Cited by 486Plant Parasitism and ResistancePlant Molecular Biology ResearchPlant and animal studies
The Pea TCP Transcription Factor PsBRC1 Acts Downstream of Strigolactones to Control Shoot Branching
Open accessBraun, Nils, de Saint Germain, Alexandre, Pillot, Jean-Paul, Boutet-Mercey, Stéphanie, Dalmais, Marion, Antoniadi, Ioanna, Li, Xin, Maia-Grondard, Alessandra +6 more·2012·PLANT PHYSIOLOGY
DOI 10.1104/pp.111.182725PMC PMC3252107
Abstract
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.
Integrity signals
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Crossref · 10.1104/pp.111.182725
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- Premisecitation
In rice, the fc1 mutant is less affected in height and tiller number than dwarf SL mutants.
“In rice, the fc1 mutant is also less affected in height and in tiller number than the dwarf (d) SL mutants (Arite et al., 2007).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites Arite et al., 2007 source
- Premisecitation
The rms2 mutant displays strong basal branching in comparison with rms1.
“The rms2 mutant displays strong basal branching in comparison with rms1 (Dun et al., 2009)”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites Dun et al., 2009 source
The Discussion states that rms2 plants branch mainly at basal nodes, and the Results describe increased basal branching in rms2 plants. Neither passage compares basal branching in rms2 with the rms1 mutant; references to RMS1 here concern gene expression, not an rms1 branching phenotype. (LLM, conf=0.86)
Experimental — accuracy not yet measured
- Premisecitation
In rice, the homolog FC1 was not transcriptionally up-regulated by SL.
“In rice, the homolog of the maize TB1 gene, FC1, was found not to be transcriptionally up-regulated by SL (Minakuchi et al., 2010).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites Minakuchi et al., 2010 source
- Premisecitation
In rice, FC1 expression strongly decreases 3 h after BAP treatment.
“In rice, similar results have been obtained, with a strong decrease of FC1 expression 3 h after BAP treatment (Minakuchi et al., 2010).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites Minakuchi et al., 2010 source
- Premisecitation
In pea, differences in auxin transport between SL-related mutants and the wild type are modest, in contrast to Arabidopsis.
“In pea, differences in auxin transport between SL-related mutants and the wild type are modest (Beveridge et al., 2000), in contrast to Arabidopsis.”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites Beveridge et al., 2000 source
- Findingown data
The low branching at upper nodes in Psbrc1 was a major difference from the rms1 mutant, which showed long branches at each node.
“The low branching at upper nodes observed in Psbrc1 Cam was particularly evident in the Psbrc1 Te mutant and was one major difference from the rms1 mutant, which showed, in comparison, long branches at each node”
Quote in the paperNot a substantial claim - Findingnone given§ 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 SL-deficient rms1 mutant exhibits a relatively dwarf phenotype.
“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.”
Quote in the paperNot a substantial claim - Findingown data
In all segregating populations, the family 4654 mutation always cosegregated with a strong branching phenotype.
“In all segregating populations, this mutation always cosegregated with a strong branching phenotype as in the BC2-F2 population (4654 3 Came ´or), where the 14 Psbrc1 mutant plants displayed a thin stem with a strong branching phenotype at nodes 1 and 2, in contrast to the nine wild-type plants, which had a small branch only at node 2”
Quote in the paperSubstantial claim - Findingown data§ 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 down-regulated by the cytokinin 6-benzylaminopurine.
“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.”
Quote in the paperSubstantial claim - Findingown data
Maximum PsBRC1 transcript levels were found in axillary buds.
“Maximum transcript levels were found in axillary buds.”
Quote in the paperSubstantial claim - Findingown data
Phylogenetic analysis placed the pea sequence in the same clade as TB1 and AtBRC1.
“Phylogenetic analysis based on the protein sequences of the TCP domain of several members of both the CYC-TB1 group and the PCF group placed the pea sequence in the same clade as TB1 and AtBRC1; consequently, the gene was named PsBRC1”
Quote in the paperNot a substantial claim - Findingown data§ 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 double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes.
“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.”
Quote in the paperSubstantial claim - Findingown data
Strong basal branching at nodes 1 and 2 was observed in the Psbrc1 Cam mutant, with often two branches at node 2.
“Strong basal branching at nodes 1 and 2 was observed in the Psbrc1 Cam mutant, with often two branches at node 2, whereas the wild-type Came ´or sometimes showed only a single branch at node 2.”
Quote in the paperNot a substantial claim - Findingown data§ 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 gene is mostly expressed in the axillary bud.
“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.”
Quote in the paperSubstantial claim - Findingown data§ 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.
Branching at upper nodes in the double rms1 Psbrc1 mutant is not significantly higher than branching in rms1.
“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.”
Quote in the paperSubstantial claim - Findingown data
PsBRC1 transcripts were only detected in axillary buds, floral buds, nodal tissue, and the shoot apex.
“Transcripts were only detected in axillary buds, floral buds, nodal tissue, and the shoot apex.”
Quote in the paperSubstantial claim - Findingown data§ 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 displays an increased shoot-branching phenotype.
“The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.”
Quote in the paperSubstantial claim- Rests on an assumption: The T195I mutation in PsBRC1 causes loss of PsBRC1 function.
- Findingown data
Floral buds, shoot apex, and nodal tissue contained 100 to 1,000 times less PsBRC1 transcript than axillary buds.
“Floral buds, shoot apex, and nodal tissue contained very low levels of transcripts, 100 to 1,000 times less than in axillary buds”
Quote in the paperSubstantial claim - Findingown data
The double mutant plants had a high number of cotyledonary branches per plant.
“A striking feature of double mutant plants was the high number of cotyledonary branches per plant.”
Quote in the paperSubstantial claim - Findingown data
The width of the main stem was reduced in both rms1 and Psbrc1 Cam mutants.
“The width of the main stem was reduced in both rms1 and Psbrc1 Cam mutants”
Quote in the paperNot a substantial claim - Findingown data§ 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 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap.
“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.”
Quote in the paperSubstantial claim - Findingown data
No inhibitory effect of GR24 was observed for the wild-type Te ́re `se and Came ́or.
“No inhibitory effect of GR24 was observed for the wild-type Te ´re `se and Came ´or, in which axillary buds are very small at this node.”
Quote in the paperNot a substantial claim - Findingown data§ 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 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length.
“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.”
Quote in the paperSubstantial claim - Findingown data
Among the six amino acid-changing mutations in PsBRC1, only one gave a clear branching phenotype cosegregating with the mutation (family 4654).
“Among these six mutations, only one gave a clear branching phenotype cosegregating with the mutation (family 4654).”
Quote in the paperSubstantial claim - Findingown data§ 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 mutation does not cause a strong increase in RMS1 transcript levels.
“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.”
Quote in the paperSubstantial claim - Findingown data§ 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 SL-deficient rms1 mutant exhibits more extensive branching at upper nodes than the Psbrc1 mutant.
“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.”
Quote in the paperNot a substantial claim - Findingown data
Branching at upper nodes (above node 3) was very low in Psbrc1 Cam.
“Branching at upper nodes (above node 3) was very low in Psbrc1 Cam”
Quote in the paperNot a substantial claim - Findingown data§ 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 does not respond to SL application.
“The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.”
Quote in the paperSubstantial claim - Findingcitation
PsBRC1 maps to pea linkage group IV close to Fa in a region where no RMS genes were ever mapped.
“PsBRC1 was then mapped using the recombinant inbred line population (Te ´re `se 3 Torsdag; Laucou et al., 1998) to the pea linkage group IV close to Fa in a region where no RMS genes were ever mapped”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Laucou et al., 1998 source
- Findingown data§ 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 transcriptionally up-regulated by direct application of the synthetic SL GR24.
“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.”
Quote in the paperSubstantial claim - Findingown data§ 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 is able to synthesize strigolactone (SL).
“The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application.”
Quote in the paperSubstantial claim - Findingown data
Decapitation of rms1 and Psbrc1 Cam mutants and their corresponding wild types resulted in increased branch lengths in all genotypes.
“Decapitation of rms1 and Psbrc1 Cam mutants and their corresponding wild types resulted in increased branch lengths in all genotypes”
Quote in the paperSubstantial claim - Findingown data
In wild-type and rms2 axillary buds, a 2-fold increase in PsBRC1 was observed 24 h after GR24 application.
“In wild-type and rms2 axillary buds, a 2-fold increase was observed 24 h after GR24 application.”
Quote in the paperSubstantial claim - Findingown data
X-CK levels in F3 rms1 Psbrc1 double mutant plants were not significantly different from X-CK levels from F3 rms1 plants.
“X-CK levels in the F3 rms1 Psbrc1 double mutant plants were not significantly different from X-CK levels from F3 rms1 plants”
Quote in the paperSubstantial claim - Findingown data
Compared with the SL-deficient rms1 mutant, the Psbrc1 mutant displayed very few long branches at upper nodes.
“When compared with the SL-deficient rms1 mutant, the Psbrc1 mutant displayed very few long branches at upper nodes.”
Quote in the paperSubstantial claim - Findingown data
In all genotypes, BAP application led to a strong reduction of PsBRC1 transcript levels.
“In all genotypes, BAP application led to a strong reduction of PsBRC1 transcript levels.”
Quote in the paperSubstantial claim - Findingown data
Amounts of fabacyl acetate were 3- to 6-fold higher from both Psbrc1 genotypes than in their corresponding wild-type background lines.
“Amounts of this compound were 3-to 6-fold higher (P , 0.05, Student's t test) from both Psbrc1 genotypes than in their corresponding wild-type background lines”
Quote in the paperSubstantial claim - Findingreasoning
The PsBRC1 transcript level was similar to the wild-type level in axillary buds of the rms2 mutant.
“In contrast, the PsBRC1 transcript level was similar to the wild-type level in axillary buds of the rms2 mutant”
Quote in the paperSubstantial claim - Findingown data
At node 4, the axillary buds of rms2 plants were only 2.3 mm long, whereas they reached 14 mm in rms1.
“at node 4, from where the buds were sampled, the axillary buds of rms2 plants were only 2.3 mm long, whereas they reached 14 mm in rms1”
Quote in the paperSubstantial claim - Findingown data
The principal SL detected in root exudates of all genotypes was fabacyl acetate.
“The principal SL detected in root exudates of all genotypes was fabacyl acetate.”
Quote in the paperNot a substantial claim - Findingown data
X-CK levels from F3 Psbrc1 and Psbrc1 Te were particularly high compared with wild-type Térèse and F3 wild-type plants.
“X-CK levels from F3 Psbrc1 and Psbrc1 Te were particularly high in comparison with wild-type Te ´re `se and F3 wild-type plants.”
Quote in the paperNot a substantial claim - Findingown data
PsBRC1 transcript levels were very low in rms1 and rms4 axillary buds compared with wild-type and Psbrc1 Te axillary buds.
“PsBRC1 transcript levels were very low in rms1 and rms4 axillary buds compared with wild-type and Psbrc1 Te axillary buds.”
Quote in the paperSubstantial claim - Findingown data
The Psbrc1 mutant height was less affected compared with rms1 and other rms mutants, which are relatively dwarf.
“Moreover, its height was less affected compared with rms1 and other rms mutants, which are relatively dwarf.”
Quote in the paperSubstantial claim - Findingown data
RMS1 mRNA was more abundant in rms1 and especially rms4 mutants compared with wild-type Térèse.
