Antagonistic CLE peptide pathways shape root meristem tissue patterning | Nature Plants

Oct 28, 2024

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Secreted CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptide ligands dimension the stem cell niche of Arabidopsis shoot meristems by signalling through redundant and cross-compensating CLAVATA1 (CLV1)-type receptor kinases. In the root meristem, the CLV1 homologues BARELY ANY MERISTEM 1 (BAM1) and BAM2 drive CLE13/16-mediated formative divisions that produce the ground tissue layers. Here we report that BAM1/2 are also required to initiate the vascular phloem lineage and that cross-compensation between CLV1-type receptors as observed in the shoot does not operate similarly in the root. Rather, we find that BAM3-mediated CLE45 signalling antagonizes BAM1/2-mediated CLE11/12/13 signalling in the phloem initials but not in the ground tissue. We further observe spatiotemporally contrasting CLE signalling requirements for phloem initiation and differentiation, which are shaped by the SHORT ROOT (SHR) pathway. Our findings thus suggest an intricate quantitative interplay between distinct and antagonistic CLE signalling pathways that organizes tissue layer formation in the Arabidopsis root meristem.

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All data needed to evaluate the conclusions are presented in the main figures and the Extended Data. Materials are available upon request. The RNA sequencing reads are deposited at the Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra) under submission ID SUB14584842, sample IDs SAMN42396145–SAMN42396186. Source data are provided with this paper.

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We thank Z. Nimchuk and X. Gou for sharing genetic materials; Z. Nimchuk for comments on the manuscript; and P. Cattaneo for technical assistance. This study was supported by Swiss National Science Foundation grant 310030_207876 awarded to C.S.H. and a CSC scholarship awarded to H.Z.

Department of Plant Molecular Biology, University of Lausanne, Lausanne, Switzerland

Hang Zhang, Qian Wang, Noel Blanco-Touriñán & Christian S. Hardtke

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H.Z. and C.S.H. designed the project and drafted the manuscript. H.Z., Q.W. and N.B.-T. performed experiments and analysed data. All authors contributed to the assembly and the revision of the manuscript.

Correspondence to Christian S. Hardtke.

The authors declare no competing interests.

Nature Plants thanks Jungmook Kim and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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a, Root length measurements for 7-day-old seedlings of the indicated genotypes. Box plots display 2nd and 3rd quartiles and the median, whiskers indicate maximum and minimum. Statistically significant differences (lower case letters) were determined by ordinary one-way ANOVA followed by Tukey’s test, two-sided, p < 0.0001. n = 8–15 independent biological replicates. b, Representative histological cross sections (toluidine blue-stained) from indicated genotypes. Orange arrowheads point out protophloem sieve element cell files.

Source data

Schematic representation of root tip expression patterns of the indicated genes, obtained from aggregation of multiple independent single-cell mRNA sequencing experiments of Arabidopsis Col-0 wildtype roots (https://rootcellatlas.org). Note the differences in expression level scales.

a, Confocal microscopy images of Col-0 wildtype root meristems grown on 20 nM CLE45 peptide in the absence or presence of ZIC2 (25 mM). b, Confocal microscopy images of Col-0 root meristems expressing the CVP2::NLS-VENUS protophloem sieve element marker, grown on ZIC2 (25 mM) as compared to control. c, Confocal microscopy images of Col-0 root meristems (propidium iodide staining, red fluorescence) expressing various markers of the SHR pathway (green fluorescence), grown on 15 nM CLE45 peptide as compared to controls. Seedlings were 5-day-old throughout.

a, Confocal microscopy images of 5-day-old root meristems of the indicated genotypes, showing optical longitudinal sections (top) and cross sections (bottom). Orange arrowheads in longitudinal sections point out protophloem sieve element cell files, yellow arrowheads indicate protophloem sieve element precursors that fail to differentiate. Colored dots in cross sections indicate cell files derived from the phloem-procambium precursor stem cell (see Fig. 1a). b, Root length measurements for 7-day-old seedlings of the indicated genotypes. Box plots display 2nd and 3rd quartiles and the median, whiskers indicate maximum and minimum. Statistically significant differences (lower case letters) were determined by ordinary one-way ANOVA followed by Tukey’s test, two-sided, p ≤ 0.0038. n = 15–79 independent biological replicates. c, Images of representative 7-day-old seedlings of the indicated genotypes.

