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Cited by 21Plant Molecular Biology ResearchPlant nutrient uptake and metabolismPlant Parasitism and Resistance

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Divergence of trafficking and polarization mechanisms for PIN auxin transporters during land plant evolution

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Tang, Han, Lu, Kuan-Ju, Zhang, YuZhou, Cheng, You-Liang, Tu, Shih-Long, Friml, Jiří·2024·Plant Communications
DOI 10.1016/j.xplc.2023.100669PMC PMC10811345

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Abstract

The phytohormone auxin, and its directional transport through tissues, plays a fundamental role in the development of higher plants. This polar auxin transport predominantly relies on PIN-FORMED (PIN) auxin exporters. Hence, PIN polarization is crucial for development, but its evolution during the rise of morphological complexity in land plants remains unclear. Here, we performed a cross-species investigation by observing the trafficking and localization of endogenous and exogenous PINs in two bryophytes, Physcomitrium patens and Marchantia polymorpha, and in the flowering plant Arabidopsis thaliana. We confirmed that the GFP fusion did not compromise the auxin export function of all examined PINs by using a radioactive auxin export assay and by observing the phenotypic changes in transgenic bryophytes. Endogenous PINs polarize to filamentous apices, while exogenous Arabidopsis PINs distribute symmetrically on the membrane in both bryophytes. In the Arabidopsis root epidermis, bryophytic PINs have no defined polarity. Pharmacological interference revealed a strong cytoskeletal dependence of bryophytic but not Arabidopsis PIN polarization. The divergence of PIN polarization and trafficking is also observed within the bryophyte clade and between tissues of individual species. These results collectively reveal the divergence of PIN trafficking and polarity mechanisms throughout land plant evolution and the co-evolution of PIN sequence-based and cell-based polarity mechanisms.

