was strongly down-regulated in the max1 to max4 mutants (Aguilar-Martı ´nez et al., 2007), suggesting that SL up-regulates transcript levels of AtBRC1 in Arabidopsis, as we observed for PsBRC1.In contrast, the other homolog, AtBRC2, is not down-regulated in the max1 and max2 mutants; FC1 and AtBRC2 appear to be similarly unresponsive to SL, while AtBRC1 and PsBRC1 are both transcriptionally up-regulated by SL. To confirm that PsBRC1 and RMS1 act in the same pathway, rms1 Psbrc1 double mutant plants were produced and their phenotype analyzed alongside the single mutant plants in a comparable genetic background.In Arabidopsis and rice, the corresponding double mutants have the same level of branching as single mutants (Aguilar-Martı ´nez et al., 2007), suggesting that the FC1/AtBRC1 TCP transcription factors act in the SL pathway.In pea, surprisingly, total branch length was strongly enhanced in the rms1 Psbrc1 double mutant plants, mainly because of strong development of the cotyledonary branches.When branching was quantified according to the position along the stem, branching of rms1 Psbrc1 was similar to branching in rms1 at upper nodes (node 3 and above) but transgressive at basal nodes (cotyledonary node and node 1).These results suggest a possible specific regulation of branching at basal nodes in pea.Unlike in Arabidopsis, these buds are differentiated very early in the embryo at the axils of particular leaves (cotyledons and scale leaves) and their development generally occurs below the soil surface.In pea, the RMS6 gene has been shown to control bud outgrowth only at these nodes (Rameau et al., 2002).Future studies will have to decipher how branching is regulated at these basal nodes and how RMS6, SL, and PsBRC1 interact to have a better understanding of the phenotype of the rms1 Psbrc1 double mutant.

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 and the RMS2-Dependent Feedback Signal

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 Acts Downstream of the SL Pathways to Control Axillary Bud Outgrowth The fact that the Psbrc1 mutant did not respond to SL application suggests that PsBRC1 may be involved in the SL signaling pathway to repress axillary bud outgrowth.In support of BRC1 acting in the SL pathway, we showed that PsBRC1 expression in axillary buds was rapidly enhanced by SL treatment.PsBRC1 transcript levels were very low in axillary buds of SLdeficient (rms1) or SL-response (rms4) mutants in comparison with wild-type buds but were rapidly up-regulated by SL application, particularly in rms1, in which it was already very low, and in the wild type, rms2, and Psbrc1.As expected for a SL-response mutant, PsBRC1 transcript levels remained very low after SL treatment in buds of the rms4 response mutant.Although RMS4 has not been proven to act in the SL pathway, it has all the features expected of a protein involved in SL signaling (branching phenotype of rms4, F-box protein, nonresponse to GR24 application of the rms4 mutant, strong feedback up-regulating RMS1 expression in the rms4 mutant).As such, the RMS4-dependent response of PsBRC1 to SL is consistent with PsBRC1 acting in the SL signaling pathway for branching inhibition.The expression of PsBRC1 in the Psbrc1 mutant was similar to its expression in the wild type, and the increase of PsBRC1 expression after GR24 application was not affected in the Psbrc1 mutant, despite the fact that the branching of this mutant was not repressed by GR24 application.These data indicate that PsBRC1 transcript levels do not simply correlate negatively with the activity of axillary buds and suggest a direct effect of the PsBRC1 transcription factor in the repression of axillary bud outgrowth.In rice, the homolog of the maize TB1 gene, FC1, was found not to be transcriptionally up-regulated by SL (Minakuchi et al., 2010).In Arabidopsis, expression of AtBRC1 was strongly down-regulated in the max1 to max4 mutants (Aguilar-Martı ´nez et al., 2007), suggesting that SL up-regulates transcript levels of AtBRC1 in Arabidopsis, as we observed for PsBRC1.In contrast, the other homolog, AtBRC2, is not down-regulated in the max1 and max2 mutants; FC1 and AtBRC2 appear to be similarly unresponsive to SL, while AtBRC1 and PsBRC1 are both transcriptionally up-regulated by SL. To confirm that PsBRC1 and RMS1 act in the same pathway, rms1 Psbrc1 double mutant plants were produced and their phenotype analyzed alongside the single mutant plants in a comparable genetic background.In Arabidopsis and rice, the corresponding double mutants have the same level of branching as single mutants (Aguilar-Martı ´nez et al., 2007), suggesting that the FC1/AtBRC1 TCP transcription factors act in the SL pathway.In pea, surprisingly, total branch length was strongly enhanced in the rms1 Psbrc1 double mutant plants, mainly because of strong development of the cotyledonary branches.When branching was quantified according to the position along the stem, branching of rms1 Psbrc1 was similar to branching in rms1 at upper nodes (node 3 and above) but transgressive at basal nodes (cotyledonary node and node 1).These results suggest a possible specific regulation of branching at basal nodes in pea.Unlike in Arabidopsis, these buds are differentiated very early in the embryo at the axils of particular leaves (cotyledons and scale leaves) and their development generally occurs below the soil surface.In pea, the RMS6 gene has been shown to control bud outgrowth only at these nodes (Rameau et al., 2002).Future studies will have to decipher how branching is regulated at these basal nodes and how RMS6, SL, and PsBRC1 interact to have a better understanding of the phenotype of the rms1 Psbrc1 double mutant.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 and the RMS2-Dependent Feedback Signal

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > CK Regulates Bud Outgrowth via PsBRC1 and Sustained Bud Growth Independently of PsBRC1

A claim is checked only against the cited paper’s full text, never its abstract, and none is available for its sources.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > The RMS2-Dependent Feedback Signal Originates between RMS4 and PsBRC1 The existence of a mobile graft-transmissible auxinindependent shoot-to-root feedback signal that up-regulates RMS1 and RMS5 transcription and downregulates X-CK export from roots was previously proposed (Beveridge et al., 2009;Dun et al., 2009).Because the rms2 mutant showed low RMS1 expression and high X-CK content in the xylem sap, in contrast to the other rms mutants (Table I), it was suggested that the shoot-to-root feedback signal was RMS2 dependent (Beveridge et al., 2000).Experiments with grafted plants bearing two shoots of different genotypes and different phenotypes indicated that the feedback signal was more likely generated by the branching shoot to suppress X-CK and up-regulate RMS1 expression, even in the presence of a nonbranching wild-type shoot.Moreover, experiments with mutants lacking axillary meristems in an rms4 background demonstrated that branching per se was not the cause of the feedback (Foo et al., 2001(Foo et al., , 2007)).It was instead suggested to be regulated by the absence of perception of SL (Foo et al., 2005;Dun et al., 2009), as various mutants unable to produce (rms1 and rms5) or to respond to (rms3 and rms4) SL have reduced export of X-CK from the roots (Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007) and increased expression of SL biosynthesis genes (Foo et al., 2005;Johnson et al., 2006;Arite et al., 2007;Hayward et al., 2009).Here, we showed that Psbrc1 Cam lacks the feedback upregulation of RMS1 gene expression and that the substantial down-regulation of X-CK content in SL biosynthesis and signaling mutants was not found in Psbrc1 Cam plants (Table I).Instead, X-CK was either nearly normal in Psbrc1 Cam or substantially increased in Psbrc1 Te mutant plants, with the response appearing to depend on genetic background in our experiments. Similarly, in rice, D10 expression was not increased in the fc1 mutant, whereas it was highly expressed in other SL-related mutants (Arite et al., 2007;Minakuchi et al., 2010).As discussed above, our results suggest that PsBRC1 acts downstream of RMS4 in the SL signaling pathway.The absence of feedback in Psbrc1 could suggest that the SL-mediated feedback regulation is generated between the action of RMS4 and PsBRC1.This is further supported by the feedback down-regulation of X-CK in Psbrc1 rms1 double and rms1 single mutants but not in Psbrc1 Te single mutants. Advances in plant hormone signaling have highlighted the role of the ubiquitin-proteasome pathway and targeted protein turnover (Santner and Estelle, 2009).Because the MAX2/RMS4 gene encodes an F-box protein (Stirnberg et al., 2002;Johnson et al., 2006), it is likely that the SL signaling pathway involves similarly targeted protein degradation.In the GA signaling pathway, DELLA proteins are repressors that act directly downstream of the GA receptor.Microarray analysis has identified early GA-and DELLA-responsive genes (Zentella et al., 2007).Among the GA-repressed and DELLA-induced targets are genes encoding GA biosynthetic enzymes, indicating the direct involvement of DELLA proteins in feedback (Domagalska and Leyser, 2011).PsBRC1 integrates the SL and CK pathways to control bud outgrowth.CK also increases bud growth via a PsBRC1-independent pathway.Auxin maintains RMS1 transcript levels, and hence SL synthesis, and down-regulates CK levels.The RMS2-dependent feedback, which upregulates SL biosynthesis and down-regulates xylem CK, is activated when there is a lack of SL signaling via RMS4 and may be independent of PsBRC1.SLs reduce PIN accumulation to the plasma membrane via RMS4 but independently of PsBRC1 and, by reducing the effectiveness of the canalization feedback loop, enhance the competition between active buds. regulation.Proteins targeted for degradation in the SL signaling pathway, still to be identified, and possibly acting as repressors of PsBRC1 may similarly regulate the feedback signal in pea controlling SL biosynthesis and X-CK.

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 Acts Downstream of the SL Pathways to Control Axillary Bud Outgrowth The fact that the Psbrc1 mutant did not respond to SL application suggests that PsBRC1 may be involved in the SL signaling pathway to repress axillary bud outgrowth.In support of BRC1 acting in the SL pathway, we showed that PsBRC1 expression in axillary buds was rapidly enhanced by SL treatment.PsBRC1 transcript levels were very low in axillary buds of SLdeficient (rms1) or SL-response (rms4) mutants in comparison with wild-type buds but were rapidly up-regulated by SL application, particularly in rms1, in which it was already very low, and in the wild type, rms2, and Psbrc1.As expected for a SL-response mutant, PsBRC1 transcript levels remained very low after SL treatment in buds of the rms4 response mutant.Although RMS4 has not been proven to act in the SL pathway, it has all the features expected of a protein involved in SL signaling (branching phenotype of rms4, F-box protein, nonresponse to GR24 application of the rms4 mutant, strong feedback up-regulating RMS1 expression in the rms4 mutant).As such, the RMS4-dependent response of PsBRC1 to SL is consistent with PsBRC1 acting in the SL signaling pathway for branching inhibition.The expression of PsBRC1 in the Psbrc1 mutant was similar to its expression in the wild type, and the increase of PsBRC1 expression after GR24 application was not affected in the Psbrc1 mutant, despite the fact that the branching of this mutant was not repressed by GR24 application.These data indicate that PsBRC1 transcript levels do not simply correlate negatively with the activity of axillary buds and suggest a direct effect of the PsBRC1 transcription factor in the repression of axillary bud outgrowth.In rice, the homolog of the maize TB1 gene, FC1, was found not to be transcriptionally up-regulated by SL (Minakuchi et al., 2010).In Arabidopsis, expression of AtBRC1 was strongly down-regulated in the max1 to max4 mutants (Aguilar-Martı ´nez et al., 2007), suggesting that SL up-regulates transcript levels of AtBRC1 in Arabidopsis, as we observed for PsBRC1.In contrast, the other homolog, AtBRC2, is not down-regulated in the max1 and max2 mutants; FC1 and AtBRC2 appear to be similarly unresponsive to SL, while AtBRC1 and PsBRC1 are both transcriptionally up-regulated by SL. To confirm that PsBRC1 and RMS1 act in the same pathway, rms1 Psbrc1 double mutant plants were produced and their phenotype analyzed alongside the single mutant plants in a comparable genetic background.In Arabidopsis and rice, the corresponding double mutants have the same level of branching as single mutants (Aguilar-Martı ´nez et al., 2007), suggesting that the FC1/AtBRC1 TCP transcription factors act in the SL pathway.In pea, surprisingly, total branch length was strongly enhanced in the rms1 Psbrc1 double mutant plants, mainly because of strong development of the cotyledonary branches.When branching was quantified according to the position along the stem, branching of rms1 Psbrc1 was similar to branching in rms1 at upper nodes (node 3 and above) but transgressive at basal nodes (cotyledonary node and node 1).These results suggest a possible specific regulation of branching at basal nodes in pea.Unlike in Arabidopsis, these buds are differentiated very early in the embryo at the axils of particular leaves (cotyledons and scale leaves) and their development generally occurs below the soil surface.In pea, the RMS6 gene has been shown to control bud outgrowth only at these nodes (Rameau et al., 2002).Future studies will have to decipher how branching is regulated at these basal nodes and how RMS6, SL, and PsBRC1 interact to have a better understanding of the phenotype of the rms1 Psbrc1 double mutant.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > CK Regulates Bud Outgrowth via PsBRC1 and Sustained Bud Growth Independently of PsBRC1