“RMS1 mRNA was more abundant in rms1 and especially in rms4 mutants compared with wild-type Te ´re `se.”
Quote in the paperSubstantial claim - Findingown data
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.
“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.”
Quote in the paperSubstantial claim - Findingown data
In rms1 mutant buds, PsBRC1 mRNA levels increased 10-fold at 6 and 24 h after GR24 treatment.
“In rms1 mutant buds, PsBRC1 mRNA levels increased 10fold at 6 and 24 h after GR24 treatment”
Quote in the paperSubstantial claim - Findingown data
PsBRC1 levels were 10 times lower in rms1 and rms4 than in the wild type.
“PsBRC1 levels were 10 times lower in rms1 and rms4 than in the wild type”
Quote in the paperSubstantial claim - Findingreasoning
PsBRC1 transcript levels remained very low after SL treatment in buds of the rms4 response mutant.
“As expected for a SL-response mutant, PsBRC1 transcript levels remained very low after SL treatment in buds of the rms4 response mutant.”
Quote in the paperSubstantial claim - Findingown data
We were unable to amplify another pea BRC homolog.
“Nevertheless, we were unable to amplify another pea BRC homolog”
Quote in the paperNot a substantial claim - Findingown data
The phenotypic response to decapitation was consistent with the CK response of Psbrc1 Cam and of the SL-deficient rms1 mutant.
“The phenotypic response to decapitation was consistent with the CK response of Psbrc1 Cam and of the SL-deficient rms1 mutant.”
Quote in the paperSubstantial claim - Findingown data
The Psbrc1 mutant did not respond to SL application.
“The fact that the Psbrc1 mutant did not respond to SL application”
Quote in the paperSubstantial claim - Findingown data
PsBRC1 expression in axillary buds was rapidly enhanced by SL treatment.
“we showed that PsBRC1 expression in axillary buds was rapidly enhanced by SL treatment.”
Quote in the paperSubstantial claim - Findingown data
PsBRC1 transcript levels decrease after direct application of CK to the axillary bud for all genotypes tested, including the SL-response rms4 and Psbrc1 mutants.
“Our results showed that PsBRC1 transcript levels decrease after direct application of CK to the axillary bud for all genotypes tested, including the SL-response rms4 and Psbrc1 mutants.”
Quote in the paperSubstantial claim - Findingreasoning
Feedback down-regulation of X-CK occurs in Psbrc1 rms1 double and rms1 single mutants but not in Psbrc1 Te single mutants.
“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.”
Quote in the paperSubstantial claim - Findingown data
Increased X-CK in Psbrc1 Te was not seen in the previous analysis with Psbrc1 Cam and its wild-type Caméor.
“increased X-CK in Psbrc1 Te in this experiment was not seen in the previous analysis with Psbrc1 Cam and its wild-type Came ´or and may relate to genetic background.”
Quote in the paperNot a substantial claim - Interpretationown data§ 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 may have a role in integrating SL and CK signals.
“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.”
Quote in the paperSubstantial claim - Interpretationreasoning§ 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, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.
“We propose that PsBRC1, acting in axillary buds, may integrate at the transcriptional level the SL and CK pathways to regulate axillary bud outgrowth.”
Quote in the paperSubstantial claim - Interpretationreasoning§ 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 is not activated in the Psbrc1 mutant.
“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.”
Quote in the paperSubstantial claim - Interpretationown data§ 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.
A PsBRC1-independent component of the CK response is implicated in sustained bud growth.
“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.”
Quote in the paperSubstantial claim - Interpretationown data§ 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 double mutant phenotype indicates a localized regulation of branching at these nodes specific to pea.
“This phenotype indicates a localized regulation of branching at these nodes specific to pea.”
Quote in the paperSubstantial claim - Interpretationown data§ 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 response to decapitation and to direct CK application of the Psbrc1 mutant suggests a PsBRC1-independent component of the CK response in pea.
“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.”
Quote in the paperSubstantial claim - Interpretationown data§ 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.
SLs act directly within the bud to regulate its outgrowth.
“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.”
Quote in the paperSubstantial claim - Interpretationown data
The T195I mutation in the PsBRC1 gene was the cause of the high-branching phenotype.
“These data strongly support the hypothesis that the T195I mutation in the PsBRC1 gene was the cause of the high-branching phenotype.”
Quote in the paperSubstantial claim- Rests on an assumption: The T195I mutation in PsBRC1 causes loss of PsBRC1 function.
- Premisecitation
Absence of SL response due to SL deficiency (in rms1 and rms5) or lack of response (in rms3 and rms4) induces a feedback signal that strongly upregulates RMS1 transcript levels.
“In particular, the absence of SL response because of SL deficiency (in rms1 and rms5) or the lack of response (in rms3 and rms4) induces a feedback signal that strongly upregulates RMS1 transcript levels”
Quote in the paperSubstantial claim0 of 3 sources checked- cites Beveridge et al., 1997a
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites Beveridge et al., 1997b
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites Beveridge, 2000 source
- Interpretationown data
The feedback signal is not activated in the Psbrc1 background.
“suggesting that the feedback signal is not activated in the Psbrc1 background.”
Quote in the paperSubstantial claim - Interpretationcitation
The milder phenotype of Psbrc1 compared with rms1 is likely not due to a leaky mutation.
“which supports the idea that the milder phenotype of Psbrc1 compared with rms1 is likely not due to a leaky mutation.”
Quote in the paperNot a substantial claim - Premisecitation
Arabidopsis max and pea rms branching mutants, except rms2, have highly depleted X-CK levels.
“This contrasts with Arabidopsis max and pea rms branching mutants, which, with the exception of rms2, have highly depleted X-CK levels”
Quote in the paperNot a substantial claim0 of 3 sources checked- cites Table I
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites Beveridge et al., 1997a
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites Foo et al., 2007 source
- Premisecitation
In tomato, the phenotype of SlBRC1b RNA interference lines is less strong than the phenotype of Slccd7 lines.
“In tomato, the phenotype of SlBRC1b RNA interference lines is also less strong than the phenotype of the Slccd7 lines (Martin-Trillo et al., 2011)”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Martin-Trillo et al., 2011 source
- Interpretationreasoning
The relatively mild branching phenotype could explain why intensive screenings for high-branching mutants in pea EMS populations previously failed to identify the Psbrc1 pea branching mutant.
“The relatively mild branching phenotype could also explain why the intensive screenings for high-branching mutants in pea ethyl methanesulfonate-mutagenized populations previously failed to identify the Psbrc1 pea branching mutant.”
Quote in the paperNot a substantial claim - Interpretationown data
CK can transcriptionally regulate PsBRC1 independently of SL.
“The CK effect on PsBRC1 expression in rms4 buds shows that CK can transcriptionally regulate PsBRC1 independently of SL.”
Quote in the paperSubstantial claim - Premisecitation
The SL biosynthesis gene RMS1/CCD8 is highly regulated at the transcriptional level.
“The SL biosynthesis gene RMS1/CCD8 is highly regulated at the transcriptional level.”
Quote in the paperNot a substantial claim0 of 3 sources checked- cites Beveridge et al., 1997a
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites Beveridge et al., 1997b
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites Beveridge, 2000 source
- Premisecitation
Experiments with grafted plants bearing two shoots of different genotypes and 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.
“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.”
Quote in the paperNot a substantial claim - Interpretationown data
PsBRC1 may be involved in the SL signaling pathway to repress axillary bud outgrowth.
“suggests that PsBRC1 may be involved in the SL signaling pathway to repress axillary bud outgrowth.”
Quote in the paperSubstantial claim - Interpretationreasoning
The difference of phenotypes between SL-deficient rms1 and Psbrc1 mutants may be explained by the very localized expression of the PsBRC1 gene, mostly in axillary buds, in contrast to the SL biosynthesis RMS1 and RMS5 genes, which are highly expressed in roots and also significantly in stems, where they are regulated by different long-distance signals.
“The difference of phenotypes between the SLdeficient rms1 and Psbrc1 mutants may be explained by the very localized expression of the PsBRC1 gene, mostly expressed in axillary buds, in contrast to the SL biosynthesis RMS1 and RMS5 genes, which are highly expressed in roots and also significantly in stems, where they are regulated by different long-distance signals.”
Quote in the paperSubstantial claim - Interpretationown data
The RMS4-dependent response of PsBRC1 to SL is consistent with PsBRC1 acting in the SL signaling pathway for branching inhibition.
“As such, the RMS4-dependent response of PsBRC1 to SL is consistent with PsBRC1 acting in the SL signaling pathway for branching inhibition.”
Quote in the paperSubstantial claim - Premisecitation
In Arabidopsis, the double Atbrc1 Atbrc2 mutant displays the same branching phenotype as the strongest Atbrc1 mutant.
“in Arabidopsis, the double Atbrc1 Atbrc2 mutant displays the same branching phenotype as the strongest Atbrc1 mutant (Aguilar-Martı ´nez et al., 2007).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Aguilar-Martı ´nez et al., 2007
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- Interpretationown data
CK controls PsBRC1 expression independently of the SL pathway.
“These results demonstrate that CK controls PsBRC1 expression independently of the SL pathway.”
Quote in the paperSubstantial claim - Premisecitation
Experiments with mutants lacking axillary meristems in an rms4 background demonstrated that branching per se was not the cause of the feedback.
“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)).”
Quote in the paperSubstantial claim0 of 2 sources checked - Interpretationown data
These results suggest possible specific regulation of branching at basal nodes in pea.
“These results suggest a possible specific regulation of branching at basal nodes in pea.”
Quote in the paperSubstantial claim - Premisecitation
In Arabidopsis, Atbrc1 mutants showed no response to decapitation.
“This result differs from that observed in Arabidopsis, where Atbrc1 mutants showed no response to decapitation (Aguilar-Martı ´nez et al., 2007).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Aguilar-Martı ´nez et al., 2007
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- Premisecitation
In Arabidopsis, expression of AtBRC1 was strongly down-regulated in max1 to max4 mutants, suggesting SL up-regulates transcript levels of AtBRC1.
“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.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Aguilar-Martı ´nez et al., 2007
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- Interpretationown data
The phenotype of Psbrc1 differs from the phenotype of the SL-deficient rms1 mutant.
“The Phenotype of Psbrc1 Differs from the Phenotype of the SL-Deficient rms1 Mutant”
Quote in the paperSubstantial claim - Interpretationown data
The absence of feedback in Psbrc1 could suggest that SL-mediated feedback regulation is generated between the action of RMS4 and PsBRC1.
“The absence of feedback in Psbrc1 could suggest that the SL-mediated feedback regulation is generated between the action of RMS4 and PsBRC1.”
Quote in the paperSubstantial claim - Interpretationown data
The study of PsBRC1 gives an example of a transcription factor with more limited phenotypic effects than its upstream signaling genes.
“The study of PsBRC1 gives an example of a transcription factor with more limited phenotypic effects than its upstream signaling genes.”
Quote in the paperSubstantial claim - Interpretationown data
The Psbrc1 Cam mutant is not strigolactone deficient.
“Grafting experiment showing that PsBRC1 acts in the shoot and that the Psbrc1 Cam mutant is not strigolactone deficient.”
Quote in the paperSubstantial claim - Interpretationown data
CK also increases bud growth via a PsBRC1-independent pathway.
“CK also increases bud growth via a PsBRC1-independent pathway.”
- Also stated in: § 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). · plain
“CK also increases bud growth via a PsBRC1-independent pathway.”