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Figures

Figure 1 Phosphorylation sites are highly conserved between bryophytic and Arabidopsis PINs. (A) Phylogenetic analysis of canonical PINs from the early-divergent plants P. patens (Pp) and M. polymorpha (Mp) and the representative angiosperm A. thaliana (At). The unrooted tree shows the relationships between different PINs in two representative bryophytes and Arabidopsis. The scale bar represents the number of changes per site. (B) Identity indexes of all PINs compared with AtPIN2 with full-length or only the HL region of coding amino acid sequences. Identity indexes of all PINs compared with AtPIN1 are shown in Supplemental Figure 1. (C) Alignment of PIN amino acid sequences. Identical amino acids are highlighted with blue columns. The red and green boxes highlight the HL regions bearing conserved phosphorylation sites, which are enlarged in (D). (D) Four conserved phosphorylation sites, labeled S1-S4, were verified in previous AtPIN2 studies and are depicted with black frames. Note that S1-S4 are conserved in every examined PIN.
Figure 1 Phosphorylation sites are highly conserved between bryophytic and Arabidopsis PINs. (A) Phylogenetic analysis of canonical PINs from the early-divergent plants P. patens (Pp) and M. polymorpha (Mp) and the representative angiosperm A. thaliana (At). The unrooted tree shows the relationships between different PINs in two representative bryophytes and Arabidopsis. The scale bar represents the number of changes per site. (B) Identity indexes of all PINs compared with AtPIN2 with full-length or only the HL region of coding amino acid sequences. Identity indexes of all PINs compared with AtPIN1 are shown in Supplemental Figure 1. (C) Alignment of PIN amino acid sequences. Identical amino acids are highlighted with blue columns. The red and green boxes highlight the HL regions bearing conserved phosphorylation sites, which are enlarged in (D). (D) Four conserved phosphorylation sites, labeled S1-S4, were verified in previous AtPIN2 studies and are depicted with black frames. Note that S1-S4 are conserved in every examined PIN.CC BY · PubMed Central
Figure 2 The HL regions in AtPIN2, PpPINA, and MpPINZ are less conserved. (A) Structural alignment of AtPIN1 and AtPIN2. The protein structures were predicted using Alphafold2 and aligned using ChimeraX. The structurally conserved regions are indicated by black arrowheads, and the non-conserved regions are indicated by white arrowheads. (B) Structural alignment of AtPIN2 with PpPINA and MpPINZ. Only the transmembrane domains and a single loop align with each other, while the majority of HL regions are not conserved. The four conserved phosphorylation sites are labeled with atomic details in ball-and-stick style. (C–E) Individual protein structures retrieved from (B). The four conserved phosphorylation sites are indicated by red arrowheads.
Figure 2 The HL regions in AtPIN2, PpPINA, and MpPINZ are less conserved. (A) Structural alignment of AtPIN1 and AtPIN2. The protein structures were predicted using Alphafold2 and aligned using ChimeraX. The structurally conserved regions are indicated by black arrowheads, and the non-conserved regions are indicated by white arrowheads. (B) Structural alignment of AtPIN2 with PpPINA and MpPINZ. Only the transmembrane domains and a single loop align with each other, while the majority of HL regions are not conserved. The four conserved phosphorylation sites are labeled with atomic details in ball-and-stick style. (C–E) Individual protein structures retrieved from (B). The four conserved phosphorylation sites are indicated by red arrowheads.CC BY · PubMed Central
Figure 3 GFP-fused PIN proteins possess auxin export activity. (A) The insertion site of GFP in the indicated PIN proteins. Numbers represent the amino acid position in the respective proteins. (B) The auxin export assay with P. patens transgenic plants. Fresh tissues were cultured in liquid medium supplied with 1μM β-estradiol to induce XVE overexpression. Tissues were then incubated with radioactive H3-IAA followed by washing. Radioactive H3-IAA exported into the new culture medium was measured using a scintillation detector after one day. Wild-type moss plants treated with the same conditions were used as a control. Ten to fifteen 10-day-old moss colonies were used for one measurement, and the graph shows the mean ± SD from four independent experiments. (C) Representative protonema cells of the indicated genetic background. The cell outline was stained with FM4-64. White arrowheads indicate the first cell division plane, and the yellow double arrow indicates the length of the subapical cell. Scale bar, 100 μm. (D and E) Quantification of the subapical cell length and division angle (ϴ) of the indicated lines. Bold horizontal lines indicate the median, and whiskers indicate the first and third quartile. ∗∗∗P < 0.001, Student’s t-test. (F) Top (top panels) and side (bottom panels) views of the indicated M. polymorpha lines. Takaragaike-1 (WT) expands its thallus horizontally on the agar surface. Overexpression of MpPINZ-, AtPIN1-, and AtPIN2-GFP showed vertical thallus growth, which generated a large angle between the lower surface of the thallus and the surface of the agar (Ɵ). Scale bar, 0.5 cm. (G) Quantification of the thallus growth angle (Ɵ) shown in (A). Bold horizontal lines indicate the median, and whiskers indicate the first and third quartile. ∗∗∗P < 0.001, Student’s t-test.