Source data

a-c, Root length measurements for 7-day-old seedlings of the indicated genotypes, grown in the presence of a set of CLE peptides (all 20 nM). Box plots display 2nd and 3rd quartiles and the median, whiskers indicate maximum and minimum. Statistically significant differences (asterisks) compared to untreated control were determined by ordinary one-way ANOVA followed by Tukey’s test, two-sided, *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001. n = 5–20 independent biological replicates. d, Root length measurements for 7-day-old seedlings of the indicated genotypes. Box plots display 2nd and 3rd quartiles and the median, whiskers indicate maximum and minimum. Statistically significant differences (lower case letters) were determined by ordinary one-way ANOVA, p < 0.0001. n = 18–35 independent biological replicates.

Source data

a-b, Confocal microscopy images of a root meristem from a bam3 brx seedling expressing a SHR::BAM3-CITRINE transgene (b) as compared to the mutant control (a). c, Confocal microscopy showing the transgenic BAM3-CITRINE signal (yellow fluorescence) by itself (top) and overlayed with calcofluor cell wall staining (bottom). d, Confocal microscopy images of a bam1 bam3 brx root meristem. e-f, Similar to b-c, for a SCR::BAM3-CITRINE transgene. g-h, Confocal microscopy images of plt1 plt2 root meristems grown in the presence of ZIC2 (h) (25 mM) as compared to untreated control (g). i, Confocal microscopy images of a bam3 plt1 plt2 root meristem. j, Confocal microscopy images of plt1 plt2 and bam3 plt1 plt2 root meristems grown in the presence of 20 nM CLE45 peptide. k-l, Root length measurements for 7-day-old seedlings of the indicated genotypes, grown in the presence of 20 nM CLE45 peptide as compared to untreated controls. Box plots display 2nd and 3rd quartiles and the median, whiskers indicate maximum and minimum. Statistically significant differences (asterisks or lower-case letters) compared to untreated control (k) or wildtype (l) were determined by ordinary one-way ANOVA followed by Tukey’s test, two-sided, p < 0.0001. n = 10–45 independent biological replicates. m, Confocal microscopy images of Col-0 wildtype root meristems grown in the presence of 20 nM CLE45 peptide applied from germination onwards or at later timepoints. Confocal images were obtained from 5-day-old seedlings. Orange and grey arrowheads in longitudinal sections point out protophloem sieve element cell files and the xylem axis, respectively, yellow arrowheads indicate protophloem sieve element precursors that fail to differentiate. Colored dots in cross sections indicate cell files derived from the phloem-procambium precursor stem cell (see Fig. 1a).

Source data

a, Amino acid sequence alignment of the prototypical CLV1-type CLE peptide receptors and the proposed alternative receptor, RPK2. Green arrowheads point out amino acid residues that have been implicated in CLE peptide interaction. b, Confocal microscopy images of 5-day-old bam3 and bam3 brx mutant root meristems (propidium iodide staining, magenta fluorescence) expressing an RPK2-CITRINE fusion protein (green fluorescence) in the phloem pole, under control of the BAM3 promoter. c, Root length measurements for 8-day-old seedlings of the indicated genotypes, grown in the presence of 20 nM CLE45 or CLE33 peptide. Box plots display 2nd and 3rd quartiles and the median, whiskers indicate maximum and minimum. Statistically significant differences (asterisks) compared to untreated control were determined by ordinary one-way ANOVA followed by Tukey’s test, two-sided, ***: p < 0.002; ****: p < 0.0001. n = 28–61 independent biological replicates.

Source data

Gene expression levels in RNAseq.

Differential expression analysis of RNAseq.

Gene ontology (GO) analysis of RNAseq.

List of mutant alleles.

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Zhang, H., Wang, Q., Blanco-Touriñán, N. et al. Antagonistic CLE peptide pathways shape root meristem tissue patterning. Nat. Plants (2024). https://doi.org/10.1038/s41477-024-01838-1

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Received: 27 April 2024

Accepted: 03 October 2024

Published: 28 October 2024

DOI: https://doi.org/10.1038/s41477-024-01838-1

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