Abstract

Apical growth in plants initiates upon seed germination, whereas radial growth is primed only during early ontogenesis in procambium cells and activated later by the vascular cambium1. Although it is not known how radial growth is organized and regulated in plants, this system resembles the developmental competence observed in some animal systems, in which pre-existing patterns of developmental potential are established early on2,3. Here we show that in Arabidopsis the initiation of radial growth occurs around early protophloem-sieve-element cell files of the root procambial tissue. In this domain, cytokinin signalling promotes the expression of a pair of mobile transcription factors—PHLOEM EARLY DOF 1 (PEAR1) and PHLOEM EARLY DOF 2 (PEAR2)—and their four homologues (DOF6, TMO6, OBP2 and HCA2), which we collectively name PEAR proteins. The PEAR proteins form a short-range concentration gradient that peaks at protophloem sieve elements, and activates gene expression that promotes radial growth. The expression and function of PEAR proteins are antagonized by the HD-ZIP III proteins, well-known polarity transcription factors4—the expression of which is concentrated in the more-internal domain of radially non-dividing procambial cells by the function of auxin, and mobile miR165 and miR166 microRNAs. The PEAR proteins locally promote transcription of their inhibitory HD-ZIP III genes, and thereby establish a negative-feedback loop that forms a robust boundary that demarks the zone of cell division. Taken together, our data establish that during root procambial development there exists a network in which a module that links PEAR and HD-ZIP III transcription factors integrates spatial information of the hormonal domains and miRNA gradients to provide adjacent zones of dividing and more-quiescent cells, which forms a foundation for further radial growth.

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Data availability

All lines and data supporting the findings of this study are available from the corresponding authors upon request. The transcriptomics data files are available at Gene Expression Omnibus (GEO), under accession number GSE115183. A list of putative direct targets of PEAR1 and/or PEAR2 with their description as well as individual P values for Tukey’s HSD test and two-sided Student’s t-test are provided as Supplementary Information.

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Acknowledgements

We thank E. Scarpella, ABRC and NASC for materials, K. Kainulainen, K. Blajecka, M. Herpola and A. Mainardi for technical assistance, N. Clark for assisting with the scanning FCS analysis, J. Jansen for technical assistance with microarray hybridizations, O. Kambur and L. Kalmbach for assistance in generating the heat map, and D. Weijers and T. Kakimoto for helpful discussions, A. Groenheide, E. Cornelissen, M. Chu, A. Korppoo and A. Rodriguez Diez for technical support. This works was supported by Finnish Centre of Excellence in Molecular Biology of Primary Producers (Academy of Finland CoE program 2014-2019) decision #271832, the Gatsby Foundation (GAT3395/PR3)), University of Helsinki (award 799992091) and the European Research Council Advanced Investigator Grant SYMDEV (No. 323052) to Y.H., a NSF-BBSRC MCSB 1517058 to R. Sozzani and Y.H., an ERC Consolidator grant (PLANTSTEMS), a Heisenberg Professorship of the German Research Foundation (DFG, GR2104/5-1) and the SFB 873 (DFG) to T.G., the Netherlands Organization for Scientific Research (NWO; VIDI-864.13.001) and The Research Foundation - Flanders (FWO; Odysseus II G0D0515N and 12D1815N) to W.S. and B.D.R., respectively, a JSPS postdoctoral fellowship for research abroad and JSPS KAKENHI Grant (17K15138) to S.M., Swiss National Science Foundation Postdoc Mobility Grant (P300P3_147894) to P.R., a JSPS Research Fellowship for Young Scientists and JSPS KAKENHI Grant (JP16J00131) to K.T., Bayer Science and Education Foundation, German Academic Exchange Service (DAAD) to B.B., The Finnish Academy of Science to J.-o.H., and Herchel Smith Postdoctoral Research Fellowship (Herchel Smith Fund) to S.O.

