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Diffusible repression of cytokinin signalling produces endodermal symmetry and passage cells

An Author Correction to this article was published on 20 June 2018

This article has been updated

Abstract

In vascular plants, the root endodermis surrounds the central vasculature as a protective sheath that is analogous to the polarized epithelium in animals, and contains ring-shaped Casparian strips that restrict diffusion1. After an initial lag phase, individual endodermal cells suberize in an apparently random fashion to produce ‘patchy’ suberization that eventually generates a zone of continuous suberin deposition2. Casparian strips and suberin lamellae affect paracellular and transcellular transport, respectively. Most angiosperms maintain some isolated cells in an unsuberized state3 as so-called ‘passage cells’, which have previously been suggested to enable uptake across an otherwise-impermeable endodermal barrier3,4. Here we demonstrate that these passage cells are late emanations of a meristematic patterning process that reads out the underlying non-radial symmetry of the vasculature. This process is mediated by the non-cell-autonomous repression of cytokinin signalling in the root meristem, and leads to distinct phloem- and xylem-pole-associated endodermal cells. The latter cells can resist abscisic acid-dependent suberization to produce passage cells. Our data further demonstrate that, during meristematic patterning, xylem-pole-associated endodermal cells can dynamically alter passage-cell numbers in response to nutrient status, and that passage cells express transporters and locally affect the expression of transporters in adjacent cortical cells.

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Figure 1: Presence and distribution of passage cells in Arabidopsis.
Figure 2: Cytokinin and auxin regulate endodermal patterning and passage cell formation.
Figure 3: Spatially restricted cytokinin repression and production underlie passage cell formation.
Figure 4: Passage cell-associated expression of PHO1;H3.
Figure 5: Overview of endodermis circumferential patterning and passage cell formation.

Change history

  • 20 June 2018

    In this Letter, the two centre panels in Extended Data Fig. 7a were identical. The original, incorrect Extended Data Fig. 7 is shown in the Supplementary Information to the accompanying Amendment. The original Letter has been corrected.

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Acknowledgements

This work was supported by funds to N.G. from an ERC Consolidator Grant (GA-N°: 616228 – ENDOFUN), an SNSF grant (31003A_156261), an IEF Marie Curie fellowship (T.G.A.) and an EMBO Long-term postdoctoral fellowship (R.U.). B.D.R., W.S. and B.W. were funded by the Netherlands Organisation for Scientific Research (NWO; VIDI-864.13.001) and The Research Foundation - Flanders (FWO; Odysseus II G0D0515N). We thank A. Paradis and the Central Imaging Facility of the University of Lausanne for support; M. Yamazaki for providing constructs; B. Müller, D. Weijers and T. Vernoux for sharing material; A. Bishopp, A. P. Mähönen, D. Weijers, S. Sabatini, V. Grieneisen, Y. Helariutta and Y. Poirier for discussions; and A. Vjestica, C. Drapek, M. Marek and M. Barberon for input to the manuscript.

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T.G.A. planned and conducted all experiments with input from N.G. and J.E.M.V. S.N. conducted initial experiments on PHO1 localization, R.U. created and tested inducible vectors, J.E.M.V. created and tested shy2-2 lines, and B.D.R, W.S. and B.W. created and selected all ARR reporter lines. T.G.A and N.G. wrote the manuscript. All authors commented on the manuscript.

Corresponding authors

Correspondence to Tonni Grube Andersen or Niko Geldner.

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The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks H. Fukuda, Y. Helariutta and A. P. Mähönen for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Passage cells and suberization in Arabidopsis.