Quote in the paperSubstantial claim - Also stated in: § 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). · plain
- Interpretationcitation
It indicates that the SL signaling pathway may be shared among multiple developmental modules, as proposed by Doebley and Lukens.
“It indicates that the SL signaling pathway may be shared among multiple developmental modules, as proposed by Doebley and Lukens (1998).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Doebley and Lukens, 1998 source
- Interpretationown data
Our results suggest that PsBRC1 acts downstream of RMS4 in the SL signaling pathway.
“As discussed above, our results suggest that PsBRC1 acts downstream of RMS4 in the SL signaling pathway.”
Quote in the paperSubstantial claim - Interpretationown data
The model that best fits the data would be one in which both regulatory systems coexist.
“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).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Domagalska and Leyser, 2011 source
- Interpretationcitation
SLs, by reducing the effectiveness of the canalization feedback loop, enhance the competition between active buds.
“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.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Domagalska and Leyser, 2011 source
- Interpretationown data
PsBRC1 integrates the SL and CK pathways to control bud outgrowth.
“PsBRC1 integrates the SL and CK pathways to control bud outgrowth.”
- Also stated in: § 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). · plain
“PsBRC1 integrates the SL and CK pathways to control bud outgrowth.”
Quote in the paperSubstantial claim - Also stated in: § 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). · plain
- Interpretationown data
The RMS2-dependent feedback signal is not activated in Psbrc1.
“The RMS2-dependent feedback signal is not activated in Psbrc1.”
Quote in the paperSubstantial claim - Interpretationown data
The RMS2-dependent feedback may be independent of PsBRC1.
“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.”
Quote in the paperSubstantial claim - Interpretationcitation
SLs reduce PIN accumulation to the plasma membrane via RMS4 but independently 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.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Domagalska and Leyser, 2011 source
- Interpretationown data
PsBRC1 acts in the shoot.
“Grafting experiment showing that PsBRC1 acts in the shoot and that the Psbrc1 Cam mutant is not strigolactone deficient.”
Quote in the paperSubstantial claim
- May combine two claims. 54
- Classified as probably not a substantial claim. 31
- The quote only partly states this claim. 1
May combine two claims.
Findingcitation§ 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 strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1.
“PsBRC1 is strongly transcriptionally up-regulated by SL, which is not the case for the rice FC1 (Minakuchi et al., 2010).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites Minakuchi et al., 2010 source
The Results section, “Hormonal control of FC1 mRNA expression,” reports that rice FC1 expression was not significantly affected by the strigolactone analog GR24. The Discussion describes strigolactone-induced BRC1 expression in Arabidopsis, but does not establish strong transcriptional up-regulation of pea PsBRC1. (LLM, conf=0.88)
Experimental — accuracy not yet measured
Classified as probably not a substantial claim. May combine two claims.
InterpretationcitationThe rms2 phenotype of inhibited buds at node 4 is explained by a (basal) branch-derived feedback signal, possibly auxin, which increases RMS1 expression and presumably SL synthesis in rms2 plants.
“The rms2 phenotype of inhibited buds at node 4 is explained by a (basal) branch-derived feedback signal, possibly auxin, which increases RMS1 expression and presumably SL synthesis in rms2 plants (Dun et al., 2009).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites Dun et al., 2009 source
The Discussion (page unavailable) explains that growing basal branches increase RMS1 expression in rms2 plants, presumably increasing strigolactones that suppress higher-node buds. It identifies auxin as a possible branch-derived signal. Our copy discusses buds at proximal higher nodes but does not specifically identify inhibited buds at node 4. (LLM, conf=0.84)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing).
Premisecitation§ 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.In pea, grafting revealed the existence of two novel long-distance signals controlling shoot branching that were different from auxin and CK.
“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),”
Quote in the paperNot a substantial claim1 of 3 sources checked- cites Beveridge et al., 2000 source
The Discussion (page not provided) says grafting provides evidence that Rms1 and Rms2 control one or more mobile signals involved in pea bud outgrowth. It does not establish that there are two distinct signals, and it calls cytokinin an unlikely candidate only for the Rms1-associated signal. (LLM, conf=0.88)
Experimental — accuracy not yet measured - cites Beveridge et al., 2009 source
- cites Beveridge, 2006 source
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Premisecitation§ 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 RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene.
“The RMS4 gene encodes an F-box protein and corresponds to the Arabidopsis MAX2 gene (Stirnberg et al., 2002;Johnson et al., 2006).”
Quote in the paperNot a substantial claim1 of 2 sources checked- cites Stirnberg et al., 2002 source
The section “MAX2 encodes an F-box leucine-rich repeat protein” (p. 10) identifies Arabidopsis MAX2 as an F-box protein. The Introduction (pp. 2–3) discusses pea RMS loci alongside MAX2 but does not identify RMS4 as the corresponding gene or establish that RMS4 encodes an F-box protein. (LLM, conf=0.88)
Experimental — accuracy not yet measured - cites Johnson et al., 2006 source
Probably not a substantial claim: may not pass S3 (load-bearing).
Premisecitation§ 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.FC1/OsTB1 acts downstream of the SL pathway in rice.
“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).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites Minakuchi et al., 2010 source
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Premisecitation§ 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 RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively.
“The RMS1 and RMS5 genes encode the CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes CCD8 and CCD7, respectively (Sorefan et al., 2003;Johnson et al., 2006).”
Quote in the paperNot a substantial claim1 of 2 sources checked- cites Sorefan et al., 2003 source
In “MAX4 belongs to the polyene chain dioxygenase family” (page unavailable), the paper identifies RMS1 as an ortholog of MAX4 and places both in the polyene dioxygenase family. It does not identify RMS1 as CCD8 or RMS5 as CCD7; it mentions CCD7 as an Arabidopsis protein, not as the product of pea RMS5. (LLM, conf=0.86)
Experimental — accuracy not yet measured - cites Johnson et al., 2006 source
Classified as probably not a substantial claim.
PremisecitationThe existence of a mobile graft-transmissible auxin-independent shoot-to-root feedback signal that up-regulates RMS1 and RMS5 transcription and downregulates X-CK export from roots was previously proposed.
“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).”
Quote in the paperSubstantial claim1 of 2 sources checkedMay combine two claims.
InterpretationcitationIf PsBRC1 integrates the SL and CK signals to control axillary bud outgrowth, the CK response of the Psbrc1 Cam mutant could be interpreted as the CK response having at least two components, one involving early bud outgrowth (PsBRC1 dependent) and the other involving sustained growth (PsBRC1 independent).
“If PsBRC1 integrates the SL and CK signals to control axillary bud outgrowth, the CK response of the Psbrc1 Cam mutant could be interpreted as the CK response having at least two components, one involving early bud outgrowth (PsBRC1 dependent) and the other involving sustained growth (PsBRC1 independent; Dun et al., 2009).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites Dun et al., 2009 source
Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.
Experimental — accuracy not yet measured
Classified as probably not a substantial claim.
PremisecitationBecause the rms2 mutant showed low RMS1 expression and high X-CK content in xylem sap, in contrast to other rms mutants, it was suggested that the shoot-to-root feedback signal was RMS2 dependent.
“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).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites Beveridge et al., 2000 source
The Abstract reports slightly elevated xylem-sap cytokinin in rms2, unlike the reduced levels described for rms1 and other rms mutants. The Discussion says Rms2 controls a mobile signal, but our copy does not report low RMS1 expression in rms2 or identify that signal as shoot-to-root feedback; it says the signal's source may be the shoot or rootstock (page unavailable). (LLM, conf=0.87)
Experimental — accuracy not yet measured
May combine two claims.
PremisecitationIn rice, D10 expression was not increased in the fc1 mutant, whereas it was highly expressed in other SL-related mutants.
“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).”
Quote in the paperSubstantial claim2 of 2 sources checkedClassified as probably not a substantial claim.
PremisecitationIt was suggested that the feedback is regulated by the absence of perception of SL, as various mutants unable to produce or respond to SL have reduced export of X-CK from roots and increased expression of SL biosynthesis genes.
“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;Ari”
Quote in the paperSubstantial claim2 of 3 sources checked- cites Foo et al., 2005 source
In “RMS1 Expression Is Feedback Regulated” (page not supplied), the paper reports elevated RMS1 expression in rms1, rms3, rms4, and rms5 mutants and suggests that feedback may respond to an actual or perceived reduction in a branching inhibitor. The Introduction (page not supplied) also reports reduced root-to-shoot cytokinin export in those mutants. The paper does not identify the inhibitor as strigolactone (SL) or establish absence of SL perception as the specific cause of the feedback. (LLM, conf=0.86)
Experimental — accuracy not yet measured - cites Dun et al., 2009 source
- cites Beveridge et al., 1997a
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationDun et al. (2009), using hypothesis-driven modeling, suggested that SL biosynthesis is tightly regulated by multiple feedback signals in both shoot and root.
“Dun et al. (2009), using a hypothesis-driven modeling approach, suggested that SL biosynthesis is tightly regulated by multiple feedback signals in both shoot and root.”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites Dun et al., 2009 source
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationTwo models are currently proposed to explain how auxin and SLs interact to control branching: the auxin transport canalization model and the second messenger model.
“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).”
Quote in the paperNot a substantial claim1 of 2 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing).
InterpretationreasoningBecause MAX2/RMS4 encodes an F-box protein, it is likely that the SL signaling pathway involves similarly targeted protein degradation.
“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.”
Quote in the paperNot a substantial claim1 of 2 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationIn the rice fc1 mutant, the level of 2'-epi-5-deoxystrigol in root exudates was slightly higher but not significantly different from the wild type.
“In the rice fc1 mutant, the level of 2#-epi-5-deoxystrigol in root exudates was slightly higher but not significantly different from the wild type (Minakuchi et al., 2010).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites Minakuchi et al., 2010 source
The Results section (‘Defects of fc1 mutants are not rescued by the application of SLs’; page unavailable) says that 2′-epi-5-deoxystrigol levels in fc1-2 root exudates were not significantly different from wild type. Our copy does not state that the mutant’s level was slightly higher. (LLM, conf=0.86)
Experimental — accuracy not yet measured
May combine two claims.
Findingown dataNo significant effect of GR24 was observed for the Psbrc1 Cam mutant, in contrast to the strong inhibition of axillary bud outgrowth in the SL-deficient rms1 mutant.
“In contrast to the strong inhibition of axillary bud outgrowth in the SL-deficient rms1 mutant, no significant effect of GR24 was observed for the Psbrc1 Cam mutant (Student's t test, P = 0.89).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataPlant height was only slightly reduced for Psbrc1 Cam and Psbrc1 Te compared with their respective wild types, whereas rms1 showed a strong reduction in internode length.
“plant height was only slightly reduced for Psbrc1 Cam and Psbrc1 Te in comparison with their respective wild types, whereas rms1 showed a strong reduction in internode length”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataAt node 2, total branch length was similar in the three genotypes, whereas at nodes below, and particularly at the cotyledonary node, branching of rms1 Psbrc1 double mutant plants was significantly higher than in either single mutant.
“At node 2, total branch length was similar in the three genotypes, whereas at nodes below, and particularly at the cotyledonary node, branching of rms1 Psbrc1 double mutant plants was significantly higher than in either single mutant”
Quote in the paperSubstantial claimMay combine two claims.
Findingcitation§ 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.In contrast to Arabidopsis, branching is increased in the pea Psbrc1 mutant in response to decapitation and to CK application.