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > PsBRC1 Acts Downstream of the SL Pathways to Control Axillary Bud Outgrowth The fact that the Psbrc1 mutant did not respond to SL application suggests that PsBRC1 may be involved in the SL signaling pathway to repress axillary bud outgrowth.In support of BRC1 acting in the SL pathway, we showed that PsBRC1 expression in axillary buds was rapidly enhanced by SL treatment.PsBRC1 transcript levels were very low in axillary buds of SLdeficient (rms1) or SL-response (rms4) mutants in comparison with wild-type buds but were rapidly up-regulated by SL application, particularly in rms1, in which it was already very low, and in the wild type, rms2, and Psbrc1.As expected for a SL-response mutant, PsBRC1 transcript levels remained very low after SL treatment in buds of the rms4 response mutant.Although RMS4 has not been proven to act in the SL pathway, it has all the features expected of a protein involved in SL signaling (branching phenotype of rms4, F-box protein, nonresponse to GR24 application of the rms4 mutant, strong feedback up-regulating RMS1 expression in the rms4 mutant).As such, the RMS4-dependent response of PsBRC1 to SL is consistent with PsBRC1 acting in the SL signaling pathway for branching inhibition.The expression of PsBRC1 in the Psbrc1 mutant was similar to its expression in the wild type, and the increase of PsBRC1 expression after GR24 application was not affected in the Psbrc1 mutant, despite the fact that the branching of this mutant was not repressed by GR24 application.These data indicate that PsBRC1 transcript levels do not simply correlate negatively with the activity of axillary buds and suggest a direct effect of the PsBRC1 transcription factor in the repression of axillary bud outgrowth.In rice, the homolog of the maize TB1 gene, FC1, was found not to be transcriptionally up-regulated by SL (Minakuchi et al., 2010).In Arabidopsis, expression of AtBRC1 was strongly down-regulated in the max1 to max4 mutants (Aguilar-Martı ´nez et al., 2007), suggesting that SL up-regulates transcript levels of AtBRC1 in Arabidopsis, as we observed for PsBRC1.In contrast, the other homolog, AtBRC2, is not down-regulated in the max1 and max2 mutants; FC1 and AtBRC2 appear to be similarly unresponsive to SL, while AtBRC1 and PsBRC1 are both transcriptionally up-regulated by SL. To confirm that PsBRC1 and RMS1 act in the same pathway, rms1 Psbrc1 double mutant plants were produced and their phenotype analyzed alongside the single mutant plants in a comparable genetic background.In Arabidopsis and rice, the corresponding double mutants have the same level of branching as single mutants (Aguilar-Martı ´nez et al., 2007), suggesting that the FC1/AtBRC1 TCP transcription factors act in the SL pathway.In pea, surprisingly, total branch length was strongly enhanced in the rms1 Psbrc1 double mutant plants, mainly because of strong development of the cotyledonary branches.When branching was quantified according to the position along the stem, branching of rms1 Psbrc1 was similar to branching in rms1 at upper nodes (node 3 and above) but transgressive at basal nodes (cotyledonary node and node 1).These results suggest a possible specific regulation of branching at basal nodes in pea.Unlike in Arabidopsis, these buds are differentiated very early in the embryo at the axils of particular leaves (cotyledons and scale leaves) and their development generally occurs below the soil surface.In pea, the RMS6 gene has been shown to control bud outgrowth only at these nodes (Rameau et al., 2002).Future studies will have to decipher how branching is regulated at these basal nodes and how RMS6, SL, and PsBRC1 interact to have a better understanding of the phenotype of the rms1 Psbrc1 double mutant.

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > SL Sampling and Analysis Pea plants were germinated in vermiculite for 6 d and then transferred to aerated hydroponic complete nutrient solution culture, with 12 plants in 6 L of solution in a growth cabinet set at 23°C, 55% relative humidity during the day and 15°C, 65% relative humidity at night, with a 16-h photoperiod and light intensity of 300 mmol m 22 s 21 provided by cool-white fluorescent lamps supplemented with tungsten lamps.At 19 d after germination, the solution was replaced with water, then 24 h later, batches of 12 plants were transferred to 900 mL of water into which exudate was collected for 24 h.Deuteriumlabeled SL internal standards (20 ng each of d 1 -orobanchol, d 1 -orobanchyl acetate, d 1 -epiorobanchyl acetate, d 1 -fabacyl acetate, d 6 -5-deoxystrigol, and d 6 -epi-5-deoxystrigol; all generous gifts of Koichi Yoneyama) were added to each sample.SLs were extracted with 0.6 volume of ethyl acetate, followed by back-extraction with 0.1 M KH 2 PO 4 .The ethyl acetate fraction was dried with anhydrous MgSO 4 , filtered, and evaporated to dryness at 35°C.Samples were redissolved in dry acetone, transferred to autosampler vials, redried, dissolved in acetonitrile:water (30:70, v/v), and filtered (0.45 mm) prior to analysis by liquid chromatography-mass spectrometry multiple reaction monitoring (MRM) in positive ion electrospray mode using the Agilent 1100 LC System and an Applied Biosystems Sciex QTrap mass spectrometer.The column was Phenomenex 3 mm C18 Luna 100 3 2 mm, heated to 40°C with a flow rate of 200 mL min 21 .The initial mobile phase was 45.5% acetonitrile in 0.1% aqueous formic acid.After 1 min, a linear gradient to 77% acetonitrile over 19 min was applied, then increased to 95% acetonitrile for 3 min.Appropriate MRM transitions were monitored for each labeled standard and corresponding unlabeled SL.For the quantitations reported here, the transitions were mass-to-charge ratio 406 to 232 for d 1 -fabacyl acetate and 405 to 231 for fabacyl acetate.Two biological replicates representing pools of 12 plants were analyzed for each genotype.SL content was calculated from MRM peak areas by the stable isotope ratio method.CYC/ TB1 CIN Class II LjCYC3 LjCYC1 LjCYC2 AmCYC AtTCP1 AtBRC2 OsTB1 ZmTB1 SbTB1 AtBRC1 PsBRC1 LjCYC5 OsPCF6 AtTCP24 AtTCP17 AtTCP4 AtTCP10 OsPCF5 AmCIN AtTCP16 87 100 35 33 99 94 59 98 89 77 87 84 82 62 64 39 78 Class I Figure S1.Phylogenetic tree of representative TCP proteins from Arabidopsis thaliana (At), PsBRC1 from pea and members of other plant species, Antirrhinum majus (Am), Lotus japonicus (Lj), rice (Os), maize (Zm), Sorghum bicolor (Sb).Evolutionary relationships were analyzed using a selection of predicted amino acid sequences of the TCP domains aligned with the program CLUSTALX ( Thompson et al., 1997).The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987) and the optimal tree was generated.Phylogenetic analyses were conducted in MEGA4 (Tamura et al., 2007).Arabidopsis and rice TCP genes named after (Martin-Trillo and Cubas, 2010); LjCYC1, LjCYC2, LjCYC3, and LjCYC5 (GenBank accession nos DQ202475, DQ202476, DQ202477 and DQ202478).0 20 40 60 80 Internode length between nodes 1 to 6 (mm) Psbrc1 Té Caméor (WT) Psbrc1 Cam Térèse (WT) rms1 C D Psbrc1 Cam Caméor WT Branch length at each node (mm). Figure: 1 Figure 1 . Figure 1.Structure of the PsBRC1 gene and phenotype of the corresponding mutant.A, Gene structure of PsBRC1 and locations of mutations.Bases are numbered from the start codon.The TCP domain (from bp 441 to 630) is shown in red, and the corresponding protein sequence is indicated below.Point mutations are indicated by triangles (black and red for the one studied here), boxes correspond to exons, and the blue area corresponds to the TILLed sequence.B, Comparison of a wild-type (WT) Te ´re `se plant (left) with rms4 (middle left), wild-type Came ´or (middle right), and Psbrc1 Cam (right).C, Branch length at each node of wild-type Came ´or, Psbrc1 Cam , wildtype Te ´re `se, rms1, and Psbrc1 Te .Data are means 6 SE (n = 12).D, Means of the number of cotyledonary branches per individual observed in a segregating F2 population of 103 plants between M3T-884 (rms1) and a Psbrc1 F2 plant (Psbrc1 Cam 3 Te ´re `se).n = the number of plants observed per genotypic class; ncot = the number of plants with at least one cotyledonary branch.Data are means 6 SE (n = 12). Figure: 2 Figure 2 . Figure 2. A, Effects of GR24 application on bud growth.Bud length at node 3 of wild-type Te ´re `se (black bars), rms1 (white bars), wild-type Came ´or (gray bars), and Psbrc1 Cam (hatched bars) was measured 10 d after treatment was applied to buds of stage 5 intact plants with solution containing 0 or 500 nM GR24.Data are means 6 SE (n = 12).B, SL levels in root exudates of wild-type and Psbrc1 plants.Exudates were collected into water for 24 h from 20-d-old hydroponically grown plants, and SLs were quantified by LC-MS using MRM transitions at mass-to-charge ratio 405 to 231 for fabacyl acetate and 406 to 232 for the d 1 -fabacyl acetate internal standard.Data are means 6 SE, based on analyses of two independent pools of 12 plants for each genotype. Figure: 3 Figure 3 . Figure 3. Effects of GR24 on PsBRC1 transcript levels.PsBRC1 transcript levels were determined relative to EF1a in axillary buds at node 4 after GR24 application (white bars) or mock treatment (black bars).RNA was extracted from dissected buds from pools of 30 plants at the six-node stage and quantified by real-time PCR.The data are representative of two to three independent experiments.A, Six and 24 h after GR24 application in wild-type Te ´re `se, rms1, rms2, and rms4 plants.The branching phenotype at node 4 after GR24 application is given below for each genotype.B, Six and 48 h after GR24 application in wild-type Te ´re `se, rms1, rms4, and Psbrc1 Te ´plants.Data are means 6 SE (n = 3). Figure: 4 Figure 4 . Figure 4. Effects of BAP on PsBRC1 transcript levels and on bud growth.A, PsBRC1 transcript levels were determined relative to EF1a in axillary buds at node 4 after BAP (50 mM) application in wild-type Came ´or, Psbrc1 Cam , wild-type Te ´re `se, rms1, and rms4.RNA was extracted from the dissected buds of 30 plants at the six-node stage and quantified by real-time PCR.The data are representative of three independent experiments.B, Effects of BAP (50 mM) treatment on bud growth at node 4 in wild-type Te ´re `se, rms4, wild-type Came ´or, and Psbrc1 Cam .Measurements were done 5 d after treatment.Data are means 6 SE (n = 12).C, Effects of decapitation above node 5 on total branch length at nodes 1 to 5. Intact and decapitated plants of Psbrc1 Cam , wild-type Came ´or, rms1, and wild-type Te ´re `se were used.Data are means 6 SE (n = 8).

