GOLVEN peptide signalling through RGI receptors and MPK6 restricts asymmetric cell division during lateral root initiation

Abstract

During lateral root initiation, lateral root founder cells undergo asymmetric cell divisions that generate daughter cells with different sizes and fates, a prerequisite for correct primordium organogenesis. An excess of the GLV6/RGF8 peptide disrupts these initial asymmetric cell divisions, resulting in more symmetric divisions and the failure to achieve lateral root organogenesis. Here, we show that loss-of-function GLV6 and its homologue GLV10 increase asymmetric cell divisions during lateral root initiation, and we identified three members of the RGF1 INSENSITIVE/RGF1 receptor subfamily as likely GLV receptors in this process. Through a suppressor screen, we found that MITOGEN-ACTIVATED PROTEIN KINASE6 is a downstream regulator of the GLV pathway. Our data indicate that GLV6 and GLV10 act as inhibitors of asymmetric cell divisions and signal through RGF1 INSENSITIVE receptors and MITOGEN-ACTIVATED PROTEIN KINASE6 to restrict the number of initial asymmetric cell divisions that take place during lateral root initiation.

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Fig. 1: CRISPR glv lines have defects in LR initiation.
Fig. 2: Identification of downstream components of GLV signalling during LR initiation.
Fig. 3: mpk6 mutants suppress the LR GLV6OE phenotype.
Fig. 4: MPK6 phosphorylation is induced shortly after GLV6p treatment.
Fig. 5: GLV6OE phenotypes and the induction of MPK6 phosphorylation are dependent on RGI receptors.
Fig. 6: Model for GLV6/10 signalling restricting ACD during LR initiation.

Data availability

The data supporting the findings in this study are available from the corresponding author upon reasonable request.

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Acknowledgements

This research was supported by FWO postdoctoral (A.F., grant no. 1293817N) and doctoral (J.J., grant no. 1168218N) fellowships, a China Scholarship Council grant (K.X., no. 201606350134) and a National Science Foundation Plant Genome Research Program Grant (Z.L.N., no. PGRP-1841917). We thank M. Njo for help with preparing the figures, V. Storme for guidance and assistance with the statistical analysis and D. Savatin for training with the MPK6 phosphorylation experiments.

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Authors

Contributions

A.I.F. designed the project. A.I.F. generated the iGLV6 line, performed the EMS mutagenesis screen and analysed the EMS mutants’ identity with help from N.V., A.D., D.O. and T.M. A.I.F. and K.X. phenotypically characterized the CRISPR glv mutants, and generated and characterized the rgi mutants and reporter lines. N.V. generated and characterized the CRISPR glv6 mutants. N.V., K.X., J.J. and S.M.-H. phenotypically characterized the mpk6 mutants. J.J., S.M.-H. and H.D.G. characterized the cross-talk between the auxin and GLV pathways. Q.Y. generated the RGI4 reporter line. B. Parizot performed the in silico expression analysis. L.A.N.C., N.V. and E.R. performed the MPK6 phosphorylation experiments. B. Peterson and Z.L.N. generated the CRISPR glv mutants. K.H., W.V. and A.M. synthesized the peptides. J.J. and A.I.F. performed the statistical analysis. A.I.F., N.V. and T.B. wrote the manuscript with input from all authors. T.B. provided guidance and advice on the project, the experiments and the analysis of the results.

Corresponding author

Correspondence to Tom Beeckman.

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The authors declare no competing interests.

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Peer review information Nature Plants thanks Melinka Butenko, Juan Xu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 GLV6 and 10 act redundantly during LR initiation.

a–c, Phenotypic characterization of CRISPR glv6 mutants compared to wild type (8 dag, n = 12). Quantification of root length (a), all primordium stages density (b) and non-emerged primordia (NE) and emerged (E) LR density (c). d, Quantification of root length in the CRISPR glv mutant compared to wild type. e, Quantification of all primordium stages density in the glv6glv10 mutant germinated on MS or on 10 nM of GLV6p/GLV10p (8 dag). Charts show mean values ± s.d. (b, e) or s.e.m (c). Significant differences compared to wild type are shown and were determined using one-way ANOVA (a, d) or a GEE model (b-c, e). In e, only significant differences in stage I primordia are displayed. For full statistical analysis see Supplementary Table 2. n.s.: no significant differences were found between mutants and wild type. f, Example of nearby primordia frequently found in glv mutants. The lower picture shows a higher magnification image of the framed area in the upper picture for each genotype. Scale bars represent 50 μm.

Extended Data Fig. 2 Suppression of the GLV6OE phenotype and LR defects in mpk6 mutants.

a, Suppression of the GLV6OE phenotype in mpk6 mutants after LR initiation was induced by gravistimulation of the primary root. This experiment was done three times with similar results. b, Quantification of all primordium stages in reported mpk6 mutants compared to wild type (8 dag). Chart represents mean values ± s.d. A GEE model was used. n.s.: no significant differences were found between mutants and wild type. For full statistical analysis see Supplementary Table 2. Scale bars represent 20 μm.

Supplementary information

Supplementary Information

Supplementary Figs. 1–9, Table 3 and methods.

Reporting Summary

Supplementary Tables 1 and 2

Table 1. Amino acid sequences predicted to be encoded in CRISPR glv6 or glv mutants. The GLV6 pre-propeptide is shown as a reference. The presumed mature GLV6 peptide sequence is highlighted in bold, and stop codons are depicted as asterisks. Amino acid sequences different from the corresponding GLV wild-type precursor are italicized in the mutants. Table 2. Statistical analysis of the primordia stages and LR density in glv and mpk6 mutants compared to controls. P values indicating significant differences are highlighted in green. See Methods for details on the statistical analysis.

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Fernandez, A.I., Vangheluwe, N., Xu, K. et al. GOLVEN peptide signalling through RGI receptors and MPK6 restricts asymmetric cell division during lateral root initiation. Nat. Plants 6, 533–543 (2020). https://doi.org/10.1038/s41477-020-0645-z

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