“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.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Aguilar-Martı ´nez et al., 2007
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
May combine two claims.
Findingown dataAt upper nodes, rms1 plants were significantly more branched than Psbrc1 Te plants, whereas the double mutant genotype was not significantly more branched than rms1 at these nodes.
“at upper nodes, rms1 plants were significantly more branched than Psbrc1 Te plants, whereas the double mutant genotype was not significantly more branched than rms1 at these nodes”
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Findingown dataGrafting experiments confirmed the SL insensitivity of Psbrc1 Cam, as branching was not inhibited when the Psbrc1 Cam mutant scion was grafted on wild-type rootstock, in contrast to the SL-deficient rms1 scion.
“Grafting experiments confirmed the SL insensitivity of Psbrc1 Cam , as branching was not inhibited when the Psbrc1 Cam mutant scion was grafted on wild-type rootstock, in contrast to the SL-deficient rms1 scion grafted on wild-type rootstock”
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Findingown dataIn the wild type and the Psbrc1 Te mutant, a 2-fold increase of PsBRC1 by GR24 was generally observed, in contrast to the noninduction in the rms4 background.
“In the wild type and the Psbrc1 Te mutant, a 2-fold increase of PsBRC1 by GR24 was generally observed, in contrast to the noninduction in the rms4 background”
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Findingown datarms2 and Psbrc1 Cam mutants contained three and six times lower RMS1 transcript levels compared with their respective wild-type lines.
“rms2 and Psbrc1 Cam mutants contained three and six times lower transcript levels compared with their respective wild-type lines”
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Findingown dataIn rms4, there was no effect of GR24 treatment, with PsBRC1 transcript levels remaining very low throughout.
“In rms4, there was no effect of GR24 treatment, with PsBRC1 transcript levels remaining very low throughout”
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Findingown dataTranscript levels increased in axillary buds of wild-type, rms1, and rms2 plants after GR24 application but not in rms4.
“Transcript levels increased in axillary buds of wild-type, rms1, and rms2 plants after GR24 applica-tion (500 nM) but not in rms4”
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FindingcitationThe Thr mutated to Ile in the studied Psbrc1 pea mutant (T195I) is located in the loop/helix II transition and appears highly conserved in the class I TCP family and in the class II CYC/TB1 clade.
“the Thr mutated to Ile in the studied Psbrc1 pea mutant (T195I) is located in the loop/helix II transition and appears highly conserved in the class I TCP family and in the class II CYC/TB1 clade”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Martı ´n-Trillo and Cubas, 2010
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
May combine two claims.
Findingown dataThe Psbrc1 mutant showed a strong branching phenotype compared with its wild-type progenitor Cameor, particularly at basal nodes; buds at upper nodes were larger or gave small branches compared with wild-type.
“The Psbrc1 mutant showed a strong branching phenotype in comparison with its wild-type progenitor Came ´or, particularly at basal nodes; buds at upper nodes were larger or gave small branches in comparison with the wild-type.”
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Findingown dataX-CK profiles and absolute amounts were very similar in two experiments, with no statistically significant differences between wild-type and Psbrc1 Cam genotypes (P = 0.49).
“The profiles of CKs and absolute amounts were very similar in both experiments, with no statistically significant differences between wild-type and Psbrc1 Cam genotypes (Student's t test, P = 0.49).”
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Findingown dataX-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.
“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.”
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Findingown dataPsBRC1 transcript levels were very low in axillary buds of SL-deficient (rms1) or SL-response (rms4) mutants compared with wild-type buds but were rapidly up-regulated by SL application, particularly in rms1, wild type, rms2, and Psbrc1.
“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.”
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Findingown dataBranching of rms1 Psbrc1 was similar to rms1 at upper nodes (node 3 and above) but transgressive at basal nodes (cotyledonary node and node 1).
“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).”
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Findingown dataPsbrc1 Cam lacks the feedback upregulation of RMS1 gene expression, and the substantial down-regulation of X-CK content in SL biosynthesis and signaling mutants was not found in Psbrc1 Cam plants.
“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).”
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Findingown dataIn pea, total branch length was strongly enhanced in rms1 Psbrc1 double mutant plants, mainly because of strong development of cotyledonary branches.
“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.”
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Findingown dataSL quantification in root exudates of Psbrc1 genotypes in comparison with their wild type shows that this mutant is not deficient in SL biosynthesis and explains the grafting results.
“SL quantification in root exudates of Psbrc1 genotypes in comparison with their wild type show that this mutant is not deficient in SL biosynthesis and explains the grafting results.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe higher level of fabacyl acetate found in root exudates of the two Psbrc1 lines compared with their wild type, despite low RMS1 expression in epicotyl, is surprising and indicates possible misregulation of SL biosynthesis in the mutant.
“The higher level of fabacyl acetate found in root exudates of the two Psbrc1 lines in comparison with their wild type, despite the low RMS1 expression in epicotyl, is surprising and indicates a possible misregulation of SL biosynthesis in the mutant.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataAt basal nodes, a transgressive phenotype was observed in the double mutant, whereas at upper nodes, branching of the double mutant was not significantly different from that observed for rms1.
“Consequently, it appeared that at basal nodes, a transgressive phenotype was observed in the double mutant, whereas at upper nodes, branching of the double mutant was not significantly different from that observed for rms1.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataPsbrc1 Cam very likely produces SL and/or the active derived compound.
“Psbrc1 Cam rootstocks were able to inhibit branching in the rms1 scion, indicating that Psbrc1 Cam very likely produces SL and/or the active derived compound”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataPsBRC1 may act downstream of the SL signaling pathway and downstream of RMS4.
“These results indicated that PsBRC1 may act downstream of the SL signaling pathway and downstream of RMS4.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataPsBRC1 transcription and its induction by GR24 are not affected in the Psbrc1 mutant.
“All these results indicate that PsBRC1 transcription and its induction by GR24 are not affected in the Psbrc1 mutant.”
Quote in the paperSubstantial claim- Rests on an assumption: The T195I mutation in PsBRC1 causes loss of PsBRC1 function.
May combine two claims.
Interpretationown dataRMS2 may control the feedback signal, as RMS1 transcript levels are not up-regulated in the rms2 mutant.
“The RMS2 gene may control this feedback signal, as RMS1 transcript levels are not up-regulated in the rms2 mutant.”
Quote in the paperNot a substantial claimMay combine two claims.
InterpretationreasoningThis SL moves up the stem and could explain why in rms2 PsBRC1 transcript levels were not low at this upper node.
“This SL moves up the stem and, because the rms2 mutant is able to synthesize and to respond to SL, it could explain why in this branching mutant PsBRC1 transcript levels were not low at this upper node.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Premisecitation§ 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.A root-to-shoot branching inhibitor was subsequently identified as a strigolactone (SL) or derived compound.
“which was subsequently identified as a strigolactone (SL) or derived compound (Gomez-Roldan et al., 2008;Umehara et al., 2008)”
Quote in the paperNot a substantial claim0 of 2 sources checkedMay combine two claims.
PremisecitationIn Arabidopsis and rice, corresponding double mutants have the same level of branching as single mutants, suggesting FC1/AtBRC1 TCP transcription factors act in the SL pathway.
“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.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Aguilar-Martı ´nez et al., 2007
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
May combine two claims.
PremisereasoningRMS4 has all the features expected of a protein involved in SL signaling: branching phenotype of rms4, F-box protein, nonresponse to GR24, and strong feedback upregulating RMS1.
“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).”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataCK may promote axillary bud outgrowth and/or growth independently of the SL signaling pathway and from PsBRC1.
“This response indicates that CK may promote axillary bud outgrowth and/or growth independently of the SL signaling pathway and from PsBRC1.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
InterpretationreasoningThe presence of a second BRC1 homolog in pea and gene redundancy could explain the weaker phenotype of Psbrc1 compared with SL mutants.
“The presence of a second BRC1 homolog in pea and gene redundancy could also explain the weaker phenotype of Psbrc1 in comparison with the SL mutants.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationIn the auxin transport theory, SL modulates polar auxin transport in the stem and axillary buds compete for exporting auxin into the PAT stream in the main stem.
“In the auxin transport theory, it is proposed that SL modulates polar auxin transport (PAT) in the stem and that axillary buds compete for exporting auxin into the PAT stream in the main stem.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationNovel roles for SL in plant architecture other than shoot branching have been suggested for dwarfism and root architecture.
“Novel roles for SL in plant architecture, other than shoot branching, have been recently suggested for dwarfism (Lin et al., 2009) and root architecture (Koltai et al., 2009;Kapulnik et al., 2011;Ruyter-Spira et al., 2011).”
Quote in the paperNot a substantial claim0 of 3 sources checkedMay combine two claims.
Interpretationown dataPsBRC1 transcript levels do not simply correlate negatively with activity of axillary buds, and PsBRC1 transcription factor has a direct effect in repression of axillary bud outgrowth.
“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.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationIn Arabidopsis, two TB1 homologs, AtBRC1 and AtBRC2, have a role in the control of shoot branching.
“In Arabidopsis, two TB1 homologs have been identified, AtBRC1 and AtBRC2, with a role in the control of shoot branching (Aguilar-Martı ´nez et al., 2007).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Aguilar-Martı ´nez et al., 2007
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
InterpretationreasoningThe relative dwarfism of rms mutants is not yet understood, but it is very likely that SL controls stem growth, and this control may be independent of PsBRC1.
“The relative dwarfism of rms mutants (in rice, the SL mutants were originally called dwarf) is not yet understood, but it is very likely that SL controls stem growth, and this control may be independent of PsBRC1.”
Quote in the paperNot a substantial claimMay combine two claims.
PremisecitationAtBRC2 is not down-regulated in max1 and max2 mutants; FC1 and AtBRC2 appear similarly unresponsive to SL, while AtBRC1 and PsBRC1 are both transcriptionally up-regulated by SL.
“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.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
InterpretationreasoningThe Psbrc1 mutant appears less branched at upper nodes than the SL-deficient rms1 mutant, which could suggest that competition between buds is higher in the Psbrc1 mutant compared with other rms mutants.
“The Psbrc1 mutant appears less branched at upper nodes than the SL-deficient rms1 mutant, which could suggest that competition between buds is higher in the Psbrc1 mutant compared with the other rms mutants.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductreasoningSimple quantification of the lengths of buds/lateral branches does not in itself distinguish the different growth stages.
“Simple quantification of the lengths of buds/lateral branches does not in itself distinguish the different growth stages.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataPsBRC1 could integrate the SL and CK pathways at the transcriptional level within the bud, and bud outgrowth would occur where PsBRC1 falls below a certain transcript level.
“Thus, PsBRC1 could integrate the SL and CK pathways at the transcriptional level within the bud and bud outgrowth would occur where PsBRC1 falls below a certain transcript level.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationIn pea, the RMS6 gene controls bud outgrowth only at basal nodes.
“In pea, the RMS6 gene has been shown to control bud outgrowth only at these nodes (Rameau et al., 2002).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Rameau et al., 2002 source
May combine two claims.
PremisecitationAuxin maintains RMS1 transcript levels, and hence SL synthesis, and down-regulates CK levels.
“Auxin maintains RMS1 transcript levels, and hence SL synthesis, and down-regulates CK levels.”
- Also stated in: § 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). · plain
“Auxin maintains RMS1 transcript levels, and hence SL synthesis, and down-regulates CK levels.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Domagalska and Leyser, 2011 source
- Also stated in: § 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). · plain
Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.