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > The RMS2-Dependent Feedback Signal Originates between RMS4 and PsBRC1 The existence of a mobile graft-transmissible auxinindependent shoot-to-root feedback signal that up-regulates RMS1 and RMS5 transcription and downregulates X-CK export from roots was previously proposed (Beveridge et al., 2009;Dun et al., 2009).Because the rms2 mutant showed low RMS1 expression and high X-CK content in the xylem sap, in contrast to the other rms mutants (Table I), it was suggested that the shoot-to-root feedback signal was RMS2 dependent (Beveridge et al., 2000).Experiments with grafted plants bearing two shoots of different genotypes and different phenotypes indicated that the feedback signal was more likely generated by the branching shoot to suppress X-CK and up-regulate RMS1 expression, even in the presence of a nonbranching wild-type shoot.Moreover, experiments with mutants lacking axillary meristems in an rms4 background demonstrated that branching per se was not the cause of the feedback (Foo et al., 2001(Foo et al., , 2007)).It was instead suggested to be regulated by the absence of perception of SL (Foo et al., 2005;Dun et al., 2009), as various mutants unable to produce (rms1 and rms5) or to respond to (rms3 and rms4) SL have reduced export of X-CK from the roots (Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007) and increased expression of SL biosynthesis genes (Foo et al., 2005;Johnson et al., 2006;Arite et al., 2007;Hayward et al., 2009).Here, we showed that Psbrc1 Cam lacks the feedback upregulation of RMS1 gene expression and that the substantial down-regulation of X-CK content in SL biosynthesis and signaling mutants was not found in Psbrc1 Cam plants (Table I).Instead, X-CK was either nearly normal in Psbrc1 Cam or substantially increased in Psbrc1 Te mutant plants, with the response appearing to depend on genetic background in our experiments. Similarly, in rice, D10 expression was not increased in the fc1 mutant, whereas it was highly expressed in other SL-related mutants (Arite et al., 2007;Minakuchi et al., 2010).As discussed above, our results suggest that PsBRC1 acts downstream of RMS4 in the SL signaling pathway.The absence of feedback in Psbrc1 could suggest that the SL-mediated feedback regulation is generated between the action of RMS4 and PsBRC1.This is further supported by the feedback down-regulation of X-CK in Psbrc1 rms1 double and rms1 single mutants but not in Psbrc1 Te single mutants. Advances in plant hormone signaling have highlighted the role of the ubiquitin-proteasome pathway and targeted protein turnover (Santner and Estelle, 2009).Because the MAX2/RMS4 gene encodes an F-box protein (Stirnberg et al., 2002;Johnson et al., 2006), it is likely that the SL signaling pathway involves similarly targeted protein degradation.In the GA signaling pathway, DELLA proteins are repressors that act directly downstream of the GA receptor.Microarray analysis has identified early GA-and DELLA-responsive genes (Zentella et al., 2007).Among the GA-repressed and DELLA-induced targets are genes encoding GA biosynthetic enzymes, indicating the direct involvement of DELLA proteins in feedback (Domagalska and Leyser, 2011).PsBRC1 integrates the SL and CK pathways to control bud outgrowth.CK also increases bud growth via a PsBRC1-independent pathway.Auxin maintains RMS1 transcript levels, and hence SL synthesis, and down-regulates CK levels.The RMS2-dependent feedback, which upregulates SL biosynthesis and down-regulates xylem CK, is activated when there is a lack of SL signaling via RMS4 and may be independent of PsBRC1.SLs reduce PIN accumulation to the plasma membrane via RMS4 but independently of PsBRC1 and, by reducing the effectiveness of the canalization feedback loop, enhance the competition between active buds. regulation.Proteins targeted for degradation in the SL signaling pathway, still to be identified, and possibly acting as repressors of PsBRC1 may similarly regulate the feedback signal in pea controlling SL biosynthesis and X-CK.

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > CONCLUSION This study shows that the PsBRC1 transcription factor acts locally in the axillary bud and strongly supports that it acts downstream of SL to repress bud outgrowth.Two models are currently proposed to explain how auxin and SLs interact to control branching: the auxin transport canalization model and the second messenger model (Brewer et al., 2009;Domagalska and Leyser, 2011).The precise comparison of the Psbrc1 Te mutant phenotype with the SL synthesis (rms1) mutant suggests that processes within the stem may be affected in SL synthesis mutants but not in Psbrc1 Te , one of these processes being PAT.The model that best fits with our data would be one in which both regulatory systems, not mutually exclusive, as indicated previously (Domagalska and Leyser, 2011), would coexist (Fig. 6).The degree of branching of a plant is an important component of its fitness, and it is very likely that different pathways are involved in its tight control according to bud position on the stem, developmental stage, and environmental conditions.SL would act locally in axillary buds via PsBRC1 and would also coordinate branching across the plant (Leyser, 2011) by controlling auxin transport independently of PsBRC1.The study of PsBRC1 gives an example of a transcription factor with more limited phenotypic effects than its upstream signaling genes.It indicates that the SL signaling pathway may be shared among multiple developmental modules, as proposed by Doebley and Lukens (1998).Future studies will decipher these different components of SL function.

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > SL Sampling and Analysis Pea plants were germinated in vermiculite for 6 d and then transferred to aerated hydroponic complete nutrient solution culture, with 12 plants in 6 L of solution in a growth cabinet set at 23°C, 55% relative humidity during the day and 15°C, 65% relative humidity at night, with a 16-h photoperiod and light intensity of 300 mmol m 22 s 21 provided by cool-white fluorescent lamps supplemented with tungsten lamps.At 19 d after germination, the solution was replaced with water, then 24 h later, batches of 12 plants were transferred to 900 mL of water into which exudate was collected for 24 h.Deuteriumlabeled SL internal standards (20 ng each of d 1 -orobanchol, d 1 -orobanchyl acetate, d 1 -epiorobanchyl acetate, d 1 -fabacyl acetate, d 6 -5-deoxystrigol, and d 6 -epi-5-deoxystrigol; all generous gifts of Koichi Yoneyama) were added to each sample.SLs were extracted with 0.6 volume of ethyl acetate, followed by back-extraction with 0.1 M KH 2 PO 4 .The ethyl acetate fraction was dried with anhydrous MgSO 4 , filtered, and evaporated to dryness at 35°C.Samples were redissolved in dry acetone, transferred to autosampler vials, redried, dissolved in acetonitrile:water (30:70, v/v), and filtered (0.45 mm) prior to analysis by liquid chromatography-mass spectrometry multiple reaction monitoring (MRM) in positive ion electrospray mode using the Agilent 1100 LC System and an Applied Biosystems Sciex QTrap mass spectrometer.The column was Phenomenex 3 mm C18 Luna 100 3 2 mm, heated to 40°C with a flow rate of 200 mL min 21 .The initial mobile phase was 45.5% acetonitrile in 0.1% aqueous formic acid.After 1 min, a linear gradient to 77% acetonitrile over 19 min was applied, then increased to 95% acetonitrile for 3 min.Appropriate MRM transitions were monitored for each labeled standard and corresponding unlabeled SL.For the quantitations reported here, the transitions were mass-to-charge ratio 406 to 232 for d 1 -fabacyl acetate and 405 to 231 for fabacyl acetate.Two biological replicates representing pools of 12 plants were analyzed for each genotype.SL content was calculated from MRM peak areas by the stable isotope ratio method.CYC/ TB1 CIN Class II LjCYC3 LjCYC1 LjCYC2 AmCYC AtTCP1 AtBRC2 OsTB1 ZmTB1 SbTB1 AtBRC1 PsBRC1 LjCYC5 OsPCF6 AtTCP24 AtTCP17 AtTCP4 AtTCP10 OsPCF5 AmCIN AtTCP16 87 100 35 33 99 94 59 98 89 77 87 84 82 62 64 39 78 Class I Figure S1.Phylogenetic tree of representative TCP proteins from Arabidopsis thaliana (At), PsBRC1 from pea and members of other plant species, Antirrhinum majus (Am), Lotus japonicus (Lj), rice (Os), maize (Zm), Sorghum bicolor (Sb).Evolutionary relationships were analyzed using a selection of predicted amino acid sequences of the TCP domains aligned with the program CLUSTALX ( Thompson et al., 1997).The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987) and the optimal tree was generated.Phylogenetic analyses were conducted in MEGA4 (Tamura et al., 2007).Arabidopsis and rice TCP genes named after (Martin-Trillo and Cubas, 2010); LjCYC1, LjCYC2, LjCYC3, and LjCYC5 (GenBank accession nos DQ202475, DQ202476, DQ202477 and DQ202478).0 20 40 60 80 Internode length between nodes 1 to 6 (mm) Psbrc1 Té Caméor (WT) Psbrc1 Cam Térèse (WT) rms1 C D Psbrc1 Cam Caméor WT Branch length at each node (mm). Figure: 1 Figure 1 . Figure 1.Structure of the PsBRC1 gene and phenotype of the corresponding mutant.A, Gene structure of PsBRC1 and locations of mutations.Bases are numbered from the start codon.The TCP domain (from bp 441 to 630) is shown in red, and the corresponding protein sequence is indicated below.Point mutations are indicated by triangles (black and red for the one studied here), boxes correspond to exons, and the blue area corresponds to the TILLed sequence.B, Comparison of a wild-type (WT) Te ´re `se plant (left) with rms4 (middle left), wild-type Came ´or (middle right), and Psbrc1 Cam (right).C, Branch length at each node of wild-type Came ´or, Psbrc1 Cam , wildtype Te ´re `se, rms1, and Psbrc1 Te .Data are means 6 SE (n = 12).D, Means of the number of cotyledonary branches per individual observed in a segregating F2 population of 103 plants between M3T-884 (rms1) and a Psbrc1 F2 plant (Psbrc1 Cam 3 Te ´re `se).n = the number of plants observed per genotypic class; ncot = the number of plants with at least one cotyledonary branch.Data are means 6 SE (n = 12). Figure: 2 Figure 2 . Figure 2. A, Effects of GR24 application on bud growth.Bud length at node 3 of wild-type Te ´re `se (black bars), rms1 (white bars), wild-type Came ´or (gray bars), and Psbrc1 Cam (hatched bars) was measured 10 d after treatment was applied to buds of stage 5 intact plants with solution containing 0 or 500 nM GR24.Data are means 6 SE (n = 12).B, SL levels in root exudates of wild-type and Psbrc1 plants.Exudates were collected into water for 24 h from 20-d-old hydroponically grown plants, and SLs were quantified by LC-MS using MRM transitions at mass-to-charge ratio 405 to 231 for fabacyl acetate and 406 to 232 for the d 1 -fabacyl acetate internal standard.Data are means 6 SE, based on analyses of two independent pools of 12 plants for each genotype. Figure: 3 Figure 3 . Figure 3. Effects of GR24 on PsBRC1 transcript levels.PsBRC1 transcript levels were determined relative to EF1a in axillary buds at node 4 after GR24 application (white bars) or mock treatment (black bars).RNA was extracted from dissected buds from pools of 30 plants at the six-node stage and quantified by real-time PCR.The data are representative of two to three independent experiments.A, Six and 24 h after GR24 application in wild-type Te ´re `se, rms1, rms2, and rms4 plants.The branching phenotype at node 4 after GR24 application is given below for each genotype.B, Six and 48 h after GR24 application in wild-type Te ´re `se, rms1, rms4, and Psbrc1 Te ´plants.Data are means 6 SE (n = 3). Figure: 4 Figure 4 . Figure 4. Effects of BAP on PsBRC1 transcript levels and on bud growth.A, PsBRC1 transcript levels were determined relative to EF1a in axillary buds at node 4 after BAP (50 mM) application in wild-type Came ´or, Psbrc1 Cam , wild-type Te ´re `se, rms1, and rms4.RNA was extracted from the dissected buds of 30 plants at the six-node stage and quantified by real-time PCR.The data are representative of three independent experiments.B, Effects of BAP (50 mM) treatment on bud growth at node 4 in wild-type Te ´re `se, rms4, wild-type Came ´or, and Psbrc1 Cam .Measurements were done 5 d after treatment.Data are means 6 SE (n = 12).C, Effects of decapitation above node 5 on total branch length at nodes 1 to 5. Intact and decapitated plants of Psbrc1 Cam , wild-type Came ´or, rms1, and wild-type Te ´re `se were used.Data are means 6 SE (n = 8).