Reviewer information

Nature thanks S. Sabatini, S. Turner and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

  1. These authors contributed equally: Shunsuke Miyashima, Pawel Roszak, Iris Sevilem

Affiliations

  1. Institute of Biotechnology, HiLIFE/Organismal and Evolutionary Biology Research Programme, Faculty of Biological and Environmental Sciences, Viikki Plant Science Centre, University of Helsinki, Helsinki, Finland

    • Shunsuke Miyashima
    • , Pawel Roszak
    • , Iris Sevilem
    • , Jung-ok Heo
    • , Hanna Help-Rinta-Rahko
    • , Ondřej Smetana
    • , Riccardo Siligato
    • , Ari Pekka Mähönen
    •  & Ykä Helariutta
  2. Graduate School of Science and Technology, Nara Institute of Science and Technology, Nara, Japan

    • Shunsuke Miyashima
    • , Kayo Hashimoto
    •  & Keiji Nakajima
  3. The Sainsbury Laboratory, University of Cambridge, Cambridge, UK

    • Pawel Roszak
    • , Koichi Toyokura
    • , Bernhard Blob
    • , Jung-ok Heo
    • , Sofia Otero
    • , Charles W. Melnyk
    •  & Ykä Helariutta
  4. Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Japan

    • Koichi Toyokura
  5. Centre for Plant Integrative Biology (CPIB) and School of Biosciences, University of Nottingham, Nottingham, UK

    • Nathan Mellor
    •  & Anthony Bishopp
  6. Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium

    • Wouter Smet
    •  & Bert De Rybel
  7. VIB Center for Plant Systems Biology, Ghent, Belgium

    • Wouter Smet
    •  & Bert De Rybel
  8. Laboratory of Biochemistry, Wageningen University, Wageningen, The Netherlands

    • Wouter Smet
    •  & Bert De Rybel
  9. Nutrition, Metabolism and Genomics Group, Division of Human Nutrition, Wageningen University, Wageningen, The Netherlands

    • Mark Boekschoten
    •  & Guido Hooiveld
  10. Graduate School of Humanities and Sciences, Nara Women’s University, Nara, Japan

    • Kayo Hashimoto
  11. Centre for Organismal Studies (COS), Heidelberg University, Heidelberg, Germany

    • Eva-Sophie Wallner
    •  & Thomas Greb
  12. Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan

    • Yuki Kondo
  13. Department of Plant Biology, Swedish University of Agricultural Sciences, Uppsala, Sweden

    • Charles W. Melnyk
  14. Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, USA

    • Rosangela Sozzani

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Contributions

S.M., P.R. and I.S. contributed equally to this work. K.T. and B.B. contributed equally to this work. S.M. characterized the molecular interactions in the PEAR and HD-ZIP III module. P.R. identified and quantified phenotypes in the PEAR loss-of-function mutants with the help of B.B. I.S. determined phloem-specific DOFs and their downstream genes with input from B.D.R., W.S., M.B. and G.H. K.T. characterized pear and hd-zip III combinatorial mutants. B.B. generated tmo6 CRISPR mutants. J.-o.H. performed in situ hybridization. N.M. and A.B. designed and performed computational modelling. H.H.-R.-R. produced the CRE1-inducible line. S.O. assisted in the microarray experiments. K.H. and K.N. produced HD-ZIP III reporter lines. O.S. and A.P.M. provided the destination vector pSm43GW. R. Siligato and A.P.M. provided the pARR5::RFPer line. E.S.W., Y.K., T.G. and C.W.M. shared informative non-published data. R. Sozzani analysed the diffusion coefficient of PEAR1–GFP with P.R. B.D.R. and Y.H. participated in experimental design. S.M. and Y.H. wrote the manuscript and all authors commented on the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Bert De Rybel or Ykä Helariutta.

Extended data figures and tables

  1. Extended Data Fig. 1 Quantification of periclinal cell division during procambial development.

    a, Schematic of root vascular tissue of Arabidopsis. Procambial cells originate from their initial cells, and periclinal cell division increases the cell files during the proliferative phase. This eventually results in a bisymmetric vascular pattern composed of a pair of phloem poles, which are separated from central xylem axis by intervening procambium. b, An example of mapping the position of periclinal cell divisions from the initial cells. From each position within the root vascular tissue (arrows), an optical cross-section image is constructed, and cells are segmented using CellSet. c, The number of periclinal cell divisions in each cell position (273 division events from 13 independent roots). d, The mean cell number in each category during procambial development. The number of events per cell in each group was calculated by dividing the number of events by the mean cell number of each group during development (see Supplementary Information).