a, A single xylem-pole-associated passage cell surrounded by suberized cells; visualized in live imaging, by expression of the suberin biosynthetic gene GPAT5 that drives expression of a plasma-membrane-localized mCitrine reporter (mCitrine–SYP122) (yellow, left) or by the suberin-specific dye fluorol yellow (green, right). In the left panel, the xylem-pole pericycle is highlighted using the promoter pXPP driving a 3mCherry reporter fused to the plasma-membrane-localized SYP122 (3mCherry-SYP122). Note that the fluorol-yellow protocol requires heating the sample to 70 °C, and is incompatible with fluorescent protein detection. b, Comparison of endodermal suberization in Col-0 and in plants expressing the GPAT5-based reporter, determined either by the suberin-specific dye fluorol yellow or live mCitrine expression, respectively. Red dots, individual data points. c, Comparison of passage-cell occurrence in Col-0 and in plants expressing the GPAT5-based reporter, determined either by the suberin-specific dye fluorol yellow or live mCitrine expression. Dots, individual data points. d, Expression of a plasma-membrane-localized mCitrine-SYP122 marker (red) driven by the xylem-pole-specific promoter pXPP, in the zone of protoxylem onset. Roots were cleared using ClearSee and stained with basic fuchsin (green) and calcofluor white (grey)32. e, Radial- and longitudinally connected passage cells in the xylem pole of the suberized zone. f, Direct quantification of passage cells residing directly above xylem pole (XP), first side cell to the xylem pole (1st) or phloem pole (PP) in five-day-old Col-0 plants. Dots, individual data points. g, Quantification of the onset of endodermal suberization in the phloem and xylem poles of roots expressing markers as in a. Dots, individual data points. h, Representative images of the patchy zone of endodermal suberization in the phloem and xylem poles of roots used for quantification in f. Yellow arrowheads, xylem pole; asterisks, passage cells. cs; Casperian strip, FY; fluorol yellow. For all stacked graphs, there are three measurements per root: unsuberized zone, white; patchy zone, grey; and suberized zone, yellow. Bar graphs represent mean ± s.d. and boxplots show median. Statistically significant differences between groups were tested using a post hoc Bonferroni-adjusted paired two-sided t-test. For more information on data plots, see Methods. a and e represent five independent lines; all stainings were repeated three times n, independent biological samples. **P < 0.01, two-tailed t-test. For individual P values, see Supplementary Table 2. Scale bars, 25 μm, unless otherwise specified.

Extended Data Figure 2 Auxin and cytokinin signalling in the suberized root zone.

a, Activity of the auxin-signalling reporter DR5 (yellow) as well as the highly sensitive DR5v2 (blue) in an area of the suberized zone of a five-day-old root with an emerging lateral root. Suberized cells were visualized based on the suberin biosynthetic gene GPAT5 driving expression of a plasma-membrane-localized 3mCherry-based reporter. b, Expression of endoplasmic-reticulum-localized GFP driven by the cytokinin signalling reporter TCSn in the phloem and xylem poles of 5-day-old roots in the suberized zone. Plants were either grown on plates containing 5 nM cytokinin (BA) or a mock treatment (DMSO). Punctured lines indicate the endodermis. c, Expression of B-type ARRs in the suberized endodermis of five-day-old roots, suberin and cellwalls are stained using a Clearsee protocol in combination with Nile red and calcofluor white, respectively32. d, Expression of A-type ARRs in the suberized endodermis of five-day-old roots, suberin and cell walls, as in c. All stainings were repeated three times. In a and b, images are representative of eight independent lines. White arrowheads, passage-cell nuclei. Asterisks, passage cells. Scale bars, 50 μm.

Extended Data Figure 3 Phenotypic analysis of hormone-treated plants and mutants.

a, Root length of five-day-old plants either germinated on increasing concentration of cytokinin (BA), auxin (NAA) or abscisic acid (ABA) (left), or of the mutants used in this study (right). Open circles, outliers. b, Emerged lateral roots per mm of ten-day-old Col-0 roots germinated in presence of DMSO, 10 nM cytokinin (BA) or 50 nM auxin (NAA) (left), or of the mutants used in this study (right). Open circles, outliers. c, Length of the root apical meristem of five-day-old Col-0 plants germinated on either DMSO, 10 nM cytokinin (BA) or 50 nM auxin (NAA) (left), or of mutants used in this study (right). Dots, individual data points. d, Onset of protoxylem and metaxylem after the elongation zone of five-day-old seedling germinated in presence of DMSO, 10 nM cytokinin (BA) or 50 nM auxin (NAA) (left), or of mutants used in this study (right). Dots, individual data points. Bar graphs represent mean ± s.d. and boxplot centres show median. Statistically significant differences between groups were tested using a post hoc Bonferroni-adjusted paired two-sided t-test. For more information on data plots, see Methods. n, independent biological samples. In a, **P < 0.01, two-tailed t-test, n = 25 independent biological replicas for each treatment.