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > The RMS2-Dependent Feedback Signal Originates between RMS4 and PsBRC1 The existence of a mobile graft-transmissible auxinindependent shoot-to-root feedback signal that up-regulates RMS1 and RMS5 transcription and downregulates X-CK export from roots was previously proposed (Beveridge et al., 2009;Dun et al., 2009).Because the rms2 mutant showed low RMS1 expression and high X-CK content in the xylem sap, in contrast to the other rms mutants (Table I), it was suggested that the shoot-to-root feedback signal was RMS2 dependent (Beveridge et al., 2000).Experiments with grafted plants bearing two shoots of different genotypes and different phenotypes indicated that the feedback signal was more likely generated by the branching shoot to suppress X-CK and up-regulate RMS1 expression, even in the presence of a nonbranching wild-type shoot.Moreover, experiments with mutants lacking axillary meristems in an rms4 background demonstrated that branching per se was not the cause of the feedback (Foo et al., 2001(Foo et al., , 2007)).It was instead suggested to be regulated by the absence of perception of SL (Foo et al., 2005;Dun et al., 2009), as various mutants unable to produce (rms1 and rms5) or to respond to (rms3 and rms4) SL have reduced export of X-CK from the roots (Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007) and increased expression of SL biosynthesis genes (Foo et al., 2005;Johnson et al., 2006;Arite et al., 2007;Hayward et al., 2009).Here, we showed that Psbrc1 Cam lacks the feedback upregulation of RMS1 gene expression and that the substantial down-regulation of X-CK content in SL biosynthesis and signaling mutants was not found in Psbrc1 Cam plants (Table I).Instead, X-CK was either nearly normal in Psbrc1 Cam or substantially increased in Psbrc1 Te mutant plants, with the response appearing to depend on genetic background in our experiments. Similarly, in rice, D10 expression was not increased in the fc1 mutant, whereas it was highly expressed in other SL-related mutants (Arite et al., 2007;Minakuchi et al., 2010).As discussed above, our results suggest that PsBRC1 acts downstream of RMS4 in the SL signaling pathway.The absence of feedback in Psbrc1 could suggest that the SL-mediated feedback regulation is generated between the action of RMS4 and PsBRC1.This is further supported by the feedback down-regulation of X-CK in Psbrc1 rms1 double and rms1 single mutants but not in Psbrc1 Te single mutants. Advances in plant hormone signaling have highlighted the role of the ubiquitin-proteasome pathway and targeted protein turnover (Santner and Estelle, 2009).Because the MAX2/RMS4 gene encodes an F-box protein (Stirnberg et al., 2002;Johnson et al., 2006), it is likely that the SL signaling pathway involves similarly targeted protein degradation.In the GA signaling pathway, DELLA proteins are repressors that act directly downstream of the GA receptor.Microarray analysis has identified early GA-and DELLA-responsive genes (Zentella et al., 2007).Among the GA-repressed and DELLA-induced targets are genes encoding GA biosynthetic enzymes, indicating the direct involvement of DELLA proteins in feedback (Domagalska and Leyser, 2011).PsBRC1 integrates the SL and CK pathways to control bud outgrowth.CK also increases bud growth via a PsBRC1-independent pathway.Auxin maintains RMS1 transcript levels, and hence SL synthesis, and down-regulates CK levels.The RMS2-dependent feedback, which upregulates SL biosynthesis and down-regulates xylem CK, is activated when there is a lack of SL signaling via RMS4 and may be independent of PsBRC1.SLs reduce PIN accumulation to the plasma membrane via RMS4 but independently of PsBRC1 and, by reducing the effectiveness of the canalization feedback loop, enhance the competition between active buds. regulation.Proteins targeted for degradation in the SL signaling pathway, still to be identified, and possibly acting as repressors of PsBRC1 may similarly regulate the feedback signal in pea controlling SL biosynthesis and X-CK.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > CONCLUSION This study shows that the PsBRC1 transcription factor acts locally in the axillary bud and strongly supports that it acts downstream of SL to repress bud outgrowth.Two models are currently proposed to explain how auxin and SLs interact to control branching: the auxin transport canalization model and the second messenger model (Brewer et al., 2009;Domagalska and Leyser, 2011).The precise comparison of the Psbrc1 Te mutant phenotype with the SL synthesis (rms1) mutant suggests that processes within the stem may be affected in SL synthesis mutants but not in Psbrc1 Te , one of these processes being PAT.The model that best fits with our data would be one in which both regulatory systems, not mutually exclusive, as indicated previously (Domagalska and Leyser, 2011), would coexist (Fig. 6).The degree of branching of a plant is an important component of its fitness, and it is very likely that different pathways are involved in its tight control according to bud position on the stem, developmental stage, and environmental conditions.SL would act locally in axillary buds via PsBRC1 and would also coordinate branching across the plant (Leyser, 2011) by controlling auxin transport independently of PsBRC1.The study of PsBRC1 gives an example of a transcription factor with more limited phenotypic effects than its upstream signaling genes.It indicates that the SL signaling pathway may be shared among multiple developmental modules, as proposed by Doebley and Lukens (1998).Future studies will decipher these different components of SL function.

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > The RMS2-Dependent Feedback Signal Originates between RMS4 and PsBRC1 The existence of a mobile graft-transmissible auxinindependent shoot-to-root feedback signal that up-regulates RMS1 and RMS5 transcription and downregulates X-CK export from roots was previously proposed (Beveridge et al., 2009;Dun et al., 2009).Because the rms2 mutant showed low RMS1 expression and high X-CK content in the xylem sap, in contrast to the other rms mutants (Table I), it was suggested that the shoot-to-root feedback signal was RMS2 dependent (Beveridge et al., 2000).Experiments with grafted plants bearing two shoots of different genotypes and different phenotypes indicated that the feedback signal was more likely generated by the branching shoot to suppress X-CK and up-regulate RMS1 expression, even in the presence of a nonbranching wild-type shoot.Moreover, experiments with mutants lacking axillary meristems in an rms4 background demonstrated that branching per se was not the cause of the feedback (Foo et al., 2001(Foo et al., , 2007)).It was instead suggested to be regulated by the absence of perception of SL (Foo et al., 2005;Dun et al., 2009), as various mutants unable to produce (rms1 and rms5) or to respond to (rms3 and rms4) SL have reduced export of X-CK from the roots (Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007) and increased expression of SL biosynthesis genes (Foo et al., 2005;Johnson et al., 2006;Arite et al., 2007;Hayward et al., 2009).Here, we showed that Psbrc1 Cam lacks the feedback upregulation of RMS1 gene expression and that the substantial down-regulation of X-CK content in SL biosynthesis and signaling mutants was not found in Psbrc1 Cam plants (Table I).Instead, X-CK was either nearly normal in Psbrc1 Cam or substantially increased in Psbrc1 Te mutant plants, with the response appearing to depend on genetic background in our experiments. Similarly, in rice, D10 expression was not increased in the fc1 mutant, whereas it was highly expressed in other SL-related mutants (Arite et al., 2007;Minakuchi et al., 2010).As discussed above, our results suggest that PsBRC1 acts downstream of RMS4 in the SL signaling pathway.The absence of feedback in Psbrc1 could suggest that the SL-mediated feedback regulation is generated between the action of RMS4 and PsBRC1.This is further supported by the feedback down-regulation of X-CK in Psbrc1 rms1 double and rms1 single mutants but not in Psbrc1 Te single mutants. Advances in plant hormone signaling have highlighted the role of the ubiquitin-proteasome pathway and targeted protein turnover (Santner and Estelle, 2009).Because the MAX2/RMS4 gene encodes an F-box protein (Stirnberg et al., 2002;Johnson et al., 2006), it is likely that the SL signaling pathway involves similarly targeted protein degradation.In the GA signaling pathway, DELLA proteins are repressors that act directly downstream of the GA receptor.Microarray analysis has identified early GA-and DELLA-responsive genes (Zentella et al., 2007).Among the GA-repressed and DELLA-induced targets are genes encoding GA biosynthetic enzymes, indicating the direct involvement of DELLA proteins in feedback (Domagalska and Leyser, 2011).PsBRC1 integrates the SL and CK pathways to control bud outgrowth.CK also increases bud growth via a PsBRC1-independent pathway.Auxin maintains RMS1 transcript levels, and hence SL synthesis, and down-regulates CK levels.The RMS2-dependent feedback, which upregulates SL biosynthesis and down-regulates xylem CK, is activated when there is a lack of SL signaling via RMS4 and may be independent of PsBRC1.SLs reduce PIN accumulation to the plasma membrane via RMS4 but independently of PsBRC1 and, by reducing the effectiveness of the canalization feedback loop, enhance the competition between active buds. regulation.Proteins targeted for degradation in the SL signaling pathway, still to be identified, and possibly acting as repressors of PsBRC1 may similarly regulate the feedback signal in pea controlling SL biosynthesis and X-CK.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > CONCLUSION This study shows that the PsBRC1 transcription factor acts locally in the axillary bud and strongly supports that it acts downstream of SL to repress bud outgrowth.Two models are currently proposed to explain how auxin and SLs interact to control branching: the auxin transport canalization model and the second messenger model (Brewer et al., 2009;Domagalska and Leyser, 2011).The precise comparison of the Psbrc1 Te mutant phenotype with the SL synthesis (rms1) mutant suggests that processes within the stem may be affected in SL synthesis mutants but not in Psbrc1 Te , one of these processes being PAT.The model that best fits with our data would be one in which both regulatory systems, not mutually exclusive, as indicated previously (Domagalska and Leyser, 2011), would coexist (Fig. 6).The degree of branching of a plant is an important component of its fitness, and it is very likely that different pathways are involved in its tight control according to bud position on the stem, developmental stage, and environmental conditions.SL would act locally in axillary buds via PsBRC1 and would also coordinate branching across the plant (Leyser, 2011) by controlling auxin transport independently of PsBRC1.The study of PsBRC1 gives an example of a transcription factor with more limited phenotypic effects than its upstream signaling genes.It indicates that the SL signaling pathway may be shared among multiple developmental modules, as proposed by Doebley and Lukens (1998).Future studies will decipher these different components of SL function.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > SL Sampling and Analysis Pea plants were germinated in vermiculite for 6 d and then transferred to aerated hydroponic complete nutrient solution culture, with 12 plants in 6 L of solution in a growth cabinet set at 23°C, 55% relative humidity during the day and 15°C, 65% relative humidity at night, with a 16-h photoperiod and light intensity of 300 mmol m 22 s 21 provided by cool-white fluorescent lamps supplemented with tungsten lamps.At 19 d after germination, the solution was replaced with water, then 24 h later, batches of 12 plants were transferred to 900 mL of water into which exudate was collected for 24 h.Deuteriumlabeled SL internal standards (20 ng each of d 1 -orobanchol, d 1 -orobanchyl acetate, d 1 -epiorobanchyl acetate, d 1 -fabacyl acetate, d 6 -5-deoxystrigol, and d 6 -epi-5-deoxystrigol; all generous gifts of Koichi Yoneyama) were added to each sample.SLs were extracted with 0.6 volume of ethyl acetate, followed by back-extraction with 0.1 M KH 2 PO 4 .The ethyl acetate fraction was dried with anhydrous MgSO 4 , filtered, and evaporated to dryness at 35°C.Samples were redissolved in dry acetone, transferred to autosampler vials, redried, dissolved in acetonitrile:water (30:70, v/v), and filtered (0.45 mm) prior to analysis by liquid chromatography-mass spectrometry multiple reaction monitoring (MRM) in positive ion electrospray mode using the Agilent 1100 LC System and an Applied Biosystems Sciex QTrap mass spectrometer.The column was Phenomenex 3 mm C18 Luna 100 3 2 mm, heated to 40°C with a flow rate of 200 mL min 21 .The initial mobile phase was 45.5% acetonitrile in 0.1% aqueous formic acid.After 1 min, a linear gradient to 77% acetonitrile over 19 min was applied, then increased to 95% acetonitrile for 3 min.Appropriate MRM transitions were monitored for each labeled standard and corresponding unlabeled SL.For the quantitations reported here, the transitions were mass-to-charge ratio 406 to 232 for d 1 -fabacyl acetate and 405 to 231 for fabacyl acetate.Two biological replicates representing pools of 12 plants were analyzed for each genotype.SL content was calculated from MRM peak areas by the stable isotope ratio method.CYC/ TB1 CIN Class II LjCYC3 LjCYC1 LjCYC2 AmCYC AtTCP1 AtBRC2 OsTB1 ZmTB1 SbTB1 AtBRC1 PsBRC1 LjCYC5 OsPCF6 AtTCP24 AtTCP17 AtTCP4 AtTCP10 OsPCF5 AmCIN AtTCP16 87 100 35 33 99 94 59 98 89 77 87 84 82 62 64 39 78 Class I Figure S1.Phylogenetic tree of representative TCP proteins from Arabidopsis thaliana (At), PsBRC1 from pea and members of other plant species, Antirrhinum majus (Am), Lotus japonicus (Lj), rice (Os), maize (Zm), Sorghum bicolor (Sb).Evolutionary relationships were analyzed using a selection of predicted amino acid sequences of the TCP domains aligned with the program CLUSTALX ( Thompson et al., 1997).The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987) and the optimal tree was generated.Phylogenetic analyses were conducted in MEGA4 (Tamura et al., 2007).Arabidopsis and rice TCP genes named after (Martin-Trillo and Cubas, 2010); LjCYC1, LjCYC2, LjCYC3, and LjCYC5 (GenBank accession nos DQ202475, DQ202476, DQ202477 and DQ202478).0 20 40 60 80 Internode length between nodes 1 to 6 (mm) Psbrc1 Té Caméor (WT) Psbrc1 Cam Térèse (WT) rms1 C D Psbrc1 Cam Caméor WT Branch length at each node (mm). Figure: 1 Figure 1 . Figure 1.Structure of the PsBRC1 gene and phenotype of the corresponding mutant.A, Gene structure of PsBRC1 and locations of mutations.Bases are numbered from the start codon.The TCP domain (from bp 441 to 630) is shown in red, and the corresponding protein sequence is indicated below.Point mutations are indicated by triangles (black and red for the one studied here), boxes correspond to exons, and the blue area corresponds to the TILLed sequence.B, Comparison of a wild-type (WT) Te ´re `se plant (left) with rms4 (middle left), wild-type Came ´or (middle right), and Psbrc1 Cam (right).C, Branch length at each node of wild-type Came ´or, Psbrc1 Cam , wildtype Te ´re `se, rms1, and Psbrc1 Te .Data are means 6 SE (n = 12).D, Means of the number of cotyledonary branches per individual observed in a segregating F2 population of 103 plants between M3T-884 (rms1) and a Psbrc1 F2 plant (Psbrc1 Cam 3 Te ´re `se).n = the number of plants observed per genotypic class; ncot = the number of plants with at least one cotyledonary branch.Data are means 6 SE (n = 12). Figure: 2 Figure 2 . Figure 2. A, Effects of GR24 application on bud growth.Bud length at node 3 of wild-type Te ´re `se (black bars), rms1 (white bars), wild-type Came ´or (gray bars), and Psbrc1 Cam (hatched bars) was measured 10 d after treatment was applied to buds of stage 5 intact plants with solution containing 0 or 500 nM GR24.Data are means 6 SE (n = 12).B, SL levels in root exudates of wild-type and Psbrc1 plants.Exudates were collected into water for 24 h from 20-d-old hydroponically grown plants, and SLs were quantified by LC-MS using MRM transitions at mass-to-charge ratio 405 to 231 for fabacyl acetate and 406 to 232 for the d 1 -fabacyl acetate internal standard.Data are means 6 SE, based on analyses of two independent pools of 12 plants for each genotype. Figure: 3 Figure 3 . Figure 3. Effects of GR24 on PsBRC1 transcript levels.PsBRC1 transcript levels were determined relative to EF1a in axillary buds at node 4 after GR24 application (white bars) or mock treatment (black bars).RNA was extracted from dissected buds from pools of 30 plants at the six-node stage and quantified by real-time PCR.The data are representative of two to three independent experiments.A, Six and 24 h after GR24 application in wild-type Te ´re `se, rms1, rms2, and rms4 plants.The branching phenotype at node 4 after GR24 application is given below for each genotype.B, Six and 48 h after GR24 application in wild-type Te ´re `se, rms1, rms4, and Psbrc1 Te ´plants.Data are means 6 SE (n = 3). Figure: 4 Figure 4 . Figure 4. Effects of BAP on PsBRC1 transcript levels and on bud growth.A, PsBRC1 transcript levels were determined relative to EF1a in axillary buds at node 4 after BAP (50 mM) application in wild-type Came ´or, Psbrc1 Cam , wild-type Te ´re `se, rms1, and rms4.RNA was extracted from the dissected buds of 30 plants at the six-node stage and quantified by real-time PCR.The data are representative of three independent experiments.B, Effects of BAP (50 mM) treatment on bud growth at node 4 in wild-type Te ´re `se, rms4, wild-type Came ´or, and Psbrc1 Cam .Measurements were done 5 d after treatment.Data are means 6 SE (n = 12).C, Effects of decapitation above node 5 on total branch length at nodes 1 to 5. Intact and decapitated plants of Psbrc1 Cam , wild-type Came ´or, rms1, and wild-type Te ´re `se were used.Data are means 6 SE (n = 8).