  2. Extended Data Fig. 2 Inhibition of symplastic connection in early PSEs results in the reduction of vascular cell number and in PSE-specific PEAR1–GFP localization.

    a, Aniline-blue-stained primary root of pPEAR1[XVE]::icals3m after 24 h of induction. Callose deposition occurs superficially in PSE cells (arrowheads, n = 10). b, c, The vascular tissue of pPEAR1[XVE]::icals3m root, in non-induced (b, n = 13) and after a three-day induction (c, n = 9). In the non-induced condition, PSE cells (white arrowheads) and their neighbouring cells—composed of metaphloem sieve element (MSE) (dark green arrows) and two lateral companion cells (orange arrowheads)—are spatially separated from the xylem axis by intervening procambium. By contrast, after a three-day induction of callose deposition in PSE cells, only a single sieve-element cell file is formed in each phloem pole (c, white arrowheads), and its neighbouring cells often touch the xylem axis (c, yellow hashtags). The number of procambial and phloem cell files is significantly reduced after a three-day induction. Box plot centres show median. For more information on box plots, see Methods. The P value was calculated by two-sided Student’s t-test. d, e, Expression of SISTER OF APL (SAPL; AT3G12730) and ATPASE 3 (AHA3; AT5G57350) in pPEAR1[XVE]::icals3m before (d, n = 10 and 4, respectively) and after 24 h of induction (e, n = 10 and 4, respectively). SAPL is expressed in companion cells (CC) and MSE in meristematic zone, and AHA3 is expressed in differentiated CC (d). After induction, expression of these genes is restricted to a single cell file, indicating that symplastic cell communication between PSEs and PSE-LNs is required for the specification of PSE-neighbouring cell identity. f, g, PEAR1–GFP localization in pCRE1[XVE]::icals3m before (f, n = 8) and after (g, n = 7) 24 h of induction. PEAR1–GFP becomes specific to the PSE cell after the induction of callose deposition in whole vascular tissue, which suggests that PEAR1–GFP moves in a short rage via plasmodesmata. White, orange and dark green arrowheads indicate PSEs, PSE-LNs and PSE-INs, respectively. Asterisks indicate protoxylem (PX) cells. In ag, n represents independent biological samples. Scale bars, 25 μm.

  3. Extended Data Fig. 3 Identification of PEAR genes.

    a, In silico analysis of transcription factors that are abundant in the early phloem. Nine transcription factors are shown to be expressed abundantly in the early phloem cell (S32 fraction), which contains four types of transcription factors—DOF-type, MADS-box, NAC-type and GATA-type transcription factors. b, A phylogenetic tree of 36 Arabidopsis DOF transcription factors drawn using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). c, Overexpression of PEAR genes (including PEAR1, PEAR2, OBP2, DOF6, HCA2 and TMO6) under the CRE1-inducible promoter enhances periclinal cell division in the vascular tissue. n represents independent biological samples. Bar graphs represent mean. Error bars are s.d. Dots, individual data points. P values were calculated by two-sided Student’s t-test. d, The expression of six PEAR genes (including PEAR1, PEAR2, DOF6, TMO6, OBP2 and HCA2) shows a similar pattern of expression to that of PEAR1, in which both mRNA and transcriptional fusion reporter exhibit a PSE-specific pattern with a broad protein localization. HCA2 translational fusion in wild-type background exhibits a weak but detectable signal in PSE-neighbouring cells (arrows), and its expression level is enhanced in the pear quintuple-mutant background. Although TMO6 mRNA is highly specific to PSE cells, its transcriptional fusion reporter shows a broad but PSE-abundant expression pattern, with a broad TMO6 protein localization. Mobility of TMO6 protein is more evident when TMO6–Venus is expressed under the PSE-specific PEAR1 promoter (pPEAR1::TMO6-VENUS) in pear sextuple mutant. The number in each panel indicates samples with similar results, of the total independent biological samples analysed. Box plot centres show median. P value calculated by two-sided Student’s t-test. Dots, individual data points. e, A quantitative analysis of PEAR transcripts in pear1 pear2 double-mutant background. Note that the level of transcripts of three PEAR genes (TMO6, DOF6 and HCA2) is elevated in the pear1 pear2 background, which suggests that a compensatory mechanism would mask the effect of pear1 pear2 loss-of function (see Supplementary Information). Bar graphs represent mean. Error bars are s.d. Dots, individual data points. P values calculated by two-sided Student’s t-test. Scale bars, 25 μm.