Extended Data Figure 4 Hormone-induced closure of passage cells.

Time-course analysis showing behaviour of passage cells, visualized by mCitrine–SYP122 driven by the promoter of the suberin synthesis marker gene GPAT5 for 9 h on 1/2 MS medium containing DMSO, cytokinin (BA), auxin (NAA) or abscisic acid (ABA). Red arrowheads point to ectopic suberization in the cortex on ABA treatment. Asterisks, passage cells. All time courses were repeated three times. Scale bars, 50 μm.

Extended Data Figure 5 Hormone-induced changes in endodermal cell lengths.

a, Expression of mCitrine–SYP122 driven by the GPAT5 promoter (yellow), in the fully suberized zone in plants that express the dominant cytokinin-signalling inhibitor AHP6 fused to GFP in the differentiated endodermis using the ELTP promoter (blue). b, Endodermal suberization in five-day-old seedlings that express AHP6–GFP or the dominant auxin signalling repressor shy2-2 in the differentiated endodermis. Red dots, individual data points. c, Occurrence of passage cells in five-day-old seedlings that express AHP6–GFP or the auxin signalling repressor shy2-2 in the differentiated endodermis. Dots, individual data points. df, Length of suberized endodermal cells measured in the suberized zone of plants expressing pGPAT5::mCitrine-SYP122 and pXPP::3mCherry-SYP122 constructs. Open circles, outliers. Plants were grown for five days on 1/2 MS medium containing depicted concentrations of abscisic acid (d), auxin and naphthalene acetic acid (e), and cytokinin and benzyl adenine (f). g, The number of meritematic cells in the phloem- and xylem-pole endodermis was counted in cleared roots stained with calcofluor white32 to highlight cell walls. Dots, individual data points. For all stacked graphs, there are three measurements per root: unsuberized zone, white; patchy zone, grey; and suberized zone, yellow. Protoxylem or -phloem was used as a marker to identify the poles. Cells were counted from the quiescent centre to the onset of elongation. Asterisks, last cell of the division zone. Blue lines, lengths of individual cells. Bar graphs represent mean ± s.d. and boxplot centres show median. Statistically significant differences between groups were tested using a post hoc Bonferroni-adjusted paired two-sided t-test. For more information on data plots, see Methods. In a, images are representative of 12 independent lines. In g, all stainings were repeated three times. n, number of measurements across 16 independent biological samples. **P < 0.01, two-tailed t-test. Scale bars, 25 μm.

Extended Data Figure 6 Manipulation of suberization through signalling repression.

a, Representative pictures of fluorol-yellow staining of suberin in lines expressing ARR10EAR–GFP in all differentiated endodermal cells, driven by the ELTP or CASP1 promotors. b, Occurrence of passage cells in lines expressing ARR10EAR–GFP in all differentiated endodermal cells, using the ELTP and CASP1 promotors. c, Behaviour of suberin in lines expressing ARR10EAR–GFP in all differentiated endodermal cells, using the ELTP and CASP1 promotors. d, Behaviour of suberin in five-day-old plants expressing ARR10EAR–GFP in all differentiated endodermal cells, using the ELTP and CASP1 promotors. Red dots, individual data points. e, Behaviour of suberin in five-day-old plants expressing ARR10EAR–GFP in all differentiated endodermal cells, using the ELTP and CASP1 promotors, when plants were germinated on either cytokinin (BA) or abscicic acid (ABA). Red dots, individual data points. For all stacked graphs, there are three measurements per root: unsuberized zone, white; patchy zone, grey; and suberized zone, yellow. Bar graphs represent mean ± s.d. and boxplots show median.Statistically significant differences between groups were tested using a post hoc Bonferroni-adjusted paired two-sided t-test. For more information on data plots, see Methods. In b, images are representative of nine independent lines. All stainings were repeated three times. n, independent biological samples. Scale bars, 50 μm.

