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  • Letter
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Diffusible repression of cytokinin signalling produces endodermal symmetry and passage cells

Nature volume 555, pages 529–533 (2018)Cite this article

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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.

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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.

References

  1. Geldner, N. The endodermis. Annu. Rev. Plant Biol. 64, 531–558 (2013)

    Article  CAS  Google Scholar 

  2. Barberon, M . et al. Adaptation of root function by nutrient-induced plasticity of endodermal differentiation. Cell 164, 447–459 (2016)

    Article  CAS  Google Scholar 

  3. Peterson, C. A. & Enstone, D. E. Functions of passage cells in the endodermis and exodermis of roots. Physiol. Plant. 97, 592–598 (1996)

    Article  CAS  Google Scholar 

  4. Kroemer, K. Wurzelhaut, Hypodermis Und Endodermis Der Angiospermenwurzel (Bibl. Bot. 59) (Erwin Nägele, 1903)

  5. Wu, H ., Jaeger, M ., Wang, M ., Li, B. & Zhang, B. G. Three-dimensional distribution of vessels, passage cells and lateral roots along the root axis of winter wheat (Triticum aestivum). Ann. Bot. 107, 843–853 (2011)

    Article  Google Scholar 

  6. Mähönen, A. P . et al. Cytokinin signaling and its inhibitor AHP6 regulate cell fate during vascular development. Science 311, 94–98 (2006)

    Article  ADS  Google Scholar 

  7. Kurakawa, T . et al. Direct control of shoot meristem activity by a cytokinin-activating enzyme. Nature 445, 652–655 (2007)

    Article  CAS  Google Scholar 

  8. De Rybel, B . et al. Integration of growth and patterning during vascular tissue formation in Arabidopsis. Science 345, 1255215 (2014)

    Article  Google Scholar 

  9. De Rybel, B ., Mähönen, A. P ., Helariutta, Y. & Weijers, D. Plant vascular development: from early specification to differentiation. Nat. Rev. Mol. Cell Biol. 17, 30–40 (2016)

    Article  CAS  Google Scholar 

  10. Bishopp, A . et al. A mutually inhibitory interaction between auxin and cytokinin specifies vascular pattern in roots. Curr. Biol. 21, 917–926 (2011)

    Article  CAS  Google Scholar 

  11. Zürcher, E . et al. A robust and sensitive synthetic sensor to monitor the transcriptional output of the cytokinin signaling network in planta. Plant Physiol. 161, 1066–1075 (2013)

    Article  Google Scholar 

  12. Hwang, I. & Sheen, J. Two-component circuitry in Arabidopsis cytokinin signal transduction. Nature 413, 383–389 (2001)

    Article  ADS  CAS  Google Scholar 

  13. Sakai, H . et al. ARR1, a transcription factor for genes immediately responsive to cytokinins. Science 294, 1519–1521 (2001)

    Article  ADS  CAS  Google Scholar 

  14. Mason, M. G . et al. Multiple type-B response regulators mediate cytokinin signal transduction in Arabidopsis. Plant Cell 17, 3007–3018 (2005)

    Article  CAS  Google Scholar 

  15. Liao, C.-Y . et al. Reporters for sensitive and quantitative measurement of auxin response. Nat. Methods 12, 207–210 (2015)

    Article  CAS  Google Scholar 

  16. Lavrekha, V. V ., Pasternak, T ., Ivanov, V. B ., Palme, K . & Mironova, V. V. 3D analysis of mitosis distribution highlights the longitudinal zonation and diarch symmetry in proliferation activity of the Arabidopsis thaliana root meristem. Plant J. 92, 834–845 (2017)

    Article  CAS  Google Scholar 

  17. Ioio, R. D . et al. A genetic framework for the control of cell division and differentiation in the root meristem. Science 322, 1380–1384 (2008)

    Article  ADS  Google Scholar 

  18. Siligato, R . et al. Multisite gateway-compatible cell type-specific gene-inducible system for plants. Plant Physiol. 170, 627–641 (2016)

    Article  CAS  Google Scholar 

  19. Besnard, F . et al. Cytokinin signalling inhibitory fields provide robustness to phyllotaxis. Nature 505, 417–421 (2014)