  4. Extended Data Fig. 4 PEAR1–GFP localization during procambial development.

    ad, PEAR1–GFP localization in wild-type background (n = 19, independent biological samples). The position of each optical section is indicated in the left panel, which shows the longitudinal section. At the position of the vascular initial cells, a weak PEAR1–GFP signal is observed in PSEs and neighbouring procambial cells but not in xylem cells (a). During an early stage of the proliferative phase, the highest PEAR1–GFP signal is detected in the PSEs, and a substantial level of PEAR1–GFP signal is observed in PSE-neighbouring cells, PSE-LNs and PSE-INs (b, c). Expression of PEAR1–GFP is maintained by the end of proliferation stage (d), which indicates that the expression pattern of PEAR1–GFP is correlated with the domain having high proliferative activity (except for PSE-Ins, in which almost no periclinal cell division is detected) (see Fig. 1b). eh, PEAR1–GFP localization in hd-zip III quadruple (phb phv cna athb8) mutant background (n = 17, independent biological samples). The position of each optical section is indicated in left panel, which shows the longitudinal section. Broad localization of PEAR1–GFP is detected at the level of vascular initial cells. Central domain is highlighted with a dotted square (e). At the early stage of proliferative phase, fluorescent signal is detected in internal procambial cells (light green arrowheads), as well as PSEs and neighbouring cells (f, g), and gradually becomes specific to PSEs and neighbours (h). i, Quantification of PEAR1–GFP signal in each cell type. Fluorescent intensity of PEAR1–GFP in internal procambial cells and PSE-INs during proliferative phase (b, c in wild-type; f, g in hd-zip III quadruple mutant, respectively) was measured and normalized to the fluorescent intensity in PSE cells, which confirms a broad distribution of PEAR1–GFP in the hd-zip III quadruple mutant. n represents individual measurements across 5 (wild type) or 4 (hd-zip III quadruple mutant) independent biological samples, respectively. Bar graphs represent mean. Error bars are s.d. Dots, individual data points. P values were calculated by two-sided Student’s t-test. White, orange, dark green and light green arrowheads indicate PSEs, PSE-LNs, PSE-INs and internal procambial cells (IPCs), respectively. Scale bars, 25 μm.

  5. Extended Data Fig. 5 Loss of function of PEAR genes.

    a, Organization of PEAR genes and CRISPR–Cas9-induced mutation in TMO6 locus. Deletions are denoted by dashes; insertions and a replacement are indicated by red letters. b, Quantification of phloem and procambium cell files in lower-order (left) and higher-order (right) pear combinatorial mutants. Tukey’s HSD test is provided for all samples in Supplementary Table 3. c, pear quintuple-mutant phenotype is suppressed by introduction of fluorescent-tagged PEAR proteins expressed under their native promoters. d, pear sextuple-mutant phenotype is significantly suppressed by the introduction of PEAR1, DOF6 and TMO6 construct, but not by the introduction of PEAR2. In the pear sextuple-mutant background, PEAR2 expression is highly reduced in the vascular tissue. e, Phloem unloading assay in wild type, pear quintuple mutant (with shortest roots) and pear sextuple mutant (n = 15, 8 and 15, respectively). Fluorescent 5(6)-carboxyfluorascein diacetate dye is loaded on the cotyledon and imaged two hours after application (see Methods). Phloem transport and unloading is not changed in the shortest roots of the pear quintuple mutant, which are strongly affected in the radial growth. pear sextuple mutant shows defects in phloem transport. f, Phenotype of pear sextuple mutant at the early developmental stage (1.5 days after germination). The cell number in vascular tissue of pear sextuple mutant is significantly reduced before the onset of phloem PSE differentiation and activation of the phloem transport (see Supplementary Information). P value was calculated by two-sided Student’s t-test. In bf, n represents independent biological samples. In bd, statistically significant differences between groups were tested using Tukey’s HSD test P < 0.05. For individual P values, see Supplementary Table 3. Box plot centres show median. For more information on box plots, see Methods. Scale bars, 25 μm.