Extended Data Figure 7 Temporal control of suberin inhibition.

Expression of temporally controlled dominant inhibitors of cytokinin (AHP6–GFP), ABA (abi1-1-GFP) or auxin (shy2-2) signalling in the differentiated endodermis, using an oestradiol-inducible system (XVE)19. ac, Fluorol-yellow staining (left) and quantification of passage cells (top right) and suberin (bottom right) in the late endodermis of plants grown on 1/2 MS medium containing DMSO for five days (a), plants grown for four days on 1/2 MS medium containing DMSO, then transferred to 5 μM oestradiol for 29 h (b) or germinated for five days on 1/2-MS-medium plates containing 5 μM oestradiol (c). Asterisks, passage cells. All dots, individual data points. For all stacked graphs, there are three measurements per root: unsuberized zone, white; patchy zone, grey; and suberized zone, yellow. n represent biologically independent samples. Bar graphs represent mean ± s.d. and boxplot centres show median. Statistically significant differences between groups were tested using a post hoc Bonferroni-adjusted paired two-sided t-test. For more information on data plots, see Methods. In ac, images are representative of nine independent lines. All stainings were repeated three times. n, independent biological samples. Scale bars, 100 μm.

Extended Data Figure 8 Cytokinin-dependent formation of passage cells.

a, Expression of AHP6 visualized by endoplasmic-reticulum-localized GFP (GFPer)19, in the root apical meristem of plants grown either in presence of a mock treatment (DMSO) or 10 nM cytokinin (BA). Arrows point to xylem-pole pericycle cells. b, Length of suberized endodermal cells in the phloem and xylem poles of five-day-old ahp6-1 and log mutants, complementing lines and stacked mutants grown on 1/2-MS-medium plates. Data partly overlap with those in Fig. 3. Open circles, outliers. c, Expression of LOG4–GFP and AHP6–3mVenus driven by their native promoters, in the root apical meristem of plants grown either in presence of mock treatment (DMSO) or 10 nM cytokinin (BA). d, Expression of LOG4 in the suberized zone of five-day-old seedlings visualized by the native LOG4 promoter driving expression of an NLS–3GFP construct. Suberized endodermal cells were highlighted by expression of the suberin biosynthetic marker pGPAT5::3mCherry-SYP122. e, Occurrence of passage cells in lines with inhibited AHP6 diffusion in combination with repressed auxin signalling (through the expression of shy2-2) in the differentiated endodermis. Dots, individual data points. f, Passage-cell occurrence in the log4 mutant complemented with a LOG4–GFP construct driven by either the native promotor (pLOG4), an early endodermis promoter (pSCR) or a differentiating endodermis promoter (pELTP). Dots, individual data points. g, Occurrence of passage cells in the suberized zone of five-day-old ahp6-1, log mutants and ahp6 log additionally complemented by non-mobile AHP6 to obtain endodermis-specific defects. Dots, individual data points. h, Behaviour of suberin in plants with or without inhibited auxin perception in the late endodermis grown on 1/2-MS-medium plates. Red dots, individual data points. For all stacked graphs, there are three measurements per root: unsuberized zone, white; patchy zone, grey; and suberized zone, yellow. Bar graphs represent mean ± s.d. and boxplot centres show median. Individual letters show significantly different groups according to a post hoc Bonferroni-adjusted paired two-sided t-test. For more information on data plots, see Methods. In c and d, images represent eight independent lines. All stainings were repeated three times. n, independent biological samples. In b, n represents measurements across 16 biologically independent samples. Scale bars, 10 μm (a) or 25 μm (c, d).