    Article  ADS  CAS  Google Scholar 

  20. Harrison-Murray, R. S. & Clarkson, D. T. Relationships between structural development and the absorption of ions by the root system of Cucurbita pepo. Planta 114, 1–16 (1973)

    Article  CAS  Google Scholar 

  21. Clarkson, D. T., Sanderson, J. & Russell, R. S. Ion uptake and root age. Nature 220, 805–806 (1968)

    Article  ADS  CAS  Google Scholar 

  22. Hamburger, D ., Rezzonico, E ., MacDonald-Comber Petétot, J ., Somerville, C. & Poirier, Y. Identification and characterization of the Arabidopsis PHO1 gene involved in phosphate loading to the xylem. Plant Cell 14, 889–902 (2002)

    Article  CAS  Google Scholar 

  23. Sasse, J . et al. Asymmetric localizations of the ABC transporter PaPDR1 trace paths of directional strigolactone transport. Curr. Biol. 25, 647–655 (2015)

    Article  CAS  Google Scholar 

  24. Khan, G. A . et al. Coordination between zinc and phosphate homeostasis involves the transcription factor PHR1, the phosphate exporter PHO1, and its homologue PHO1;H3 in Arabidopsis. J. Exp. Bot. 65, 871–884 (2014)

    Article  CAS  Google Scholar 

  25. Naseer, S. et al. Casparian strip diffusion barrier in Arabidopsis is made of a lignin polymer without suberin. Proc. Natl Acad. Sci. USA 109, 10101–10106 (2012)

    Article  ADS  CAS  Google Scholar 

  26. Müller, B. & Sheen, J. Cytokinin and auxin interplay in root stem-cell specification during early embryogenesis. Nature 453, 1094–1097 (2008)

    Article  ADS  Google Scholar 

  27. Wendrich, J. R., Liao, C.-Y., van den Berg, W. A. M., De Rybel, B. & Weijers, D. Ligation-independent cloning for plant research. Methods Mol. Biol. 1284, 421–431 (2015)

    Article  CAS  Google Scholar 

  28. Karimi, M., Bleys, A., Vanderhaeghen, R. & Hilson, P. Building blocks for plant gene assembly. Plant Physiol. 145, 1183–1191 (2007)

    Article  CAS  Google Scholar 

  29. Kurihara, D., Mizuta, Y., Sato, Y. & Higashiyama, T. ClearSee: a rapid optical clearing reagent for whole-plant fluorescence imaging. Development 142, 4168–4179 (2015)

    Article  CAS  Google Scholar 

  30. Pfister, A. et al. A receptor-like kinase mutant with absent endodermal diffusion barrier displays selective nutrient homeostasis defects. eLife 3, e03115 (2014)

    Article  Google Scholar 

  31. Roppolo, D. et al. A novel protein family mediates Casparian strip formation in the endodermis. Nature 473, 380–383 (2011)

    Article  ADS  CAS  Google Scholar 

  32. Ursache, R., Andersen, T. G., Marhavy, P. & Geldner, N. A protocol for combining fluorescent proteins with histological stains for diverse cell wall components. Plant J. 93, 399–412 (2017)

    Article  Google Scholar 

  33. R Development Core Team. R: A Language And Environment For Statistical Computing (R Foundation for Statistical Computing, 2015)

Download references

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.

Author information

Author notes

  1. Joop E. M. Vermeer

    Present address: Department of Plant and Microbial Biology, University of Zürich, 8008, Zürich, Switzerland

Authors and Affiliations

  1. Department of Plant Molecular Biology, University of Lausanne, Lausanne, 1015, Switzerland

    Tonni Grube Andersen, Sadaf Naseer, Robertas Ursache, Joop E. M. Vermeer & Niko Geldner

  2. Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, 9052, Belgium

    Brecht Wybouw, Wouter Smet & Bert De Rybel

  3. VIB Center for Plant Systems Biology, Ghent, 9052, Belgium

    Brecht Wybouw, Wouter Smet & Bert De Rybel

  4. Wageningen University, Laboratory of Biochemistry, Wageningen, 6708 WE, The Netherlands

    Wouter Smet & Bert De Rybel

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Contributions

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.

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Correspondence to Tonni Grube Andersen or Niko Geldner.

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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. d–f, 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. a–c, 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 a–c, 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 x–z 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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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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