Study design and conductnone givenThree biological repeats were analyzed in duplicate for quantitative RT-PCR.
“Three biological repeats were analyzed in duplicate.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Study design and conductnone givenRNA was quantified using NanoDrop 1000 and migrated on gels to check RNA nondegradation.
“RNA was quantified using NanoDrop 1000 and migrated on gels to check RNA nondegradation.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenThe absence of contamination with genomic DNA was checked using 35 cycles of PCR with RMS1 primers.
“The absence of contamination with genomic DNA was checked using 35 cycles of PCR with RMS1 primers (see below).”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductcitationTranscript levels for the different genes were expressed relative to the expression of the EF1a gene.
“Transcript levels for the different genes were expressed relative to the expression of the EF1a gene (Johnson et al., 2006).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Johnson et al., 2006 source
May combine two claims.
Interpretationown dataThis 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.
“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.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing). May combine two claims.
Interpretationnone givenThe degree of branching is an important component of fitness, and it is very likely that different pathways are involved in its tight control according to bud position, developmental stage, and environmental conditions.
“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.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).
InterpretationreasoningProteins 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.
“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.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationAdvances in plant hormone signaling have highlighted the role of the ubiquitin-proteasome pathway and targeted protein turnover.
“Advances in plant hormone signaling have highlighted the role of the ubiquitin-proteasome pathway and targeted protein turnover (Santner and Estelle, 2009).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Santner and Estelle, 2009 source
Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.
InterpretationreasoningThe RMS2-dependent feedback upregulates SL biosynthesis and down-regulates xylem CK.
“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.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing), S4 (based).
Interpretationnone givenThe RMS2-dependent feedback is activated when there is a lack of SL signaling via RMS4.
“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.”
Quote in the paperNot a substantial claimMay combine two claims.
InterpretationreasoningThe 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.
“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.”
Quote in the paperSubstantial claimMay combine two claims.
InterpretationcitationSL would act locally in axillary buds via PsBRC1 and would also coordinate branching across the plant by controlling auxin transport independently of PsBRC1.
“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.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Leyser, 2011 source
May combine two claims.
Interpretationown dataThe feedback regulation of X-CK is restored in the Psbrc1 rms1 double mutant but is absent or misregulated in Psbrc1 single mutants.
“Overall, these results indicate that the feedback regulation of X-CK is restored in the Psbrc1 rms1 double mutant but is absent or misregulated in Psbrc1 single mutants.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Interpretationown dataThe difference in X-CK between Psbrc1 Te and previous Psbrc1 Cam analysis may relate to genetic background.
“increased X-CK in Psbrc1 Te in this experiment was not seen in the previous analysis with Psbrc1 Cam and its wild-type Came ´or and may relate to genetic background.”
Quote in the paperNot a substantial claimMay combine two claims.
InterpretationreasoningSLs reduce PIN accumulation to the plasma membrane via RMS4 but independently of PsBRC1 and, by reducing the effectiveness of the canalization feedback loop, enhance competition between active buds.
“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.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataThe 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 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.”
Quote in the paperSubstantial claim
73 of 73
The paper relies on this without support.
The T195I mutation in PsBRC1 causes loss of PsBRC1 function.
“These data strongly support the hypothesis that the T195I mutation in the PsBRC1 gene was the cause of the high-branching phenotype.”
1 of 1
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
Orthologs of TB1 and TAC1 were identified in dicots, and now key genes for genetic engineering or for use as genetic markers for classical breeding of monocot and dicot crops are available.
“However, orthologs of TB1 and TAC1 were soon also identified in dicots (Aguilar-Martinez et al., 2007; Martin-Trillo et al., 2011; Braun et al., 2012; Dardick et al., 2013). Now, with this knowledge, key genes for genetic engineering or for use as genetic markers for classical breeding of monocot and dicot crops are available.”
From Shaping plant architecture (2015) · cites this paper as Braun et al., 2012
The Results section (page unavailable) identifies pea PsBRC1 as a homolog of maize TB1, supporting the identification of a TB1-related gene in a dicot. The cited paper does not identify a dicot TAC1 ortholog. The subsequent statement about engineering and breeding is the citing authors’ own conclusion, not a claim directly attached to this citation. (LLM, conf=0.86)
Experimental — accuracy not yet measuredCKs down-regulate the FINE CULM1/PsBRANCHED1 (FC1/PsBRC1) gene specifically expressed in axillary buds in rice and pea.
“In rice and pea, CKs down-regulate the FINE CULM1/PsBRANCHED1 (FC1/PsBRC1) gene specifically expressed in axillary buds”
From Multiple pathways regulate shoot branching (2015) · cites this paper as Braun et al., 2012
Direct application of cytokinin on buds reduces BRC1 transcript levels in pea.
“Direct application of cytokinin on buds reduced BRC1 transcript levels in pea (Braun et al., 2012; Dun et al., 2012).”
From Shaping plant architecture (2015) · cites this paper as Braun et al., 2012
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Genetic Regulation of Shoot Architecture
Wang, B., Smith, S. M., & Li, J. (2018) · Annual Review of Plant Biology · doi:10.1146/annurev-arplant-042817-040422 · Cited by 771 · see citing works
“The TB1 ortholog is known as OsTB1 or FINECULM1 in rice and as BRANCHED1 (BRC1) in Arabidopsis, pea, and tomato (2, 17, 104, 110).”
BackgroundStrigolactone Signaling and Evolution
Waters, M. T., Gutjahr, C., Bennett, T., & Nelson, D. C. (2017) · Annual Review of Plant Biology · doi:10.1146/annurev-arplant-042916-040925 · Cited by 655 · see citing works
“First, there are qualitative differences in branching between brc1 and SL mutants in Arabidopsis and pea, and these mutations have additive effects on branching in Arabidopsis (28, 39).”
Antagonistic action of strigolactone and cytokinin in bud outgrowth control
Dun, E. A., de Saint Germain, A., Rameau, C., & Beveridge, C. A. (2012) · Plant Physiology · doi:10.1104/pp.111.186783 · Cited by 466 · see citing works
Self-citationA Pivotal Role of DELLAs in Regulating Multiple Hormone Signals
Davière, J., & Achard, P. (2015) · Molecular Plant · doi:10.1016/j.molp.2015.09.011 · Cited by 459 · see citing works
Shinohara, N., Taylor, C., & Leyser, O. (2013) · PLoS Biology · doi:10.1371/journal.pbio.1001474 · Cited by 438 · see citing works
“Up-regulation of BRC1 by strigolactone addition to pea buds has been shown to be independent of new translation, but so far it has only been measured after 6 h [26,48], and no such upregulation was detected in a similar experiment in rice after 3 h of treatment [49].”
BackgroundWang, L., Wang, B., Jiang, L., Liu, X., Li, X., Lu, Z., Meng, X., Wang, Y., Smith, S. M., & Li, J. (2015) · The Plant Cell · doi:10.1105/tpc.15.00605 · Cited by 434 · see citing works
Self-citationDiverse Roles of Strigolactones in Plant Development
Brewer, P. B., Koltai, H., & Beveridge, C. A. (2012) · Molecular Plant · doi:10.1093/mp/sss130 · Cited by 387 · see citing works
“in garden pea, the actions of strigolactones and cytokinins appear to converge on the bud-specific target gene BRANCHED1 (BRC1), which encodes a transcription factor that represses bud outgrowth (aguilar-Martínez et al., 2007; Finlayson, 2007; Braun et al., 2012; Dun et al., 2012).”
BackgroundTranscriptional regulation of strigolactone signalling in Arabidopsis
Wang, L., Wang, B., Yu, H. J., Guo, H., Lin, T., Kou, L., Wang, A., Shao, N., Ma, H., Xiong, G., et al. (2020) · Nature · doi:10.1038/s41586-020-2382-x · Cited by 364 · see citing works
Self-citationAn Update on the Signals Controlling Shoot Branching
Barbier, F. F., Dun, E. A., Kerr, S. C., Chabikwa, T. G., & Beveridge, C. A. (2019) · Trends in Plant Science · doi:10.1016/j.tplants.2018.12.001 · Cited by 372 · see citing works
Multiple pathways regulate shoot branching
Rameau, C., Bertheloot, J., Leduc, N., Andrieu, B., Foucher, F., & Sakr, S. (2015) · Frontiers in Plant Science · doi:10.3389/fpls.2014.00741 · Cited by 357 · see citing works
Self-citationRuyter-Spira, C. P., Al‐Babili, S., van der Krol, A. R., & Bouwmeester, H. J. (2012) · Trends in Plant Science · doi:10.1016/j.tplants.2012.10.003 · Cited by 353 · see citing works
Wang, Y., Sun, S., Zhu, W., Jia, K., Yang, H., & Wang, X. (2013) · Developmental Cell · doi:10.1016/j.devcel.2013.11.010 · Cited by 319 · see citing works
Song, X., Lu, Z., Yu, H. J., Shao, G., Xiong, J., Meng, X., Jing, Y., Liu, G., Xiong, G., Duan, J., et al. (2017) · Cell Research · doi:10.1038/cr.2017.102 · Cited by 315 · see citing works
The Role of Trehalose 6-Phosphate (Tre6P) in Plant Metabolism and Development
Fichtner, F., & Lunn, J. E. (2021) · Annual Review of Plant Biology · doi:10.1146/annurev-arplant-050718-095929 · Cited by 314 · see citing works
Barbier, F. F., Péron, T., Lecerf, M., Pérez-Garcia, M.-D., Barrière, Q., Rolc̆ı́k, J., Boutet‐Mercey, S., Citerne, S., Lemoine, R., Porcheron, B., et al. (2015) · Journal of Experimental Botany · doi:10.1093/jxb/erv047 · Cited by 295 · see citing works
“Strigolactones modulate BRC1 expression, a gene coding for a transcription factor that inhibits bud outgrowth and with expression well related to bud outgrowth state (Aguilar-Martínez et al., 2007; Braun et al., 2012).”
BackgroundSelf-citationAn histidine covalent receptor and butenolide complex mediates strigolactone perception
Germain, A. D. S., Clavé, G., Badet‐Denisot, M., Pillot, J.-P., Cornu, D., Caër, J. L., Bürger, M., Pélissier, F., Retailleau, P., Turnbull, C. G. N., et al. (2016) · Nature Chemical Biology · doi:10.1038/nchembio.2147 · Cited by 276 · see citing works
“Plants were grown in a greenhouse under long days as described 37 .”
Self-citationBrewer, P. B., Yoneyama, K., Filardo, F. F., Meyers, E., Scaffidi, A., Frickey, T., Akiyama, K., Seto, Y., Dun, E. A., Cremer, J. E., et al. (2016) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1601729113 · Cited by 275 · see citing works
“The active complex targets proteins for degradation, such as SMAX1-like proteins (SMXL6 – SMXL8), and promotes expression of transcription factor genes, such as BRANCHED1 , directly in buds (9 – 13).”
Background<i>BRANCHED1</i> Promotes Axillary Bud Dormancy in Response to Shade in <i>Arabidopsis</i>
González-Grandío, E., Poza-Carrión, C., Sorzano, C. O. S., & Cubas, P. (2013) · The Plant Cell · doi:10.1105/tpc.112.108480 · Cited by 272 · see citing works
Regulation of Leaf Maturation by Chromatin-Mediated Modulation of Cytokinin Responses
Efroni, I., Han, S., Kim, H. J., Wu, M., Steiner, E., Birnbaum, K. D., Hong, J. C., Eshed, Y., & Wagner, D. (2013) · Developmental Cell · doi:10.1016/j.devcel.2013.01.019 · Cited by 266 · see citing works
“Consistent with our findings, the BRANCHED class II CIN-TCP gene, which is specifically expressed in axillary meristems, dampens apical dominance release—a classical CK response (Braun et al., 2012).”