Figure 3 GFP-fused PIN proteins possess auxin export activity. (A) The insertion site of GFP in the indicated PIN proteins. Numbers represent the amino acid position in the respective proteins. (B) The auxin export assay with P. patens transgenic plants. Fresh tissues were cultured in liquid medium supplied with 1μM β-estradiol to induce XVE overexpression. Tissues were then incubated with radioactive H3-IAA followed by washing. Radioactive H3-IAA exported into the new culture medium was measured using a scintillation detector after one day. Wild-type moss plants treated with the same conditions were used as a control. Ten to fifteen 10-day-old moss colonies were used for one measurement, and the graph shows the mean ± SD from four independent experiments. (C) Representative protonema cells of the indicated genetic background. The cell outline was stained with FM4-64. White arrowheads indicate the first cell division plane, and the yellow double arrow indicates the length of the subapical cell. Scale bar, 100 μm. (D and E) Quantification of the subapical cell length and division angle (ϴ) of the indicated lines. Bold horizontal lines indicate the median, and whiskers indicate the first and third quartile. ∗∗∗P < 0.001, Student’s t-test. (F) Top (top panels) and side (bottom panels) views of the indicated M. polymorpha lines. Takaragaike-1 (WT) expands its thallus horizontally on the agar surface. Overexpression of MpPINZ-, AtPIN1-, and AtPIN2-GFP showed vertical thallus growth, which generated a large angle between the lower surface of the thallus and the surface of the agar (Ɵ). Scale bar, 0.5 cm. (G) Quantification of the thallus growth angle (Ɵ) shown in (A). Bold horizontal lines indicate the median, and whiskers indicate the first and third quartile. ∗∗∗P < 0.001, Student’s t-test.CC BY · PubMed Central
Figure 4 Endogenous PINs present different localization patterns in different tissues. (A) The localization of PpPINA-GFP in tissues with different complexities. PpPINA-GFP is polarized to the tip of initial and elongated protonema cells. The initial protonema cell is regenerated from a detached leaf, and the representative image shows the maximum projection with a 5-μm-thick Z section. Autofluorescence from the chloroplasts is indicated by asterisks. In elongated protonema cells, the polarity of PpPINA-GFP is plotted using an intensity measurement along the PM, as represented by the yellow arrows. The same measurement is applied to (B) and (C). In complex tissues composed of multiple cell layers (e.g. young leaves in P. patens), PpPINA-GFP is polarized at apical and basal domains (white arrowheads). Scale bars, 10 μm. (B) The localization of MpPINZ-GFP in emerging rhizoids, young rhizoids, and gemma epidermal cells. A representative image with a 5-μm-thick Z section at the gemma surface shows the accumulation of MpPINZ-GFP at the tips of emerging rhizoids, as indicated by yellow arrowheads in rhizoid precursor cells (asterisks). MpPINZ-GFP is polarized at the tip of young rhizoids (center). A middle section image shows the even distribution of MpPINZ-GFP on the PM of gemmae composed of multiple cell layers. Scale bars, 10 μm. (C) AtPIN2-GFP shows a polarized signal at the tip of the initial root hair, but the polarized signal is not observed in elongated root hairs. All imaging details are described in the Methods. The pixel values ranging from 0-255 are represented by the rainbow color. In Arabidopsis epidermal cells, AtPIN2-GFP shows apical polarization, as indicated by white arrowheads. Scale bars, 10 μm for root hairs and 1 cm for epidermis.
Figure 4 Endogenous PINs present different localization patterns in different tissues. (A) The localization of PpPINA-GFP in tissues with different complexities. PpPINA-GFP is polarized to the tip of initial and elongated protonema cells. The initial protonema cell is regenerated from a detached leaf, and the representative image shows the maximum projection with a 5-μm-thick Z section. Autofluorescence from the chloroplasts is indicated by asterisks. In elongated protonema cells, the polarity of PpPINA-GFP is plotted using an intensity measurement along the PM, as represented by the yellow arrows. The same measurement is applied to (B) and (C). In complex tissues composed of multiple cell layers (e.g. young leaves in P. patens), PpPINA-GFP is polarized at apical and basal domains (white arrowheads). Scale bars, 10 μm. (B) The localization of MpPINZ-GFP in emerging rhizoids, young rhizoids, and gemma epidermal cells. A representative image with a 5-μm-thick Z section at the gemma surface shows the accumulation of MpPINZ-GFP at the tips of emerging rhizoids, as indicated by yellow arrowheads in rhizoid precursor cells (asterisks). MpPINZ-GFP is polarized at the tip of young rhizoids (center). A middle section image shows the even distribution of MpPINZ-GFP on the PM of gemmae composed of multiple cell layers. Scale bars, 10 μm. (C) AtPIN2-GFP shows a polarized signal at the tip of the initial root hair, but the polarized signal is not observed in elongated root hairs. All imaging details are described in the Methods. The pixel values ranging from 0-255 are represented by the rainbow color. In Arabidopsis epidermal cells, AtPIN2-GFP shows apical polarization, as indicated by white arrowheads. Scale bars, 10 μm for root hairs and 1 cm for epidermis.CC BY · PubMed Central