Extended Data Figure 9 The PHO1 transporter gene family as marker for passage cells.

a, Expression of PHO1 family members in the cortex and/or epidermis (left) and inside the stele (right) within the suberized zone of five-day-old roots grown on 1/2 MS medium. Individual nuclei showing expression of the respective NLS reporter were counted in similar 3D stacks, taken either in a fully suberized area (red bars) or in an area containing one passage cell (white bars). Open circles, outliers. b, Expression of PHO1 in five-day-old seedlings in the late endodermis of wild type, or a suberin-degradation line with virtually absent suberin (pCASP1::CDEF1). c, Representative images of the expression of each member of the PHO1 family in roots of five-day-old seedlings, highlighted by the NLS–3mVenus reporter. Seedlings were stained with Nile red for suberin and calcofluor white for cell walls. Scale bars in xz projections represent 10 μm. White arrowheads, passage-cell nuclei; yellow arrowheads point towards nuclei of cortex and/or epidermal cells. Asterisks, passage cells. n.d., not detected. Boxplot centres show median. **P < 0.01, two-sided t-test. For more information on data plots, see Methods. In b and c, images represent 15 and 11 independent lines, respectively. All stainings were repeated three times. n, independent biological samples. Unless otherwise stated, scale bars represent 25 μm. For inserts, scale bars represent 10 μm.

Extended Data Figure 10 Nutrient deficiency responses of suberization in the xylem pole.

a, Expression of the suberin marker GPAT5 and the xylem-pole-pericycle marker XPP using a mCitrine–SYP122 or a 3mCherry–SYP122 plasma-membrane-anchored reporter, respectively. Plants were germinated for five days on 1/2 MS medium (1/2 MS), plates with low (10 μM) inorganic phosphate (low Pi), no zinc (−Zn) or no iron (−Fe). Images representative of four independent lines. b, Expression of the suberin marker GPAT5 and PHO1;H3 using a mCitrine–SYP122 or a NLS–3mScarlet reporter, respectively, in the suberized zone of Col-0 plants or plants expressing the dominant cytokinin signalling repressor ARR10–GFP, in all differentiating endodermal cells. Each image is representative of eight independent lines. c, Expression of the suberin marker GPAT5 and PHO1;H3 using a mCitrine–SYP122 or a NLS–3mVenus reporter, respectively, in the suberized zone of plants expressing oestradiol-inducible (XVE) versions of the dominant cytokinin signalling repressor AHP6–GFP or the dominant ABA signalling repressor abi1-1, in all differentiated endodermal cells. Plants were grown for five days in presence of DMSO or 5 μM oestradiol (+E2D), all treatments were repeated three times. Each image is representative of five independent lines. d, Expression of PHO1;H3 and the suberin marker GPAT5 in roots of five-day-old Col-0 plants grown on 1/2 MS medium, plates containing no iron, no zinc or with low (10 μM) inorganic phosphate. Each image is representative of nine independent lines. All treatments were repeated three times. e, Transcriptional analysis of PHO1;H3 expression in roots of five-day-old Col-0 plants grown on 1/2 MS medium, plates containing no iron, no zinc or with low (10 μM) inorganic phosphate, in plants expressing the dominant cytokinin signalling repressor ARR10–GFP in all differentiated endodermal cells, or in plants with strongly reduced suberin deposition by expression of a suberin-degrading enzyme (pCASP1::CDEF). Expression was normalized to that of UBQ10. Dots, individual data points. For all stacked graphs, there are three measurements per root: unsuberized zone, white; patchy zone, grey; and suberized zone, yellow. Asterisks highlight unsuberized xylem-pole endodermal cells. Boxplot centres show median. Statistically significant differences between groups were tested using a post hoc Bonferroni-adjusted paired two-sided t-test. For more information on data plots, see Methods. n, independent biological samples. Scale bars, 25 μm.

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Supplementary Table 1

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This table contains individual P-values for all statistical analyses. (XLSX 102 kb)

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Andersen, T., Naseer, S., Ursache, R. et al. Diffusible repression of cytokinin signalling produces endodermal symmetry and passage cells. Nature 555, 529–533 (2018). https://doi.org/10.1038/nature25976

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