  6. Extended Data Fig. 6 Identification of genes that act downstream of PEAR1 and PEAR2.

    a, Venn diagram that shows the genes upregulated by overexpression of PEAR1 or PEAR2, with and without cycloheximide (CHX). The analysis revealed 212 and 435 upregulated genes in the respective experiments. Heat map showing the predicted spatio-temporal expression patterns of all genes induced by PEAR1 or PEAR2. bi, Expression patterns of eight selected genes that respond to PEAR2 overexpression. In control conditions, all genes exhibit a broad expression pattern; five of them are transcribed both in phloem and procambial cells (bd and i), and the rest of them are transcribed in PSEs and neighbouring cells, in which PEAR proteins accumulate abundantly (eh). Whereas expression of SMXL3, AT1G09460 and AT1G15080 are maintained even in pear sextuple mutant (b, c, f), the expression level of the remaining five genes are attenuated (d, e, gi). The number in each panel indicates samples with similar results, of the total independent biological samples analysed. j, Number of vascular cells after a three-day induction of overexpression of each gene that acts downstream of PEAR1 and PEAR2. In each case, several lines were analysed in parallel for a phenotypic change. Of the genes tested, only SMXL3 overexpression can increase the vascular cell number (confirmed in three independent lines). Box plot centres show median. For more information on box plots, see Methods. n represents independent biological samples. Dots, individual data points. P value was calculated by two-sided Student’s t-test. Scale bars, 25 μm.

  7. Extended Data Fig. 7 Cytokinin controls PEAR expression.

    a, Expression of auxin (pIAA2::GFP-GUS)- and cytokinin (pARR5::RFPer)-response genes (n = 15). Auxin response is restricted to the xylem cells at initial stage (a′) and maintained during development (a′′, a′′′). A high cytokinin response is activated initially and maintained in PSEs and neighbouring cells (a′ a′′), and later becomes concentrated into the intervening procambial cells that flank xylem cells (a′′′). b, Exogenous cytokinin application rapidly promotes the transcript level of PEAR genes, including PEAR2, DOF6 and TMO6. Asterisks indicate significant (P < 0.05) upregulation, as determined by a two-sided t-test on three biological replicates. Bar graphs represent mean. Error bars are s.e.m. For individual P values, see Supplementary Table 3. c, Sustained cytokinin application leads to ectopic transcription of PEAR genes. The optical cross-section images were obtained after a 4-day (for PEAR1, PEAR2 and HCA2) or 1-day (DOF6 and OBP2) treatment of 1 μM of 6-benzylaminopurine. d, Conditional induction of CRE1 expression restores TMO6 transcription in wol root. In the absence of a cytokinin response, TMO6 transcription is increased in the pericycle and attenuated in the vascular tissue (control, hashtags), and is restored in the vascular tissue after CRE1 induction (est). el, Expression pattern of auxin (pIAA2::GFP-GUS)- and cytokinin (pARR5::RFPer)-response reporters during embryogenesis in wild-type (eh, n = 29 (e), 13 (f), 13 (g) and 11 (h)) and wol (il, n = 12 (i), 8 (j), 6 (k) and 10 (l)) embryos. At the globular stage, the auxin response is activated among provascular cells both in wild type (e) and wol (i) embryos. At the early heart stage, the cytokinin response is activated in cells positioned below the shoot apical meristem (f, upper lower tier (ult), arrowheads), and the stripe of the cytokinin-response domain is formed by mid-heart stage (g, ult, arrowheads). Simultaneously, the auxin response becomes concentrated in the cells proximal to the cotyledon (g, ult, asterisks), which results in a bisymmetric hormonal-response pattern. During the torpedo stage, the cytokinin-response domain reaches to the lower lower tier (llt) (h, llt, arrowheads). In wol embryos, the activation of the cytokinin response in vascular tissue does not occur, and a radial auxin-response pattern is maintained (il). mr, Expression of ARR5 and PEAR1 during embryogenesis. In the wild-type embryo (mo, n = 17 (m), 25 (n) and 10 (o)), PEAR1 is broadly transcribed among provascular cells in both the ult and llt, with a radial symmetric pattern at the globular stage (m). At the heart stage, PEAR1 transcription is enhanced in ult cells underneath the shoot apical meristem, which correlates with the activation of the cytokinin response in this domain (n, arrowheads). The expression of both ARR5 and PEAR1 extends rootward and reaches to llt, and becomes more concentrated within the cell files in which phloem is specified post-embryonically (o, arrowheads). In wol embryos (pr, n = 19 (p), 13 (q) and 13 (r)), PEAR1 transcription is initiated among provascular cells at the globular embryo stage (p), similar to wild type (m), but neither the cytokinin response nor PEAR1 transcription occurs in ult at the heart stage (q) and PEAR1 expression is gradually attenuated by the torpedo stage (r). In a, er, n represents independent biological samples. In c, d, the number in each panel indicates samples with similar results, of the total independent biological samples analysed. Scale bars, 25 μm.