Figure 5 Exogenous PINs are PM-localized with no defined polarity. (A) Overexpressed PpPINA-GFP is polarized to the tip of protonemata, but overexpressed AtPIN1-GFP is evenly distributed on the PM with intracellular puncta. The PM is stained with the membrane dye FM4-64. The occurrence frequency is indicated in the bottom left corner of each image. Scale bars, 10 μm. (B) Overexpressed MpPINZ-GFP is polarized to the tip of young rhizoids, while overexpressed AtPIN1-GFP shows strong cytosolic signals and weak PM localization with no polarity. The PM is stained with the membrane dye FM4-64. Scale bars, 10 μm. (C) AtPIN2-GFP under its native promoter localizes to the apical side of epidermal cells, but PpPINA-GFP and MpPINZ-GFP are mislocalized to the basal and lateral sites, as indicated by white arrowheads. Scale bar, 1 cm. (D) Intensity plots of representative images for PpPINA-GFP, AtPIN1-GFP in P. patens protonema cells, and MpPINZ-GFP and AtPIN1-GFP in M. polymorpha young rhizoids. (E) Polarity index (ratio of signal intensity at the apical PM/signal intensity at the lateral PM) for apical localization of the indicated PIN-GFP in Arabidopsis epidermal cells. PpPINA-GFP and MpPINZ-GFP are significantly lower on the polarity index. Shown are 12-22 cells from three roots in three independent experiments for each line. ∗∗∗P < 0.001, Student’s t-test.
Figure 5 Exogenous PINs are PM-localized with no defined polarity. (A) Overexpressed PpPINA-GFP is polarized to the tip of protonemata, but overexpressed AtPIN1-GFP is evenly distributed on the PM with intracellular puncta. The PM is stained with the membrane dye FM4-64. The occurrence frequency is indicated in the bottom left corner of each image. Scale bars, 10 μm. (B) Overexpressed MpPINZ-GFP is polarized to the tip of young rhizoids, while overexpressed AtPIN1-GFP shows strong cytosolic signals and weak PM localization with no polarity. The PM is stained with the membrane dye FM4-64. Scale bars, 10 μm. (C) AtPIN2-GFP under its native promoter localizes to the apical side of epidermal cells, but PpPINA-GFP and MpPINZ-GFP are mislocalized to the basal and lateral sites, as indicated by white arrowheads. Scale bar, 1 cm. (D) Intensity plots of representative images for PpPINA-GFP, AtPIN1-GFP in P. patens protonema cells, and MpPINZ-GFP and AtPIN1-GFP in M. polymorpha young rhizoids. (E) Polarity index (ratio of signal intensity at the apical PM/signal intensity at the lateral PM) for apical localization of the indicated PIN-GFP in Arabidopsis epidermal cells. PpPINA-GFP and MpPINZ-GFP are significantly lower on the polarity index. Shown are 12-22 cells from three roots in three independent experiments for each line. ∗∗∗P < 0.001, Student’s t-test.CC BY · PubMed Central
Figure 6 Cytoskeletal networks are important for the polarization of bryophytic PINs. (A) PpPINA-GFP is polarized to the tip of protonemata, and disruption of actin filaments with latrunculin (LatB) induced its hyperpolarization (white arrowhead). Disruption of microtubules by oryzalin (Ory) disturbed the polarization of PpPINA-GFP (yellow arrowhead). The occurrence frequency is indicated in the bottom right corner of each image. For P. patens and A. thaliana, tissues were treated with 20 μM LatB or 5 μM Ory for 4 h. Scale bars, 10 μm. (B) MpPINZ-GFP is polarized to the tip of young rhizoids (white arrowheads), and its polarization was only abolished by Ory treatment (yellow arrowhead). The gemmae were treated with 2 μM LatB or 10 μM Ory for 2 h. Scale bars, 10 μm. (C) AtPIN2-GFP is polarized to the tip of initial root hair cells, and disruption of either actin filaments or microtubules did not change its polarization (white arrowheads) but did attenuate the peak signal at the tips, as shown in (F). The pixel values ranging from 0-255 are represented by the rainbow color. Scale bars, 10 μm. (D) The hyperpolarity index of PpPINA-GFP treated with DMSO or LatB was calculated by dividing the intensity at the very tip by the intensity at the curvature side of the tip. (E and F) Intensity plots of representative images for MpPINZ-GFP and AtPIN2-GFP along the tips of the rhizoids in M. polymorpha and root hairs in A. thaliana.
Figure 6 Cytoskeletal networks are important for the polarization of bryophytic PINs. (A) PpPINA-GFP is polarized to the tip of protonemata, and disruption of actin filaments with latrunculin (LatB) induced its hyperpolarization (white arrowhead). Disruption of microtubules by oryzalin (Ory) disturbed the polarization of PpPINA-GFP (yellow arrowhead). The occurrence frequency is indicated in the bottom right corner of each image. For P. patens and A. thaliana, tissues were treated with 20 μM LatB or 5 μM Ory for 4 h. Scale bars, 10 μm. (B) MpPINZ-GFP is polarized to the tip of young rhizoids (white arrowheads), and its polarization was only abolished by Ory treatment (yellow arrowhead). The gemmae were treated with 2 μM LatB or 10 μM Ory for 2 h. Scale bars, 10 μm. (C) AtPIN2-GFP is polarized to the tip of initial root hair cells, and disruption of either actin filaments or microtubules did not change its polarization (white arrowheads) but did attenuate the peak signal at the tips, as shown in (F). The pixel values ranging from 0-255 are represented by the rainbow color. Scale bars, 10 μm. (D) The hyperpolarity index of PpPINA-GFP treated with DMSO or LatB was calculated by dividing the intensity at the very tip by the intensity at the curvature side of the tip. (E and F) Intensity plots of representative images for MpPINZ-GFP and AtPIN2-GFP along the tips of the rhizoids in M. polymorpha and root hairs in A. thaliana.CC BY · PubMed Central