TheArabidopsis thalianaTCP transcription factors: A broadening horizon beyond development
Li, S. (2015) · Plant Signaling & Behavior · doi:10.1080/15592324.2015.1044192 · Cited by 247 · see citing works
“TCP proteins from various species throughout the plant kingdom have been demonstrated to regulate a large variety of biological processes, such as floral asymmetry [23-34], plant architectures including branching [35-39], lateral organ development [40-55], seed germination [56,57], gametophyte development [58,59], lea…”
BackgroundTCP factors: new kids on the signaling block
Nicolas, M., & Cubas, P. (2016) · Current Opinion in Plant Biology · doi:10.1016/j.pbi.2016.05.006 · Cited by 250 · see citing works
“Moreover, double mutants in both brc1 and SL-related genes do not display overall additive or synergistic phenotypes (although in some cases phenotypes are slightly stronger that those of the single mutants [47,49]) indicating that they act, at least in part, through the same pathway [45,47,49,54].”
BackgroundChevalier, F., Nieminen, K. M., Sánchez-Ferrero, J. C., Rodríguez, M. L., Chagoyen, M., Hardtke, C. S., & Cubas, P. (2014) · The Plant Cell · doi:10.1105/tpc.114.122903 · Cited by 239 · see citing works
A genetic network mediating the control of bud break in hybrid aspen
Singh, R. K., Maurya, J. P., Azeez, A., Miskolczi, P., Tylewicz, S., Stojkovič, K., Delhomme, N., Busov, V. B., & Bhalerao, R. P. (2018) · Nature Communications · doi:10.1038/s41467-018-06696-y · Cited by 249 · see citing works
Strigolactones and the control of plant development: lessons from shoot branching
Waldie, T., McCulloch, H., & Leyser, O. (2014) · The Plant Journal · doi:10.1111/tpj.12488 · Cited by 242 · see citing works
Teichmann, T., & Muhr, M. (2015) · Frontiers in Plant Science · doi:10.3389/fpls.2015.00233 · Cited by 228 · see citing works
TCP transcription factors: architectures of plant form
Manassero, N. G. U., Viola, I. L., Welchen, E., & González, D. H. (2013) · BioMolecular Concepts · doi:10.1515/bmc-2012-0051 · Cited by 227 · see citing works
Danisman, S. (2016) · Frontiers in Plant Science · doi:10.3389/fpls.2016.01930 · Cited by 204 · see citing works
“This fits to the observation that it is not the formation of axillary meristems but the outgrowth of these that is affected by TCPs (Braun et al., 2012).”
BackgroundCheng, X., Ruyter-Spira, C. P., & Bouwmeester, H. J. (2013) · Frontiers in Plant Science · doi:10.3389/fpls.2013.00199 · Cited by 200 · see citing works
Abscisic Acid Is a General Negative Regulator of Arabidopsis Axillary Bud Growth
Yao, C., & Finlayson, S. A. (2015) · PLANT PHYSIOLOGY · doi:10.1104/pp.15.00682 · Cited by 199 · see citing works
“…branching in intact wild-type Arabidopsis (Gomez-Roldan et al., 2008; Nelson et al., 2011), O. sativa (Umehara et al., 2008), petunia (Petunia hybrida; Hamiaux et al., 2012; Kretzschmar et al., 2012), pea (Braun et al., 2012), tomato (Koltai et al., 2010), and maize (Zea mays; Guan et al., 2012).”
BackgroundCytokinin Targets Auxin Transport to Promote Shoot Branching
Waldie, T., & Leyser, O. (2018) · PLANT PHYSIOLOGY · doi:10.1104/pp.17.01691 · Cited by 198 · see citing works
“There is good evidence to support this model, since in both pea (Pisum sativum) and Arabidopsis, BRC1 expression is down-regulated by CK (Braun et al., 2012; Dun et al., 2012; Seale et al., 2017).”
BackgroundInfluentialDuan, J., Yu, H. J., Yuan, K., et al. (2019) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1810980116 · Cited by 194 · see citing works
“SL and CK have been shown to function antagonistically in pea bud elongation (7, 10) and rice mesocotyl elongation in darkness (8); however, compared with the well-studied interactions between auxin and CK and between auxin and SL, relatively little is known about the interaction between CK and SL, especially in monoc…”
Boyer, F., Germain, A. D. S., Pillot, J.-P., Pouvreau, J., Chen, V. X., Ramos, S., Stévenin, A., Simier, P., Delavault, P. M., Beau, J., et al. (2012) · PLANT PHYSIOLOGY · doi:10.1104/pp.112.195826 · Cited by 188 · see citing works
“Trends Plant Sci 12: 224–230 Braun N, de Saint Germain A, Pillot JP, Boutet-Mercey S, Dalmais M, Antoniadi I, Li X, Maia-Grondard A, Le Signor C, Bouteiller N, et al (2012) The pea TCP transcription factor PsBRC1 acts downstream of strigolactones to control shoot branching.”
BackgroundMethodInfluentialSelf-citationBRANCHED1: A Key Hub of Shoot Branching
Wang, M., Moigne, M.-A. L., Bertheloot, J., Crespel, L., Pérez-Garcia, M.-D., Ogé, L., Demotes‐Mainard, S., Hamama, L., Davière, J., & Sakr, S. (2019) · Frontiers in Plant Science · doi:10.3389/fpls.2019.00076 · Cited by 184 · see citing works
Xu, J., Zha, M., Li, Y., Ding, Y., Chen, L., Ding, C., & Wang, S. (2015) · Plant Cell Reports · doi:10.1007/s00299-015-1815-8 · Cited by 185 · see citing works
Gibberellin Promotes Shoot Branching in the Perennial Woody PlantJatropha curcas
Ni, J., Gao, C., Chen, M.-S., Pan, B.-Z., Ye, K., & Xu, Z. (2015) · Plant and Cell Physiology · doi:10.1093/pcp/pcv089 · Cited by 179 · see citing works
“BRC1 acts as a local integrator controlling bud outgrowth that is positively regulated by SLs, and down-regulated by CK treatment in Arabidopsis and pea (Aguilar-Martı́nez et al. 2007, Braun et al. 2012).”
BackgroundCytokinin is required for escape but not release from auxin mediated apical dominance
Müller, D., Waldie, T., Miyawaki, K. N., To, J. P. C., Melnyk, C. W., Kieber, J. J., Kakimoto, T., & Leyser, O. (2015) · The Plant Journal · doi:10.1111/tpj.12862 · Cited by 175 · see citing works
“This is proposed to be mediated by the opposite effects of SL and CK on the expression of BRANCHED1 (Braun et al., 2012; Dun et al., 2012), a member of the TEOSINTE BRANCHED1, CYCLOIDEA and PROLIFERATING CELL FACTORS 1 and -2 (TCP) gene family with a well-supported role in inhibiting bud activity (Doebley et al.”
BackgroundInfluentialGrasses provide new insights into regulation of shoot branching
Kebrom, T. H., Spielmeyer, W., & Finnegan, E. J. (2012) · Trends in Plant Science · doi:10.1016/j.tplants.2012.07.001 · Cited by 162 · see citing works
Strigolactones stimulate internode elongation independently of gibberellins
de Saint Germain, A., Ligerot, Y., Dun, E. A., Pillot, J.-P., Ross, J. J., Beveridge, C. A., & Rameau, C. (2013) · Plant Physiology · doi:10.1104/pp.113.220541 · Cited by 166 · see citing works
“For hydroponics treatments, culture was as described by Braun et al. (2012), except that 33-”
BackgroundMethodInfluentialSelf-citationRegulation of shoot branching in arabidopsis by trehalose 6‐phosphate
Fichtner, F., Barbier, F. F., Annunziata, M. G., Feil, R., Olas, J. J., Mueller‐Roeber, B., Stitt, M., Beveridge, C. A., & Lunn, J. E. (2020) · New Phytologist · doi:10.1111/nph.17006 · Cited by 164 · see citing works
“…synthesis of strigolactones, which inhibit axillary bud growth (Shimizu-Sato et al., 2009; Domagalska and Leyser, 2011), partly via induction of the BRANCHED1 (BRC1) transcription factor, which is a repressor of branching (Aguilar-Martinez et al., 2007; Dun et al., 2009, 2012; Braun et al., 2012).”
BackgroundShen, J., Zhang, Y., Ge, D., Wang, Z., Song, W., Gu, R., Che, G., Cheng, Z., Liu, R., & Zhang, X. (2019) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1907968116 · Cited by 162 · see citing works
Sugar availability suppresses the auxin‐induced strigolactone pathway to promote bud outgrowth
Bertheloot, J., Barbier, F. F., Boudon, F., Pérez-Garcia, M.-D., Péron, T., Citerne, S., Dun, E. A., Beveridge, C. A., Godin, C., & Sakr, S. (2019) · New Phytologist · doi:10.1111/nph.16201 · Cited by 158 · see citing works
Strigolactone regulates shoot development through a core signalling pathway
Bennett, T., Liang, Y., Seale, M., Ward, S. P., Müller, D., & Leyser, O. (2016) · Biology Open · doi:10.1242/bio.021402 · Cited by 155 · see citing works
“of genetic additivity in some, but not all, brc1 max double mutants (Aguilar-Martinez et al., 2007; Braun et al., 2012; Chevalier et al., 2014).”
BackgroundInfluentialReady, steady, go! A sugar hit starts the race to shoot branching
Barbier, F. F., Lunn, J. E., & Beveridge, C. A. (2015) · Current Opinion in Plant Biology · doi:10.1016/j.pbi.2015.04.004 · Cited by 153 · see citing works
Guan, J., Koch, K. E., Suzuki, M., Wu, S., Latshaw, S. P., Petruff, T. A., Goulet, C., Klee, H. J., & McCarty, D. R. (2012) · PLANT PHYSIOLOGY · doi:10.1104/pp.112.204503 · Cited by 153 · see citing works
“Auxin up-regulates CCD7 and CCD8 genes in Arabidopsis (Hayward et al., 2009), pea (Brewer et al., 2009), and rice (Arite et al., 2007).”
BackgroundAuxin Interactions with Other Hormones in Plant DevelopmentPreprint
Mazzoni, S. M., Brumós, J., Zhao, C., Alonso, J. M., & Stepanova, A. N. (2021) · Cold Spring Harbor Perspectives in Biology · doi:10.1101/cshperspect.a039990 · Cited by 157 · see citing works
Liang, Y., Ward, S. P., Li, P., Bennett, T., & Leyser, O. (2016) · The Plant Cell · doi:10.1105/tpc.16.00286 · Cited by 141 · see citing works
Thilakarathne, A. S., Liu, F., & Zou, Z. (2025) · Plants · doi:10.3390/plants14071070 · Cited by 150 · see citing works
Emerging Roles of Strigolactones in Plant Responses to Stress and Development
Pandey, A., Sharma, M., & Pandey, G. K. (2016) · Frontiers in Plant Science · doi:10.3389/fpls.2016.00434 · Cited by 135 · see citing works
Roles of Auxin in the Growth, Development, and Stress Tolerance of Horticultural Plants
Zhang, Q., Gong, M., Xu, X., et al. (2022) · Cells · doi:10.3390/cells11172761 · Cited by 135 · see citing works
“On the contrary, auxin inhibits cytokinin (CK) biosynthesis to promote transcription of TB1/BRC1 and repress shoot branching in pea plants [43].”