  8. Extended Data Fig. 8 HD-ZIP III proteins restrict periclinal cell divisions during procambial development.

    ac, Periclinal cell divisions in cells that are not adjacent to the pericycle—including PSE-INs (a), internal procambial cells (b) and xylem cells (c)—occurs in hd-zip III quadruple mutant (phb phv cna athb8). n = 8. d, e, The number of periclinal cell divisions in cells adjacent (d) or non-adjacent (e) to the pericycle in wild type, pear and hd-zip III combinatorial mutants. In the analysis of pear quintuple mutant and pear hd-zip III nonuple mutant, a population of slowly elongating roots is selected, as described in Fig. 2f. Box plot centres show median. Statistically significant differences between groups were tested using Tukey’s HSD test P < 0.05. For individual P values, see Supplementary Table 3. fi, Localization of PHB–GFP (f, n = 12), CNA–GFP (g, n = 10), REV–GFP (h, n = 7) and ATHB8–GFP (i, n = 5). j, k, Protein localization of PHB–GFP (j, n = 7) and CNA–GFP (k, n = 4) during procambial development. In the initial cells (j′, k′), both proteins are localized in metaxylem cells but not in PSEs (white arrowheads). During the proliferation stage, PSE-INs (green arrowheads)—which are produced by periclinal cell division in PSEs—acquire expression of both PHB–GFP (j′′, j′′′) and CNA (k′′, k′′′). lo, Overexpression of PEAR1–Venus under the CRE1-inducible promoter in wild type (l, n, n = 5 (l) and 3 (n)) and heterozygous phb-1d background (phb-1d/+, m, o, n = 5). After 18 h of induction, PEAR1–Venus signal is detected in both backgrounds (l, m); however, enhanced periclinal divisions are only observed in wild type (l), and not in phb-1d (m). Longer induction of PEAR1 overexpression induces divisions even in phb-1d background (n, o). pr, pPEAR1-PEAR1-GFP expression is reduced in heterozygous phb-1d/+ background. Most phb-1d heterozygotes exhibit a single PEAR1–GFP-expressing pole (q, 72% of n = 11), and the expression of PEAR1–GFP is almost completely abolished in some roots of phb-1d plants (r, 18% of n = 11). s, t, The expression of pPEAR1::GFPer in wild-type (s, n = 10) and shr-2 plants (t, n = 9). The fluorescent signal is below the limit of detection in shr-2 plants. uw, Expression of pPEAR1::GFP-GUS in wild type (u, n = 19) and hd-zip III quadruple mutant (w, n = 11). In aw, n represents independent biological samples. White, orange and dark green arrowheads indicate PSEs, PSE-LNs and PSE-Ins, respectively. Scale bars, 25 μm.

  9. Extended Data Fig. 9 Overexpression of PEAR1 enhances the transcription of HD-ZIP III genes.

    ah, The transcription patterns of four HD-ZIP III genes—PHB (a, b), CNA (c, d), REV (e, f) and ATHB8 (g, h)—are visualized using their transcriptional fusion constructs. A longitudinal section is shown in the left panel, and the optical cross-sections associated with the longitudinal section are shown in the right panels (the position of each section is indicated in the left panel). a, b, Transcription pattern of PHB (pPHB::GV>UAS::GFPer) in pCRE1[XVE]::PEAR1 plant before (a) and after (b) 24 h of induction of PEAR1 overexpression. PHB transcription is observed in whole vascular tissue at the initial and proliferative phase, with peaks in xylem cells (a′), and its expression becomes concentrated into protoxylem cells (a′′ and a′′′; asterisks indicate protoxylem cell). After the induction of PEAR1 overexpression, PHB expression in the central domain of the vascular tissue is maintained at the later stage, which results in the radially symmetric PHB transcription pattern (b′ and b′′). c, d, Transcription of CNA (pCNA::GV>UAS::GFPer) in pCRE1[XVE]::PEAR1-RFP plant before (c) and after (d) 24 h of induction. CNA transcription is observed mainly in the xylem lineage at initial cells (c′), and becomes broader in whole vascular tissue. CNA transcription peaks in procambial tissue, including PSE-neighbouring cells (c′′); eventually its expression is gradually reduced in PSEs and metaxylem, but is maintained in procambium, PSE-neighbouring cells and protoxylem cells (c′′′). In a similar manner to PHB, CNA transcription in the central domain of the vascular tissue is maintained at the later stage when PEAR1-RFP is overexpressed (d′′′). e, f, Transcription of REV (pREV::RFPer) in pCRE1[XVE]::PEAR1-VENUS plant before (e) and after (f) 24 h of induction. REV exhibits a distinct transcriptional pattern in which its expression is initially uniform in vascular tissue (e′), and the highest expression is localized in PSEs, decreasing towards the xylem axis (e′′ and e′′′). When PEAR1–Venus is overexpressed (f), the transcription pattern of REV is also activated in the central domain of vascular tissue, which results in the radially symmetric REV transcription pattern. g, h, The expression pattern of pATHB8::HTA6-YFP is highly specific to xylem cells (g), and its expression is enhanced after 24 h of induction of PEAR1 overexpression, with a broad expression domain (h). The number in each panel indicates samples with similar results, of the total independent biological samples analysed.