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > The RMS2-Dependent Feedback Signal Originates between RMS4 and PsBRC1 The existence of a mobile graft-transmissible auxinindependent shoot-to-root feedback signal that up-regulates RMS1 and RMS5 transcription and downregulates X-CK export from roots was previously proposed (Beveridge et al., 2009;Dun et al., 2009).Because the rms2 mutant showed low RMS1 expression and high X-CK content in the xylem sap, in contrast to the other rms mutants (Table I), it was suggested that the shoot-to-root feedback signal was RMS2 dependent (Beveridge et al., 2000).Experiments with grafted plants bearing two shoots of different genotypes and different phenotypes indicated that the feedback signal was more likely generated by the branching shoot to suppress X-CK and up-regulate RMS1 expression, even in the presence of a nonbranching wild-type shoot.Moreover, experiments with mutants lacking axillary meristems in an rms4 background demonstrated that branching per se was not the cause of the feedback (Foo et al., 2001(Foo et al., , 2007)).It was instead suggested to be regulated by the absence of perception of SL (Foo et al., 2005;Dun et al., 2009), as various mutants unable to produce (rms1 and rms5) or to respond to (rms3 and rms4) SL have reduced export of X-CK from the roots (Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007) and increased expression of SL biosynthesis genes (Foo et al., 2005;Johnson et al., 2006;Arite et al., 2007;Hayward et al., 2009).Here, we showed that Psbrc1 Cam lacks the feedback upregulation of RMS1 gene expression and that the substantial down-regulation of X-CK content in SL biosynthesis and signaling mutants was not found in Psbrc1 Cam plants (Table I).Instead, X-CK was either nearly normal in Psbrc1 Cam or substantially increased in Psbrc1 Te mutant plants, with the response appearing to depend on genetic background in our experiments. Similarly, in rice, D10 expression was not increased in the fc1 mutant, whereas it was highly expressed in other SL-related mutants (Arite et al., 2007;Minakuchi et al., 2010).As discussed above, our results suggest that PsBRC1 acts downstream of RMS4 in the SL signaling pathway.The absence of feedback in Psbrc1 could suggest that the SL-mediated feedback regulation is generated between the action of RMS4 and PsBRC1.This is further supported by the feedback down-regulation of X-CK in Psbrc1 rms1 double and rms1 single mutants but not in Psbrc1 Te single mutants. Advances in plant hormone signaling have highlighted the role of the ubiquitin-proteasome pathway and targeted protein turnover (Santner and Estelle, 2009).Because the MAX2/RMS4 gene encodes an F-box protein (Stirnberg et al., 2002;Johnson et al., 2006), it is likely that the SL signaling pathway involves similarly targeted protein degradation.In the GA signaling pathway, DELLA proteins are repressors that act directly downstream of the GA receptor.Microarray analysis has identified early GA-and DELLA-responsive genes (Zentella et al., 2007).Among the GA-repressed and DELLA-induced targets are genes encoding GA biosynthetic enzymes, indicating the direct involvement of DELLA proteins in feedback (Domagalska and Leyser, 2011).PsBRC1 integrates the SL and CK pathways to control bud outgrowth.CK also increases bud growth via a PsBRC1-independent pathway.Auxin maintains RMS1 transcript levels, and hence SL synthesis, and down-regulates CK levels.The RMS2-dependent feedback, which upregulates SL biosynthesis and down-regulates xylem CK, is activated when there is a lack of SL signaling via RMS4 and may be independent of PsBRC1.SLs reduce PIN accumulation to the plasma membrane via RMS4 but independently of PsBRC1 and, by reducing the effectiveness of the canalization feedback loop, enhance the competition between active buds. regulation.Proteins targeted for degradation in the SL signaling pathway, still to be identified, and possibly acting as repressors of PsBRC1 may similarly regulate the feedback signal in pea controlling SL biosynthesis and X-CK.