BackgroundAltering Plant Architecture to Improve Performance and Resistance
Guo, W., Chen, L., Herrera‐Estrella, L., et al. (2020) · Trends in Plant Science · doi:10.1016/j.tplants.2020.05.009 · Cited by 130 · see citing works
Habets, M. E. J., & Offringa, R. (2014) · New Phytologist · doi:10.1111/nph.12831 · Cited by 130 · see citing works
An, J., Liu, Y., Zhang, X., Bi, S., Wang, X., You, C., & Hao, Y. (2020) · Journal of Experimental Botany · doi:10.1093/jxb/eraa056 · Cited by 129 · see citing works
“There is evidence that TCP proteins play broad roles in plant growth and development, including cell proliferation, gametophyte development, seed germination, leaf and flower development, branching, regulation of the circadian clock, and multiple hormone signals (Corley et al., 2005; Tatematsu et al., 2008; Guo et al.…”
BackgroundRegulation of axillary shoot development
Janssen, B. J., Drummond, R., & Snowden, K. C. (2013) · Current Opinion in Plant Biology · doi:10.1016/j.pbi.2013.11.004 · Cited by 126 · see citing works
A Dual Role of Strigolactones in Phosphate Acquisition and Utilization in Plants
Czarnecki, O., Yang, J., Weston, D. J., Tuskan, G. A., & Chen, J. (2013) · International Journal of Molecular Sciences · doi:10.3390/ijms14047681 · Cited by 128 · see citing works
“Consistent with this view, it recently was found that the Pisum sativum TCP transcription factor PsBRC1, a homolog of the maize TEOSINTE BRANCHED1 [120] and the Arabidopsis BRANCHED1 (AtBRC1) [121], acts downstream of MAX2 to control shoot branching [122].”
Science and application of strigolactones
Aliche, E. B., Screpanti, C., Mesmaeker, A. D., Munnik, T., & Bouwmeester, H. J. (2020) · New Phytologist · doi:10.1111/nph.16489 · Cited by 126 · see citing works
Cytokinins Are Initial Targets of Light in the Control of Bud Outgrowth
Roman, H., Girault, T., Barbier, F. F., Péron, T., Brouard, N., Pěnčík, A., Novák, O., Vian, A., Sakr, S., Lothier, J., et al. (2016) · PLANT PHYSIOLOGY · doi:10.1104/pp.16.00530 · Cited by 125 · see citing works
“It should be pointed out that such negative effects of CK on RwMAX2 transcripts also may contribute indirectly to the repression of RhBRC1 that is promoted by the SL signal (Aguilar-Martínez et al., 2007; Braun et al., 2012).”
BackgroundStrigolactone biosynthesis and signaling in plant development
Lopez‐Obando, M., Ligerot, Y., Bonhomme, S., Boyer, F., & Rameau, C. (2015) · Development · doi:10.1242/dev.120006 · Cited by 124 · see citing works
“Because SL-deficient mutants are more branched than brc1 mutants (Braun et al., 2012; Minakuchi et al., 2010), there is very likely a BRC1-independent effect of SLs on shoot branching.”
BackgroundSelf-citationContribution of strigolactone in plant physiology, hormonal interaction and abiotic stresses
Bhoi, A., Yadu, B., Chandra, J., & Keshavkant, S. (2021) · Planta · doi:10.1007/s00425-021-03678-1 · Cited by 124 · see citing works
Seale, M., Bennett, T., & Leyser, O. (2017) · Development · doi:10.1242/dev.145649 · Cited by 121 · see citing works
Luo, L., Li, W., Miura, K., Ashikari, M., & Kyozuka, J. (2012) · Plant and Cell Physiology · doi:10.1093/pcp/pcs122 · Cited by 117 · see citing works
“…as Arabidopsis BRANCHED 1 (AtBRC1), rice FINE CULM1 (FC1/OsTB1) and pea BRANCHED1 (PsBRC1), have been identified as downstream regulators of SL (Mashiguchi et al. 2009, Minakuchi et al. 2010, Braun et al. 2012); however, the mechanisms by which the SLs control bud outgrowth are poorly understood.”
BackgroundA Recently Evolved Alternative Splice Site in the BRANCHED1a Gene Controls Potato Plant Architecture
Nicolas, M., Rodríguez‐Buey, M. L., Franco‐Zorrilla, J. M., & Cubas, P. (2015) · Current Biology · doi:10.1016/j.cub.2015.05.053 · Cited by 117 · see citing works
“This indicated that the role of potato BRC1a is similar to that of other BRC1-like genes and controls development of aerial and underground lateral shoots [6, 11, 15].”
Sucrose promotes D53 accumulation and tillering in rice
Patil, S. B., Barbier, F. F., Zhao, J., Zafar, S. A., Uzair, M., Sun, Y., Fang, J., Pérez-Garcia, M.-D., Bertheloot, J., Sakr, S., et al. (2021) · New Phytologist · doi:10.1111/nph.17834 · Cited by 115 · see citing works
Self-citationYuan, K., Zhang, H., Yu, C., et al. (2023) · Molecular Plant · doi:10.1016/j.molp.2023.09.022 · Cited by 114 · see citing works
Light Signaling in Bud Outgrowth and Branching in Plants
Leduc, N., Roman, H., Barbier, F. F., Péron, T., Huché‐Thélier, L., Lothier, J., Demotes‐Mainard, S., & Sakr, S. (2014) · Plants · doi:10.3390/plants3020223 · Cited by 112 · see citing works
“BRC1 expression is also repressed by CKs [55,96,176],”
BackgroundInfluentialStrigolactones and their crosstalk with other phytohormones
Omoarelojie, L. O., Kulkarni, M. G., Finnie, J. F., & Staden, J. V. (2019) · Annals of Botany · doi:10.1093/aob/mcz100 · Cited by 110 · see citing works
Dynamics of Strigolactone Function and Shoot Branching Responses in Pisum sativum
Dun, E. A., Germain, A. D. S., Rameau, C., & Beveridge, C. A. (2012) · Molecular Plant · doi:10.1093/mp/sss131 · Cited by 107 · see citing works
Self-citationThe role of the potato ( S olanum tuberosum ) CCD8 gene in stolon and tuber development
Pasare, S. A., Ducreux, L. J. M., Morris, W. L., Campbell, R., Sharma, S. K., Roumeliotis, E., Kohlen, W., van der Krol, S., Bramley, P. M., Roberts, A. G., et al. (2013) · New Phytologist · doi:10.1111/nph.12217 · Cited by 108 · see citing works
The molecular and genetic regulation of shoot branching
Luo, Z., Janssen, B. J., & Snowden, K. C. (2021) · PLANT PHYSIOLOGY · doi:10.1093/plphys/kiab071 · Cited by 104 · see citing works
Strigolactones positively regulate chilling tolerance in pea and in Arabidopsis
Cooper, J. W., Hu, Y., Beyyoudh, L., et al. (2018) · Plant Cell & Environment · doi:10.1111/pce.13147 · Cited by 102 · see citing works
Hd3a promotes lateral branching in rice
Tsuji, H., Tachibana, C., Tamaki, S., Taoka, K., Kyozuka, J., & Shimamoto, K. (2015) · The Plant Journal · doi:10.1111/tpj.12811 · Cited by 100 · see citing works
Strigolactone Inhibition of Branching Independent of Polar Auxin Transport
Brewer, P. B., Dun, E. A., Gui, R., Mason, M. G., & Beveridge, C. A. (2015) · PLANT PHYSIOLOGY · doi:10.1104/pp.15.00014 · Cited by 100 · see citing works
“In pea, BRC1 is also regulated by cytokinins and Suc (Braun et al., 2012; Dun et al., 2012; Mason et al., 2014), and it is possible that cell cycle progression is repressed by BRC1 in the bud (Martín-Trillo and Cubas, 2010).”
BackgroundInfluentialSchneider, A., Godin, C., Boudon, F., Demotes‐Mainard, S., Sakr, S., & Bertheloot, J. (2019) · Frontiers in Plant Science · doi:10.3389/fpls.2019.01296 · Cited by 102 · see citing works
“, Cournede, P. H., Mathieu, A., de Reffye, P., and Constant, T. (2008). Parametric identification of a functional-structural tree growth model and application to beech trees (Fagus sylvatica).”
BackgroundDistinct gibberellin functions during and after grapevine bud dormancy release
Zheng, C., Acheampong, A. K., Shi, Z., et al. (2018) · Journal of Experimental Botany · doi:10.1093/jxb/ery022 · Cited by 96 · see citing works
“A positive role for CK in regulation of paradormant bud outgrowth is also well established (Domagalska and Leyser, 2011; Müller and Leyser, 2011; Braun et al., 2012; Dun et al., 2012).”
BackgroundStrigolactone signalling: standing on the shoulders of DWARFs
Bennett, T., & Leyser, O. (2014) · Current Opinion in Plant Biology · doi:10.1016/j.pbi.2014.08.001 · Cited by 95 · see citing works
Strigolactone and Cytokinin Act Antagonistically in Regulating Rice Mesocotyl Elongation in Darkness
Hu, Z., Yamauchi, T., Yang, J., Jikumaru, Y., Tsuchida‐Mayama, T., Ichikawa, H., Takamure, I., Nagamura, Y., Tsutsumi, N., Yamaguchi, S., et al. (2013) · Plant and Cell Physiology · doi:10.1093/pcp/pct150 · Cited by 94 · see citing works
“Indeed, CK down-regulates and SL up-regulates the expression of PsBRC1 in buds (Braun et al. 2012, Dun et al. 2012).”
InfluentialAuxin and cytokinin coordinate the dormancy and outgrowth of axillary bud in strawberry runner
Qiu, Y., Guan, S., Wen, C., Li, P., Gao, Z., & Chen, X. (2019) · BMC Plant Biology · doi:10.1186/s12870-019-2151-x · Cited by 91 · see citing works
Strigolactones spatially influence lateral root development through the cytokinin signaling network
Jiang, L., Matthys, C., Márquez‐García, B., Cuyper, C. D., Smet, L. D., Keyser, A. D., Boyer, F., Beeckman, T., Depuydt, S., & Goormachtig, S. (2015) · Journal of Experimental Botany · doi:10.1093/jxb/erv478 · Cited by 92 · see citing works
“When MAX2 function was restored specifically in the root endodermis of max2 mutants, their insensitivity could be partially complemented (Koren et al., 2013).”