  10. Extended Data Fig. 10 The boundary between HD-ZIP III and PEAR proteins forms within the PSE-INs.

    a, Summarizing the results of the pattern of auxin–cytokinin (data shown in Extended Data Fig. 7a), HD-ZIP III (CNA, data shown in Extended Data Fig. 8g, k) and PEAR (PEAR1, data shown in Fig. 1d and Extended Data Fig. 4a–d) proteins in procambium. In the simulation, we simulate the concentration of HD-ZIP III proteins and PEAR1 proteins along the axis between metaxylem (MX, brown arrowheads) and PSE (white arrowheads). b, Summarizing the results of procambial development. The number of cells between metaxylem and PSEs increases during procambial development (data shown in Extended Data Fig. 1). Therefore, the model is defined as a line in one spatial dimension that represents 3, 4 or 5 cells from the centre of the xylem axis to the outer edge of the PSE cell. c, The regulatory network embedded within each cell. Regulatory interactions that are shown using a black line have previously been published; interactions shown using a green line are described here for the first time. d, Predicted concentration gradient of all elements in 3, 4 or 5 cells (from left to right). Within different root geometries that correspond to different growth stages in Arabidopsis, both PEAR1 and HD-ZIP III proteins are co-localized in PSE-INs, forming a sharp concentration boundary within this cell. In fh, only PEAR and HD-ZIP III protein concentrations are shown, whereas in d all model components are shown in all cases. e, Quantification of expression level of CNA–GFP and PEAR1–GFP in a four-cell region in wild-type background. n represents individual measurements across 3 (CNA–GFP) or 4 (PEAR1–GFP) independent biological samples. Bar graphs represent mean. Error bars are s.d. Dots, individual data points. f, The inclusion of all three interactions labelled in c in the model results in the formation of sharp concentration gradients of PEAR1 (black line) and HD-ZIP III proteins (blue line) with the boundary that forms in the PSE-INs. g, In simulations in which HD-ZIP III proteins do not regulate PEAR1 diffusion (interaction (3) in c), the PEAR1 protein is predicted to spread into the procambium and metaxylem, as shown in Fig. 4k–m and Extended Data Fig. 4. h, In simulations in which PEAR1 does not activate HD-ZIP III transcription (interaction (1) in c), the concentration of HD-ZIP III proteins is reduced in the PSE-IN cell, as shown in Fig. 4h–j. i, A regulatory mechanism forming the boundary between a dividing and a non-dividing cell during procambial development. White, orange, dark green and light green arrowheads indicate PSEs, PSE-LNs, PSE-INs and internal procambial cells, respectively. Scale bars, 25 μm.

Supplementary information

  1. Supplementary Information

    This file contains Supplementary Notes: (1) description of mapping of the position of periclinal cell divisions, (2) description of redundancy of PEAR genes, (3) notes on uncoupled cell division and cell differentiation effects of the pear mutants, (4) interaction of PEAR1 and cytokinin signalling during embryogenesis and (5) analysis of PEAR1/2 downstream targets. Supplementary modelling information: model aims and philosophy, model description, alternate cases and model implementation.

  2. Reporting Summary

  3. Supplementary Table 1

    A list of primers and plasmids used in this study.

  4. Supplementary Table 2

    A list of putative PEAR1/PEAR2 direct targets and their descriptions.

  5. Supplementary Table 3

    Individual P-values for Tukey’s HSD test (for Fig. 2f, Fig. 4g-i and Extended Data Fig. 5b-d) and for two-sided Student’s t-test (for Extended Data Fig. 7b).

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https://doi.org/10.1038/s41586-018-0839-y

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