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > SL Sampling and Analysis Pea plants were germinated in vermiculite for 6 d and then transferred to aerated hydroponic complete nutrient solution culture, with 12 plants in 6 L of solution in a growth cabinet set at 23°C, 55% relative humidity during the day and 15°C, 65% relative humidity at night, with a 16-h photoperiod and light intensity of 300 mmol m 22 s 21 provided by cool-white fluorescent lamps supplemented with tungsten lamps.At 19 d after germination, the solution was replaced with water, then 24 h later, batches of 12 plants were transferred to 900 mL of water into which exudate was collected for 24 h.Deuteriumlabeled SL internal standards (20 ng each of d 1 -orobanchol, d 1 -orobanchyl acetate, d 1 -epiorobanchyl acetate, d 1 -fabacyl acetate, d 6 -5-deoxystrigol, and d 6 -epi-5-deoxystrigol; all generous gifts of Koichi Yoneyama) were added to each sample.SLs were extracted with 0.6 volume of ethyl acetate, followed by back-extraction with 0.1 M KH 2 PO 4 .The ethyl acetate fraction was dried with anhydrous MgSO 4 , filtered, and evaporated to dryness at 35°C.Samples were redissolved in dry acetone, transferred to autosampler vials, redried, dissolved in acetonitrile:water (30:70, v/v), and filtered (0.45 mm) prior to analysis by liquid chromatography-mass spectrometry multiple reaction monitoring (MRM) in positive ion electrospray mode using the Agilent 1100 LC System and an Applied Biosystems Sciex QTrap mass spectrometer.The column was Phenomenex 3 mm C18 Luna 100 3 2 mm, heated to 40°C with a flow rate of 200 mL min 21 .The initial mobile phase was 45.5% acetonitrile in 0.1% aqueous formic acid.After 1 min, a linear gradient to 77% acetonitrile over 19 min was applied, then increased to 95% acetonitrile for 3 min.Appropriate MRM transitions were monitored for each labeled standard and corresponding unlabeled SL.For the quantitations reported here, the transitions were mass-to-charge ratio 406 to 232 for d 1 -fabacyl acetate and 405 to 231 for fabacyl acetate.Two biological replicates representing pools of 12 plants were analyzed for each genotype.SL content was calculated from MRM peak areas by the stable isotope ratio method.CYC/ TB1 CIN Class II LjCYC3 LjCYC1 LjCYC2 AmCYC AtTCP1 AtBRC2 OsTB1 ZmTB1 SbTB1 AtBRC1 PsBRC1 LjCYC5 OsPCF6 AtTCP24 AtTCP17 AtTCP4 AtTCP10 OsPCF5 AmCIN AtTCP16 87 100 35 33 99 94 59 98 89 77 87 84 82 62 64 39 78 Class I Figure S1.Phylogenetic tree of representative TCP proteins from Arabidopsis thaliana (At), PsBRC1 from pea and members of other plant species, Antirrhinum majus (Am), Lotus japonicus (Lj), rice (Os), maize (Zm), Sorghum bicolor (Sb).Evolutionary relationships were analyzed using a selection of predicted amino acid sequences of the TCP domains aligned with the program CLUSTALX ( Thompson et al., 1997).The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987) and the optimal tree was generated.Phylogenetic analyses were conducted in MEGA4 (Tamura et al., 2007).Arabidopsis and rice TCP genes named after (Martin-Trillo and Cubas, 2010); LjCYC1, LjCYC2, LjCYC3, and LjCYC5 (GenBank accession nos DQ202475, DQ202476, DQ202477 and DQ202478).0 20 40 60 80 Internode length between nodes 1 to 6 (mm) Psbrc1 Té Caméor (WT) Psbrc1 Cam Térèse (WT) rms1 C D Psbrc1 Cam Caméor WT Branch length at each node (mm). Figure: 1 Figure 1 . Figure 1.Structure of the PsBRC1 gene and phenotype of the corresponding mutant.A, Gene structure of PsBRC1 and locations of mutations.Bases are numbered from the start codon.The TCP domain (from bp 441 to 630) is shown in red, and the corresponding protein sequence is indicated below.Point mutations are indicated by triangles (black and red for the one studied here), boxes correspond to exons, and the blue area corresponds to the TILLed sequence.B, Comparison of a wild-type (WT) Te ´re `se plant (left) with rms4 (middle left), wild-type Came ´or (middle right), and Psbrc1 Cam (right).C, Branch length at each node of wild-type Came ´or, Psbrc1 Cam , wildtype Te ´re `se, rms1, and Psbrc1 Te .Data are means 6 SE (n = 12).D, Means of the number of cotyledonary branches per individual observed in a segregating F2 population of 103 plants between M3T-884 (rms1) and a Psbrc1 F2 plant (Psbrc1 Cam 3 Te ´re `se).n = the number of plants observed per genotypic class; ncot = the number of plants with at least one cotyledonary branch.Data are means 6 SE (n = 12). Figure: 2 Figure 2 . Figure 2. A, Effects of GR24 application on bud growth.Bud length at node 3 of wild-type Te ´re `se (black bars), rms1 (white bars), wild-type Came ´or (gray bars), and Psbrc1 Cam (hatched bars) was measured 10 d after treatment was applied to buds of stage 5 intact plants with solution containing 0 or 500 nM GR24.Data are means 6 SE (n = 12).B, SL levels in root exudates of wild-type and Psbrc1 plants.Exudates were collected into water for 24 h from 20-d-old hydroponically grown plants, and SLs were quantified by LC-MS using MRM transitions at mass-to-charge ratio 405 to 231 for fabacyl acetate and 406 to 232 for the d 1 -fabacyl acetate internal standard.Data are means 6 SE, based on analyses of two independent pools of 12 plants for each genotype. Figure: 3 Figure 3 . Figure 3. Effects of GR24 on PsBRC1 transcript levels.PsBRC1 transcript levels were determined relative to EF1a in axillary buds at node 4 after GR24 application (white bars) or mock treatment (black bars).RNA was extracted from dissected buds from pools of 30 plants at the six-node stage and quantified by real-time PCR.The data are representative of two to three independent experiments.A, Six and 24 h after GR24 application in wild-type Te ´re `se, rms1, rms2, and rms4 plants.The branching phenotype at node 4 after GR24 application is given below for each genotype.B, Six and 48 h after GR24 application in wild-type Te ´re `se, rms1, rms4, and Psbrc1 Te ´plants.Data are means 6 SE (n = 3). Figure: 4 Figure 4 . Figure 4. Effects of BAP on PsBRC1 transcript levels and on bud growth.A, PsBRC1 transcript levels were determined relative to EF1a in axillary buds at node 4 after BAP (50 mM) application in wild-type Came ´or, Psbrc1 Cam , wild-type Te ´re `se, rms1, and rms4.RNA was extracted from the dissected buds of 30 plants at the six-node stage and quantified by real-time PCR.The data are representative of three independent experiments.B, Effects of BAP (50 mM) treatment on bud growth at node 4 in wild-type Te ´re `se, rms4, wild-type Came ´or, and Psbrc1 Cam .Measurements were done 5 d after treatment.Data are means 6 SE (n = 12).C, Effects of decapitation above node 5 on total branch length at nodes 1 to 5. Intact and decapitated plants of Psbrc1 Cam , wild-type Came ´or, rms1, and wild-type Te ´re `se were used.Data are means 6 SE (n = 8).

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > SL Sampling and Analysis Pea plants were germinated in vermiculite for 6 d and then transferred to aerated hydroponic complete nutrient solution culture, with 12 plants in 6 L of solution in a growth cabinet set at 23°C, 55% relative humidity during the day and 15°C, 65% relative humidity at night, with a 16-h photoperiod and light intensity of 300 mmol m 22 s 21 provided by cool-white fluorescent lamps supplemented with tungsten lamps.At 19 d after germination, the solution was replaced with water, then 24 h later, batches of 12 plants were transferred to 900 mL of water into which exudate was collected for 24 h.Deuteriumlabeled SL internal standards (20 ng each of d 1 -orobanchol, d 1 -orobanchyl acetate, d 1 -epiorobanchyl acetate, d 1 -fabacyl acetate, d 6 -5-deoxystrigol, and d 6 -epi-5-deoxystrigol; all generous gifts of Koichi Yoneyama) were added to each sample.SLs were extracted with 0.6 volume of ethyl acetate, followed by back-extraction with 0.1 M KH 2 PO 4 .The ethyl acetate fraction was dried with anhydrous MgSO 4 , filtered, and evaporated to dryness at 35°C.Samples were redissolved in dry acetone, transferred to autosampler vials, redried, dissolved in acetonitrile:water (30:70, v/v), and filtered (0.45 mm) prior to analysis by liquid chromatography-mass spectrometry multiple reaction monitoring (MRM) in positive ion electrospray mode using the Agilent 1100 LC System and an Applied Biosystems Sciex QTrap mass spectrometer.The column was Phenomenex 3 mm C18 Luna 100 3 2 mm, heated to 40°C with a flow rate of 200 mL min 21 .The initial mobile phase was 45.5% acetonitrile in 0.1% aqueous formic acid.After 1 min, a linear gradient to 77% acetonitrile over 19 min was applied, then increased to 95% acetonitrile for 3 min.Appropriate MRM transitions were monitored for each labeled standard and corresponding unlabeled SL.For the quantitations reported here, the transitions were mass-to-charge ratio 406 to 232 for d 1 -fabacyl acetate and 405 to 231 for fabacyl acetate.Two biological replicates representing pools of 12 plants were analyzed for each genotype.SL content was calculated from MRM peak areas by the stable isotope ratio method.CYC/ TB1 CIN Class II LjCYC3 LjCYC1 LjCYC2 AmCYC AtTCP1 AtBRC2 OsTB1 ZmTB1 SbTB1 AtBRC1 PsBRC1 LjCYC5 OsPCF6 AtTCP24 AtTCP17 AtTCP4 AtTCP10 OsPCF5 AmCIN AtTCP16 87 100 35 33 99 94 59 98 89 77 87 84 82 62 64 39 78 Class I Figure S1.Phylogenetic tree of representative TCP proteins from Arabidopsis thaliana (At), PsBRC1 from pea and members of other plant species, Antirrhinum majus (Am), Lotus japonicus (Lj), rice (Os), maize (Zm), Sorghum bicolor (Sb).Evolutionary relationships were analyzed using a selection of predicted amino acid sequences of the TCP domains aligned with the program CLUSTALX ( Thompson et al., 1997).The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987) and the optimal tree was generated.Phylogenetic analyses were conducted in MEGA4 (Tamura et al., 2007).Arabidopsis and rice TCP genes named after (Martin-Trillo and Cubas, 2010); LjCYC1, LjCYC2, LjCYC3, and LjCYC5 (GenBank accession nos DQ202475, DQ202476, DQ202477 and DQ202478).0 20 40 60 80 Internode length between nodes 1 to 6 (mm) Psbrc1 Té Caméor (WT) Psbrc1 Cam Térèse (WT) rms1 C D Psbrc1 Cam Caméor WT Branch length at each node (mm). Figure: 1 Figure 1 . Figure 1.Structure of the PsBRC1 gene and phenotype of the corresponding mutant.A, Gene structure of PsBRC1 and locations of mutations.Bases are numbered from the start codon.The TCP domain (from bp 441 to 630) is shown in red, and the corresponding protein sequence is indicated below.Point mutations are indicated by triangles (black and red for the one studied here), boxes correspond to exons, and the blue area corresponds to the TILLed sequence.B, Comparison of a wild-type (WT) Te ´re `se plant (left) with rms4 (middle left), wild-type Came ´or (middle right), and Psbrc1 Cam (right).C, Branch length at each node of wild-type Came ´or, Psbrc1 Cam , wildtype Te ´re `se, rms1, and Psbrc1 Te .Data are means 6 SE (n = 12).D, Means of the number of cotyledonary branches per individual observed in a segregating F2 population of 103 plants between M3T-884 (rms1) and a Psbrc1 F2 plant (Psbrc1 Cam 3 Te ´re `se).n = the number of plants observed per genotypic class; ncot = the number of plants with at least one cotyledonary branch.Data are means 6 SE (n = 12). Figure: 2 Figure 2 . Figure 2. A, Effects of GR24 application on bud growth.Bud length at node 3 of wild-type Te ´re `se (black bars), rms1 (white bars), wild-type Came ´or (gray bars), and Psbrc1 Cam (hatched bars) was measured 10 d after treatment was applied to buds of stage 5 intact plants with solution containing 0 or 500 nM GR24.Data are means 6 SE (n = 12).B, SL levels in root exudates of wild-type and Psbrc1 plants.Exudates were collected into water for 24 h from 20-d-old hydroponically grown plants, and SLs were quantified by LC-MS using MRM transitions at mass-to-charge ratio 405 to 231 for fabacyl acetate and 406 to 232 for the d 1 -fabacyl acetate internal standard.Data are means 6 SE, based on analyses of two independent pools of 12 plants for each genotype. Figure: 3 Figure 3 . Figure 3. Effects of GR24 on PsBRC1 transcript levels.PsBRC1 transcript levels were determined relative to EF1a in axillary buds at node 4 after GR24 application (white bars) or mock treatment (black bars).RNA was extracted from dissected buds from pools of 30 plants at the six-node stage and quantified by real-time PCR.The data are representative of two to three independent experiments.A, Six and 24 h after GR24 application in wild-type Te ´re `se, rms1, rms2, and rms4 plants.The branching phenotype at node 4 after GR24 application is given below for each genotype.B, Six and 48 h after GR24 application in wild-type Te ´re `se, rms1, rms4, and Psbrc1 Te ´plants.Data are means 6 SE (n = 3). Figure: 4 Figure 4 . Figure 4. Effects of BAP on PsBRC1 transcript levels and on bud growth.A, PsBRC1 transcript levels were determined relative to EF1a in axillary buds at node 4 after BAP (50 mM) application in wild-type Came ´or, Psbrc1 Cam , wild-type Te ´re `se, rms1, and rms4.RNA was extracted from the dissected buds of 30 plants at the six-node stage and quantified by real-time PCR.The data are representative of three independent experiments.B, Effects of BAP (50 mM) treatment on bud growth at node 4 in wild-type Te ´re `se, rms4, wild-type Came ´or, and Psbrc1 Cam .Measurements were done 5 d after treatment.Data are means 6 SE (n = 12).C, Effects of decapitation above node 5 on total branch length at nodes 1 to 5. Intact and decapitated plants of Psbrc1 Cam , wild-type Came ´or, rms1, and wild-type Te ´re `se were used.Data are means 6 SE (n = 8).