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Metadata

Authors
Tang, H., Lu, K.-J., Zhang, Y., Cheng, Y.-L., Tu, S.-L., & Friml, J.
Affiliations
  • Institute of Science and Technology Austria (AT)
  • National Chung Hsing University (TW)
  • Northwest A&F University (CN)
  • Institute of Plant and Microbial Biology, Academia Sinica (TW)
ORCID
Venue
Plant Communications
Publisher
Elsevier BV
Published
2024-01
Volume
5
Issue
1
Pages
100669
ISSN
2590-3462 (ISSN-L)
DOI
10.1016/j.xplc.2023.100669
PMC
PMC10811345
OpenAlex
W4385431171
Cited by
21
References
65
Citations by year
2026: 4 · 2025: 5 · 2024: 11
FWCI
3.31
Type
journal article
Language
en
Open access
Licence
Locations
  • Plant Communications (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • Ministry of Science and Technology (110-2636-B-005-001)
  • European Research Council (ETAP-742985)
  • Taiwan Ministry of Science and Technology
Topics
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Molecular Biology Research
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant nutrient uptake and metabolism
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Parasitism and Resistance
Keywords
PIN auxin transporters, polar auxin transport, land plant evolution, bryophytes, Physcomitrella patens, Marchantia polymorpha, Arabidopsis thaliana, cytoskeleton, GFP fusion protein, root epidermis, apical polarity, exogenous auxin
MeSH terms
Indoleacetic Acids, Arabidopsis, Plant Roots, Membrane Transport Proteins, Arabidopsis Proteins

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