BackgroundNew Strigolactone Analogs as Plant Hormones with Low Activities in the Rhizosphere
Boyer, F., Germain, A. D. S., Pouvreau, J., Clavé, G., Pillot, J.-P., Roux, A., Rasmussen, A., Depuydt, S., Lauressergues, D., Frey, N. F. D., et al. (2013) · Molecular Plant · doi:10.1093/mp/sst163 · Cited by 93 · see citing works
Self-citationThe miR164‐GhCUC2‐GhBRC1 module regulates plant architecture through abscisic acid in cotton
Zhan, J., Chu, Y., Wang, Y., Diao, Y., Zhao, Y., Liu, L., Xi, W., Meng, Y., Li, F., & Ge, X. (2021) · Plant Biotechnology Journal · doi:10.1111/pbi.13599 · Cited by 90 · see citing works
“…been called a branching integrator because it acts downstream of many other pathways that influence branching, including the strigolactone pathway (Braun et al., 2011), auxin (AguilarMart ınez et al., 2007), ABA (Gonz alez-Grand ıo et al., 2017), cytokinins (Stes et al., 2015), decapitation (Gao…”
BackgroundFellowship of the rings: a saga of strigolactones and other small signals
Machin, D. C., Hamon‐Josse, M., & Bennett, T. (2019) · New Phytologist · doi:10.1111/nph.16135 · Cited by 87 · see citing works
“…factor is the clearest candidate, given reduced BRC1 expression in strigolactone signalling mutants, increased BRC1 expression in smxl6 smxl7 smxl8 triple mutants, and rapid transcriptional responses to rac-GR24 (Braun et al., 2012; Dun et al., 2012; Soundappan et al., 2015; Seale et al., 2017).”
BackgroundCostes, E., Crespel, L., Denoyes-Rothan, B., Morel, P., Demene, M.-N., Lauri, P., & Wenden, B. (2014) · Frontiers in Plant Science · doi:10.3389/fpls.2014.00666 · Cited by 89 · see citing works
“The BRANCHED 1 (BRC1) gene likely integrates these multiple bud outgrowth pathways (Braun et al., 2012).”
BackgroundInfluentialMehrnia, M., Balazadeh, S., Zanor, M. I., & Mueller‐Roeber, B. (2013) · PLANT PHYSIOLOGY · doi:10.1104/pp.113.214049 · Cited by 88 · see citing works
“Previously, transcription factors of the TCP family, including BRC1 and BRC2 in Arabidopsis, were shown to play a critical role in shoot branching (Aguilar-Martínez et al., 2007; Finlayson, 2007; Martín-Trillo et al., 2011; Braun et al., 2012).”
BackgroundStrigolactones, karrikins and beyond
Cuyper, C. D., Struk, S., Braem, L., Gevaert, K., Jaeger, G. D., & Goormachtig, S. (2017) · Plant Cell & Environment · doi:10.1111/pce.12996 · Cited by 87 · see citing works
“All rights reserved. because tir3-1 mutant plants with a deficient PIN1 accumulation have no increased BRC1 expression levels and brc1-2brc2-1 double mutants have no altered PIN1 distribution or auxin flow (Bennett et al. 2016).”
InfluentialPossible Roles of Strigolactones during Leaf Senescence
Yamada, Y., & Umehara, M. (2015) · Plants · doi:10.3390/plants4030664 · Cited by 86 · see citing works
“In shoot branching regulation, strigolactones and cytokinins are thought to be transported acropetally through the xylem and act directly to control axillary bud outgrowth through joint regulation of a TCP (for TB1, CYCLOIDEA, PCF domain) transcription factor BRC1 [61,62].”
BackgroundBiological Functions of Strigolactones and Their Crosstalk With Other Phytohormones
Wu, F., Gao, Y., Yang, W., Sui, N., & Zhu, J. (2022) · Frontiers in Plant Science · doi:10.3389/fpls.2022.821563 · Cited by 86 · see citing works
“This process involves SL-induced upregulation of the TCP transcription factor BRANCHED1 (BRC1) that suppresses bud activity (Braun et al., 2012; Dun et al., 2013).”
BackgroundCao, D., Chabikwa, T. G., Barbier, F. F., Dun, E. A., Fichtner, F., Dong, L., Kerr, S. C., & Beveridge, C. A. (2023) · PLANT PHYSIOLOGY · doi:10.1093/plphys/kiad034 · Cited by 90 · see citing works
“…lack of SL antagonism of CK response during bud release (shortly after inductive treatments) contrasts with the many findings regarding the antagonism of SL and CK during bud outgrowth which is thought to occur through regulation of BRC1 (Braun et al., 2012; Dun et al., 2012; Kerr et al., 2020).”
BackgroundResultInfluentialRoles of miR319-regulated TCPs in plant development and response to abiotic stress
Fang, Y., Zheng, Y., Lu, W., Li, J., Duan, Y., Zhang, S., & Wang, Y. (2020) · The Crop Journal · doi:10.1016/j.cj.2020.07.007 · Cited by 87 · see citing works
Strigolactone: An Emerging Growth Regulator for Developing Resilience in Plants
Alvi, A. F., Sehar, Z., Fatma, M., Masood, A., & Khan, N. A. (2022) · Plants · doi:10.3390/plants11192604 · Cited by 86 · see citing works
Cheng, J., Zhang, M., Tan, B., Jiang, Y., Zheng, X., Ye, X., Guo, Z., Xiong, T., Wang, W., Li, J., et al. (2019) · Plant Biotechnology Journal · doi:10.1111/pbi.13094 · Cited by 81 · see citing works
“Teosinte branched1 (Tb1), a TCP transcription factor, is a key component in the SL signalling pathway and plays a role in repressing the growth of lateral branches in Arabidopsis, pea, poplar and rice (Braun et al., 2012; Finlayson et al., 2010; Minakuchi et al., 2010; Muhr et al., 2016).”
BackgroundKorek, M., & Marzec, M. (2023) · BMC Plant Biology · doi:10.1186/s12870-023-04332-6 · Cited by 88 · see citing works
Hu, J., Ji, Y., Hu, X., Sun, S., & Wang, X. (2019) · Plant Communications · doi:10.1016/j.xplc.2019.100014 · Cited by 83 · see citing works
“In addition, in pea and rice, Psbrc1/Osfc1 mutants are insensitive to GR24 treatment and genetically function downstream of SL signaling to inhibit branching (or tillering in rice) (Aguilar-Martinez et al., 2007; Braun et al., 2012; Dun et al., 2012; Guan et al., 2012; Lu et al., 2013).”
BackgroundBao, S., Owens, R. A., Sun, Q., et al. (2019) · PLoS Pathogens · doi:10.1371/journal.ppat.1008110 · Cited by 81 · see citing works
Environmental Control of Branching in Petunia
Drummond, R., Janssen, B. J., Luo, Z., Oplaat, C., Ledger, S., Wohlers, M. W., & Snowden, K. C. (2015) · PLANT PHYSIOLOGY · doi:10.1104/pp.15.00486 · Cited by 80 · see citing works
“A TCP ortholog from pea, PsBRC1, is responsive to SL (Braun et al., 2012; Dun et al., 2012), with PsBRC1 mRNA levels regulated in a translation-independent manner, suggesting the SL-induced degradation of a transcriptional repressor of PsBRC1.”
BackgroundChomicki, G., Coiro, M., & Renner, S. S. (2017) · Annals of Botany · doi:10.1093/aob/mcx113 · Cited by 81 · see citing works
The importance of strigolactone transport regulation for symbiotic signaling and shoot branching
Borghi, L., Liu, G., Emonet, A., Kretzschmar, T., & Martinoia, E. (2016) · Planta · doi:10.1007/s00425-016-2503-9 · Cited by 76 · see citing works
“…in vitro with TOPLESS (TPL) and TPL-related co-repressors (Jiang et al. 2013; Zhou et al. 2013), which possibly regulate downstream transcription factors such as FINE CULM1/ BRANCHED1 (FC1/BRC1) (Minakuchi et al. 2010; Braun et al. 2012), involved in SL-induced repression of lateral bud outgrowth.”
BackgroundInfluentialKebrom, T. H., & Mullet, J. E. (2016) · PLANT PHYSIOLOGY · doi:10.1104/pp.16.00014 · Cited by 78 · see citing works
“These genes also help mediate bud growth inhibition caused by SL (Aguilar-Martínez et al., 2007; Finlayson, 2007; Braun et al., 2012).”
Backgroundvan Rongen, M., Bennett, T., Ticchiarelli, F., & Leyser, O. (2019) · PLoS Genetics · doi:10.1371/journal.pgen.1008023 · Cited by 74 · see citing works
Liu, M., Wang, M.-M., Yang, J., Wen, J., Guo, P.-C., Wu, Y., Ke, Y.-Z., Li, P.-F., Li, J., & Du, H. (2019) · International Journal of Molecular Sciences · doi:10.3390/ijms20143591 · Cited by 77 · see citing works
“Pisum sativum PsBRC1 Regulate shoot branching [57].”
BackgroundResultKnockout of VvCCD8 gene in grapevine affects shoot branching
Ren, C., Guo, Y., Kong, J., Lecourieux, F., Dai, Z., Li, S., & Liang, Z. (2020) · BMC Plant Biology · doi:10.1186/s12870-020-2263-3 · Cited by 75 · see citing works
Lessons from a century of apical dominance research
Beveridge, C. A., Rameau, C., & Yapa, A. W. (2023) · Journal of Experimental Botany · doi:10.1093/jxb/erad137 · Cited by 80 · see citing works
“…several species, when comparing the shoot branching of brc1 mutants with that of SL-related mutants, a strong difference in intensity of branching and plant height can be observed, with the SL-related mutants being more dwarf and bushy than brc1 mutants (Braun et al., 2012; Bennett et al., 2016).”
InfluentialSelf-citation
- Authors
- Braun, N., de Saint Germain, A., Pillot, J.-P., Boutet-Mercey, S., Dalmais, M., Antoniadi, I., Li, X., Maia-Grondard, A., Le Signor, C., Bouteiller, N., Luo, D., Bendahmane, A., Turnbull, C., & Rameau, C.
- Affiliations
- Centre National de la Recherche Scientifique (FR)
- Institut Jean-Pierre Bourgin, INRA UMR1318 INRA-AgroParisTech, F–78000 Versailles, France (N. Braun, A.d.S.G., J.-P.P., S.B.-M., A.M.-G., C.R.); School of Life Sciences, Sun Yat Sen University, Guangzhou 510275, China (X.L., D.L.); Unité de Recherche en Génomique Végétale, INRA/CNRS, 91057 Evry cedex, France (M.D., N. Bouteiller, A.B.); INRA Dijon, 21065 Dijon cedex, France (C.L.S.); Division of
- ORCID
- Venue
- PLANT PHYSIOLOGY
- Publisher
- Oxford University Press (OUP)
- Published
- 2011-11-01
- Volume
- 158
- Issue
- 1
- Pages
- 225-238
- ISSN
- 0032-0889 (ISSN-L)
- 1532-2548
- PMC
- PMC3252107
- OpenAlex
- W2154110613
- Cited by
- 486
- References
- 83
- Citations by year
- 2026: 22 · 2025: 30 · 2024: 33 · 2023: 30 · 2022: 65 · 2021: 36 · 2020: 38 · 2019: 41 · 2018: 21 · 2017: 28 · 2016: 32 · 2015: 30 · 2014: 14 · 2013: 26 · 2012: 12
- FWCI
- 21.53
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model (vor)
- Locations
- PLANT PHYSIOLOGY (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (submittedVersion)
- HAL (Le Centre pour la Communication Scientifique Directe) (submittedVersion, other-oa)
- Spiral (Imperial College London) (submittedVersion)
- Topics
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Parasitism and Resistance
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Molecular Biology Research
- Life Sciences › Agricultural and Biological Sciences › Ecology, Evolution, Behavior and Systematics › Plant and animal studies
- Keywords
- strigolactones, shoot branching, TEOSINTE BRANCHED1, cytokinin signaling, axillary bud outgrowth, GR24, benzyladenine, zeatin riboside
- MeSH terms
- Benzyl Compounds, Cytokinins, Isopentenyladenosine
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