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > SL Sampling and Analysis Pea plants were germinated in vermiculite for 6 d and then transferred to aerated hydroponic complete nutrient solution culture, with 12 plants in 6 L of solution in a growth cabinet set at 23°C, 55% relative humidity during the day and 15°C, 65% relative humidity at night, with a 16-h photoperiod and light intensity of 300 mmol m 22 s 21 provided by cool-white fluorescent lamps supplemented with tungsten lamps.At 19 d after germination, the solution was replaced with water, then 24 h later, batches of 12 plants were transferred to 900 mL of water into which exudate was collected for 24 h.Deuteriumlabeled SL internal standards (20 ng each of d 1 -orobanchol, d 1 -orobanchyl acetate, d 1 -epiorobanchyl acetate, d 1 -fabacyl acetate, d 6 -5-deoxystrigol, and d 6 -epi-5-deoxystrigol; all generous gifts of Koichi Yoneyama) were added to each sample.SLs were extracted with 0.6 volume of ethyl acetate, followed by back-extraction with 0.1 M KH 2 PO 4 .The ethyl acetate fraction was dried with anhydrous MgSO 4 , filtered, and evaporated to dryness at 35°C.Samples were redissolved in dry acetone, transferred to autosampler vials, redried, dissolved in acetonitrile:water (30:70, v/v), and filtered (0.45 mm) prior to analysis by liquid chromatography-mass spectrometry multiple reaction monitoring (MRM) in positive ion electrospray mode using the Agilent 1100 LC System and an Applied Biosystems Sciex QTrap mass spectrometer.The column was Phenomenex 3 mm C18 Luna 100 3 2 mm, heated to 40°C with a flow rate of 200 mL min 21 .The initial mobile phase was 45.5% acetonitrile in 0.1% aqueous formic acid.After 1 min, a linear gradient to 77% acetonitrile over 19 min was applied, then increased to 95% acetonitrile for 3 min.Appropriate MRM transitions were monitored for each labeled standard and corresponding unlabeled SL.For the quantitations reported here, the transitions were mass-to-charge ratio 406 to 232 for d 1 -fabacyl acetate and 405 to 231 for fabacyl acetate.Two biological replicates representing pools of 12 plants were analyzed for each genotype.SL content was calculated from MRM peak areas by the stable isotope ratio method.CYC/ TB1 CIN Class II LjCYC3 LjCYC1 LjCYC2 AmCYC AtTCP1 AtBRC2 OsTB1 ZmTB1 SbTB1 AtBRC1 PsBRC1 LjCYC5 OsPCF6 AtTCP24 AtTCP17 AtTCP4 AtTCP10 OsPCF5 AmCIN AtTCP16 87 100 35 33 99 94 59 98 89 77 87 84 82 62 64 39 78 Class I Figure S1.Phylogenetic tree of representative TCP proteins from Arabidopsis thaliana (At), PsBRC1 from pea and members of other plant species, Antirrhinum majus (Am), Lotus japonicus (Lj), rice (Os), maize (Zm), Sorghum bicolor (Sb).Evolutionary relationships were analyzed using a selection of predicted amino acid sequences of the TCP domains aligned with the program CLUSTALX ( Thompson et al., 1997).The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987) and the optimal tree was generated.Phylogenetic analyses were conducted in MEGA4 (Tamura et al., 2007).Arabidopsis and rice TCP genes named after (Martin-Trillo and Cubas, 2010); LjCYC1, LjCYC2, LjCYC3, and LjCYC5 (GenBank accession nos DQ202475, DQ202476, DQ202477 and DQ202478).0 20 40 60 80 Internode length between nodes 1 to 6 (mm) Psbrc1 Té Caméor (WT) Psbrc1 Cam Térèse (WT) rms1 C D Psbrc1 Cam Caméor WT Branch length at each node (mm). Figure: 1 Figure 1 . Figure 1.Structure of the PsBRC1 gene and phenotype of the corresponding mutant.A, Gene structure of PsBRC1 and locations of mutations.Bases are numbered from the start codon.The TCP domain (from bp 441 to 630) is shown in red, and the corresponding protein sequence is indicated below.Point mutations are indicated by triangles (black and red for the one studied here), boxes correspond to exons, and the blue area corresponds to the TILLed sequence.B, Comparison of a wild-type (WT) Te ´re `se plant (left) with rms4 (middle left), wild-type Came ´or (middle right), and Psbrc1 Cam (right).C, Branch length at each node of wild-type Came ´or, Psbrc1 Cam , wildtype Te ´re `se, rms1, and Psbrc1 Te .Data are means 6 SE (n = 12).D, Means of the number of cotyledonary branches per individual observed in a segregating F2 population of 103 plants between M3T-884 (rms1) and a Psbrc1 F2 plant (Psbrc1 Cam 3 Te ´re `se).n = the number of plants observed per genotypic class; ncot = the number of plants with at least one cotyledonary branch.Data are means 6 SE (n = 12). Figure: 2 Figure 2 . Figure 2. A, Effects of GR24 application on bud growth.Bud length at node 3 of wild-type Te ´re `se (black bars), rms1 (white bars), wild-type Came ´or (gray bars), and Psbrc1 Cam (hatched bars) was measured 10 d after treatment was applied to buds of stage 5 intact plants with solution containing 0 or 500 nM GR24.Data are means 6 SE (n = 12).B, SL levels in root exudates of wild-type and Psbrc1 plants.Exudates were collected into water for 24 h from 20-d-old hydroponically grown plants, and SLs were quantified by LC-MS using MRM transitions at mass-to-charge ratio 405 to 231 for fabacyl acetate and 406 to 232 for the d 1 -fabacyl acetate internal standard.Data are means 6 SE, based on analyses of two independent pools of 12 plants for each genotype. Figure: 3 Figure 3 . Figure 3. Effects of GR24 on PsBRC1 transcript levels.PsBRC1 transcript levels were determined relative to EF1a in axillary buds at node 4 after GR24 application (white bars) or mock treatment (black bars).RNA was extracted from dissected buds from pools of 30 plants at the six-node stage and quantified by real-time PCR.The data are representative of two to three independent experiments.A, Six and 24 h after GR24 application in wild-type Te ´re `se, rms1, rms2, and rms4 plants.The branching phenotype at node 4 after GR24 application is given below for each genotype.B, Six and 48 h after GR24 application in wild-type Te ´re `se, rms1, rms4, and Psbrc1 Te ´plants.Data are means 6 SE (n = 3). Figure: 4 Figure 4 . Figure 4. Effects of BAP on PsBRC1 transcript levels and on bud growth.A, PsBRC1 transcript levels were determined relative to EF1a in axillary buds at node 4 after BAP (50 mM) application in wild-type Came ´or, Psbrc1 Cam , wild-type Te ´re `se, rms1, and rms4.RNA was extracted from the dissected buds of 30 plants at the six-node stage and quantified by real-time PCR.The data are representative of three independent experiments.B, Effects of BAP (50 mM) treatment on bud growth at node 4 in wild-type Te ´re `se, rms4, wild-type Came ´or, and Psbrc1 Cam .Measurements were done 5 d after treatment.Data are means 6 SE (n = 12).C, Effects of decapitation above node 5 on total branch length at nodes 1 to 5. Intact and decapitated plants of Psbrc1 Cam , wild-type Came ´or, rms1, and wild-type Te ´re `se were used.Data are means 6 SE (n = 8).

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching 1[W] The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated.The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes.The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine.The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth.However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth.In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant.Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1.This phenotype indicates a localized regulation of branching at these nodes specific to pea. Early studies on shoot branching were based on decapitation experiments that emphasized the role of the shoot apex in the inhibition of axillary bud outgrowth (Thimann and Skoog, 1933).In the classical theory of apical dominance, auxin from the apex was proposed to act indirectly to suppress bud outgrowth, while cytokinin (CK) coming from the roots promoted bud outgrowth (Snow, 1937;Sachs and Thimann, 1967;Cline, 1991).More than a decade ago, with the identification and characterization of high-branching mutants in pea (Pisum sativum), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Petunia hybrida, longdistance signaling was shown to be an important process in the control of shoot branching (Ongaro and Leyser, 2008;Beveridge et al., 2009;McSteen, 2009;Beveridge and Kyozuka, 2010).In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK (Beveridge et al., 2000(Beveridge et al., , 2009;;Beveridge, 2006): a root-to-shoot branching inhibitor (Beveridge et al., 1997b;Morris et al., 2001), which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008), and a shoot-to-root feedback signal, which was shown to be auxin independent and has still to be identified (Beveridge et al., 1997a(Beveridge et al., , 2000)).To date, the genetic and physiological model of branching control in pea includes five RAMOSUS genes (RMS1-RMS5).Branching of the pea SL-deficient rms1 and rms5 mutants is suppressed when scions of these mutants are grafted on wild-type rootstock or when the syn-thetic SL GR24 is applied on axillary buds.In contrast, the rms3 and rms4 SL-response mutants are not rescued when grafted to wild-type rootstocks, and they do not respond to GR24 application (Table I; Beveridge et al., 1996Beveridge et al., , 2009;;Dun et al., 2009).All rms mutants, except rms2, have high levels of RMS1 transcripts in epicotyls compared with the wild type (Foo et al., 2005).Moreover, these branching mutants, with the exception of rms2, have greatly reduced amounts of CK in xylem sap (X-CK) compared with wild-type plants (Table I; Beveridge et al., 1997a;Morris et al., 2001;Foo et al., 2007).The reduced X-CK in several rms branching mutants appears to be mediated by a shootto-root mobile signal (Beveridge et al., 1997a;Beveridge, 2000).Because rms2 is the only rms mutant that does not show down-regulation of X-CK, it has been proposed that RMS2 may play a role in the generation of this feedback signal.It was hypothesized that the same signal may also regulate RMS1 transcript levels (Foo et al., 2005), because X-CK and RMS1 transcript levels are typically anticorrelated (Dun et al., 2009).The rms2 mutant does respond to SL (Dun et al., 2009).This mutant, having low transcript levels of RMS1 and slightly elevated X-CK in comparison with the wild type, may branch because of low SL levels and/or high CK content. Among these five RMS genes, three have been cloned and correspond to branching genes identified in other species.The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).While RMS4 transcripts are found in all tissues, the SL biosynthesis RMS1 and RMS5 genes are highly expressed in roots and in the basal stem (Foo et al., 2005;Johnson et al., 2006) and were shown to be finely regulated along the stem (Dun et al., 2009). In the current model of shoot branching in pea, auxin, originating from the main shoot apex, regulates SL levels by maintaining RMS1 and RMS5 transcript levels (Foo et al., 2005;Johnson et al., 2006) and downregulates CK in both xylem sap (Bangerth, 1994;Li et al., 1995) and the stem (Tanaka et al., 2006).Auxin regulation of SL synthesis genes, and bud outgrowth inhibition by SL application in decapitated plants, suggest that SL is the second messenger by which auxin controls branching in decapitation experiments (Brewer et al., 2009).SL and CK would act locally within the axillary bud to control its outgrowth.Another hypothesis has been proposed where SLs would act upstream of auxin by modulating its transport in the main shoot by limiting the accumulation of PIN auxin efflux carrier protein on the plasma membrane of cells involved in the polar auxin transport stream (PATS; Domagalska and Leyser, 2011).Consistent with this, the Arabidopsis branching max mutants show increased polar PIN accumulation and increased auxin transport (Bennett et al., 2006;Prusinkiewicz et al., 2009;Crawford et al., 2010).The rice SL-deficient d27 mutant also shows increased auxin transport (Lin et al., 2009), but this difference is not always observed in pea (Beveridge, 2000(Beveridge, , 2006;;Beveridge et al., 2000;Dun et al., 2006).In the auxin transport model, the auxin exported from active apices, moving in the PATS, would prevent auxin export from dormant buds and, therefore, would block their outgrowth, this process being amplified by the canalization positive feedback.Buds would compete to export auxin into the main stem, and SL, by dampening auxin transport in the PATS, would enhance this competition (Crawford et al., 2010;Domagalska and Leyser, 2011). How SL and CK interact to control axillary bud outgrowth is still not understood, and the role of auxin is still a matter of debate.However, the discovery of SL as a plant hormone allows novel approaches.In particular, the discovery of genes responding to SL application and deciphering SL signaling pathways are essential for a better understanding of the control of branching.Axillary buds have to integrate many factors that influence switching between dormant and growing states during plant ontogeny, and they can respond differently according to their position along the main stem (Cline, 1991).It is very likely that several pathways control axillary bud outgrowth and that important molecular processes within the bud are involved in this control, in particular to integrate the multiple long-distance signals.In this paper, PsBRC1, the pea homolog of TEOSINTE BRANCHED1 (TB1) from maize (Zea mays; Doebley et al., 1997) and of BRANCHED1 (AtBRC1) from Arabidopsis (Aguilar-Martı ´nez et al., 2007;Finlayson, 2007), was integrated into the pea model.This gene is almost exclusively expressed in the axillary bud and may provide the link between systemic signaling and events occurring within the axillary bud to control bud outgrowth. Table I.Summary of the physiological characteristics of the different pea branching mutants RMS1 expression indicates RMS1 mRNA accumulation in epicotyl, and Response to GR24 indicates the inhibition effect of GR24 on bud outgrowth.Characteristic rms1 rms5 rms3 rms4 rms2 Psbrc1 RMS1 expression High High High High Low Low Response to GR24 Yes Yes No No Yes No X-CK Low Low Low Low High Wild type/high Plant height Dwarf Dwarf Dwarf Dwarf Dwarf Near wild type The TB1 gene from maize, which affects plant and inflorescence architecture, is a well-known target for artificial selection during maize domestication from its wild and highly branched ancestor teosinte (Zea mays ssp.parviglumis).It is a striking example of how human selection modified gene expression to change plant architecture.In maize, the repression of branching results from higher TB1 transcript levels in axillary buds in comparison with those in teosinte (Doebley et al., 1997;Wang et al., 1999).Recently, it has been demonstrated that the corresponding gene in barley (Hordeum vulgare; INTERMEDIUM-C) has also been targeted in human selection to regulate not only tillering but also fertility of the lateral spikelet (Ramsay et al., 2011).In monocots, only one homolog of the TB1/CYCLOIDEA (CYC) clade has been identified that regulates axillary bud outgrowth, whereas in Arabidopsis, two homologs (AtBRC1 and AtBRC2) were shown to control shoot branching.These genes belong to the TCP family of transcription factors specific to plants and named for the first three identified members, TB1, CYC in Antirrhinum majus, and PCF-coding genes in rice.Phylogenetic analysis of this family comprising 24 members in Arabidopsis (Martı ´n-Trillo and Cubas, 2010) has identified two distinct classes, classes I and II, with roles in plant development and morphogenesis processes as diverse as the establishment of floral symmetry, plant architecture (Doebley et al., 1997;Aguilar-Martı ´nez et al., 2007), leaf morphogenesis (Nath et al., 2003;Palatnik et al., 2003;Ori et al., 2007) and senescence (Schommer et al., 2008), embryo growth (Tatematsu et al., 2008), and circadian rhythm (Pruneda-Paz et al., 2009;Giraud et al., 2010).Globally, class I TCP proteins appear to promote cell division, whereas class II proteins, containing TB1/AtBRC1, repress organ growth by inhibiting cell proliferation (Martı ´n-Trillo and Cubas, 2010).For example, class I TCP proteins, PCF1 and PCF2, were shown to promote Proliferating Cell Nuclear Antigen (PCNA) gene expression, activating the G1-to-S transition by binding to cis-acting elements in the promoter of PCNA (Kosugi and Ohashi, 1997).Class II TCP proteins, CINCINNATA in Antirrhinum and TCP4 in Arabidopsis, control leaf morphology by repressing cell proliferation specifically in the leaf margins (Nath et al., 2003;Palatnik et al., 2003;Crawford et al., 2004).In yeast, TCP4 has been shown to function in a dosedependent manner and to block the cell cycle at the G1-to-S transition (Aggarwal et al., 2011). In rice, the high-tillering fine culm1 (fc1) mutant is mutated in the rice homolog of TB1 (Takeda et al., 2003;Arite et al., 2007).Both the absence of response of the fc1 mutant to GR24 application and the similarity of the phenotype of the fc1 d17 double mutant to the SL-deficient d17 mutant phenotype suggested that FC1/OsTB1 acts downstream of the SL pathway in rice (Minakuchi et al., 2010).Here, we show that the regulation of pea PsBRC1 exhibits several similarities to and differences from those described in rice and Arabidopsis.PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).In contrast to Arabidopsis (Aguilar-Martı ´nez et al., 2007), branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.However, the response to decapitation and to direct CK application of the Psbrc1 mutant also suggests a PsBRC1-independent component of the CK response in pea. > SL Sampling and Analysis Pea plants were germinated in vermiculite for 6 d and then transferred to aerated hydroponic complete nutrient solution culture, with 12 plants in 6 L of solution in a growth cabinet set at 23°C, 55% relative humidity during the day and 15°C, 65% relative humidity at night, with a 16-h photoperiod and light intensity of 300 mmol m 22 s 21 provided by cool-white fluorescent lamps supplemented with tungsten lamps.At 19 d after germination, the solution was replaced with water, then 24 h later, batches of 12 plants were transferred to 900 mL of water into which exudate was collected for 24 h.Deuteriumlabeled SL internal standards (20 ng each of d 1 -orobanchol, d 1 -orobanchyl acetate, d 1 -epiorobanchyl acetate, d 1 -fabacyl acetate, d 6 -5-deoxystrigol, and d 6 -epi-5-deoxystrigol; all generous gifts of Koichi Yoneyama) were added to each sample.SLs were extracted with 0.6 volume of ethyl acetate, followed by back-extraction with 0.1 M KH 2 PO 4 .The ethyl acetate fraction was dried with anhydrous MgSO 4 , filtered, and evaporated to dryness at 35°C.Samples were redissolved in dry acetone, transferred to autosampler vials, redried, dissolved in acetonitrile:water (30:70, v/v), and filtered (0.45 mm) prior to analysis by liquid chromatography-mass spectrometry multiple reaction monitoring (MRM) in positive ion electrospray mode using the Agilent 1100 LC System and an Applied Biosystems Sciex QTrap mass spectrometer.The column was Phenomenex 3 mm C18 Luna 100 3 2 mm, heated to 40°C with a flow rate of 200 mL min 21 .The initial mobile phase was 45.5% acetonitrile in 0.1% aqueous formic acid.After 1 min, a linear gradient to 77% acetonitrile over 19 min was applied, then increased to 95% acetonitrile for 3 min.Appropriate MRM transitions were monitored for each labeled standard and corresponding unlabeled SL.For the quantitations reported here, the transitions were mass-to-charge ratio 406 to 232 for d 1 -fabacyl acetate and 405 to 231 for fabacyl acetate.Two biological replicates representing pools of 12 plants were analyzed for each genotype.SL content was calculated from MRM peak areas by the stable isotope ratio method.CYC/ TB1 CIN Class II LjCYC3 LjCYC1 LjCYC2 AmCYC AtTCP1 AtBRC2 OsTB1 ZmTB1 SbTB1 AtBRC1 PsBRC1 LjCYC5 OsPCF6 AtTCP24 AtTCP17 AtTCP4 AtTCP10 OsPCF5 AmCIN AtTCP16 87 100 35 33 99 94 59 98 89 77 87 84 82 62 64 39 78 Class I Figure S1.Phylogenetic tree of representative TCP proteins from Arabidopsis thaliana (At), PsBRC1 from pea and members of other plant species, Antirrhinum majus (Am), Lotus japonicus (Lj), rice (Os), maize (Zm), Sorghum bicolor (Sb).Evolutionary relationships were analyzed using a selection of predicted amino acid sequences of the TCP domains aligned with the program CLUSTALX ( Thompson et al., 1997).The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987) and the optimal tree was generated.Phylogenetic analyses were conducted in MEGA4 (Tamura et al., 2007).Arabidopsis and rice TCP genes named after (Martin-Trillo and Cubas, 2010); LjCYC1, LjCYC2, LjCYC3, and LjCYC5 (GenBank accession nos DQ202475, DQ202476, DQ202477 and DQ202478).0 20 40 60 80 Internode length between nodes 1 to 6 (mm) Psbrc1 Té Caméor (WT) Psbrc1 Cam Térèse (WT) rms1 C D Psbrc1 Cam Caméor WT Branch length at each node (mm). Figure: 1 Figure 1 . Figure 1.Structure of the PsBRC1 gene and phenotype of the corresponding mutant.A, Gene structure of PsBRC1 and locations of mutations.Bases are numbered from the start codon.The TCP domain (from bp 441 to 630) is shown in red, and the corresponding protein sequence is indicated below.Point mutations are indicated by triangles (black and red for the one studied here), boxes correspond to exons, and the blue area corresponds to the TILLed sequence.B, Comparison of a wild-type (WT) Te ´re `se plant (left) with rms4 (middle left), wild-type Came ´or (middle right), and Psbrc1 Cam (right).C, Branch length at each node of wild-type Came ´or, Psbrc1 Cam , wildtype Te ´re `se, rms1, and Psbrc1 Te .Data are means 6 SE (n = 12).D, Means of the number of cotyledonary branches per individual observed in a segregating F2 population of 103 plants between M3T-884 (rms1) and a Psbrc1 F2 plant (Psbrc1 Cam 3 Te ´re `se).n = the number of plants observed per genotypic class; ncot = the number of plants with at least one cotyledonary branch.Data are means 6 SE (n = 12). Figure: 2 Figure 2 . Figure 2. A, Effects of GR24 application on bud growth.Bud length at node 3 of wild-type Te ´re `se (black bars), rms1 (white bars), wild-type Came ´or (gray bars), and Psbrc1 Cam (hatched bars) was measured 10 d after treatment was applied to buds of stage 5 intact plants with solution containing 0 or 500 nM GR24.Data are means 6 SE (n = 12).B, SL levels in root exudates of wild-type and Psbrc1 plants.Exudates were collected into water for 24 h from 20-d-old hydroponically grown plants, and SLs were quantified by LC-MS using MRM transitions at mass-to-charge ratio 405 to 231 for fabacyl acetate and 406 to 232 for the d 1 -fabacyl acetate internal standard.Data are means 6 SE, based on analyses of two independent pools of 12 plants for each genotype. Figure: 3 Figure 3 . Figure 3. Effects of GR24 on PsBRC1 transcript levels.PsBRC1 transcript levels were determined relative to EF1a in axillary buds at node 4 after GR24 application (white bars) or mock treatment (black bars).RNA was extracted from dissected buds from pools of 30 plants at the six-node stage and quantified by real-time PCR.The data are representative of two to three independent experiments.A, Six and 24 h after GR24 application in wild-type Te ´re `se, rms1, rms2, and rms4 plants.The branching phenotype at node 4 after GR24 application is given below for each genotype.B, Six and 48 h after GR24 application in wild-type Te ´re `se, rms1, rms4, and Psbrc1 Te ´plants.Data are means 6 SE (n = 3). Figure: 4 Figure 4 . Figure 4. Effects of BAP on PsBRC1 transcript levels and on bud growth.A, PsBRC1 transcript levels were determined relative to EF1a in axillary buds at node 4 after BAP (50 mM) application in wild-type Came ´or, Psbrc1 Cam , wild-type Te ´re `se, rms1, and rms4.RNA was extracted from the dissected buds of 30 plants at the six-node stage and quantified by real-time PCR.The data are representative of three independent experiments.B, Effects of BAP (50 mM) treatment on bud growth at node 4 in wild-type Te ´re `se, rms4, wild-type Came ´or, and Psbrc1 Cam .Measurements were done 5 d after treatment.Data are means 6 SE (n = 12).C, Effects of decapitation above node 5 on total branch length at nodes 1 to 5. Intact and decapitated plants of Psbrc1 Cam , wild-type Came ´or, rms1, and wild-type Te ´re `se were used.Data are means 6 SE (n = 8).