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A network of CLAVATA receptors buffers auxin-dependent meristem maintenance

Nature Plants volume 9, pages 1306–1317 (2023)Cite this article

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Abstract

Plant body plans are elaborated in response to both environmental and endogenous cues. How these inputs intersect to promote growth and development remains poorly understood. During reproductive development, central zone stem cell proliferation in inflorescence meristems is negatively regulated by the CLAVATA3 (CLV3) peptide signalling pathway. In contrast, floral primordia formation on meristem flanks requires the hormone auxin. Here we show that CLV3 signalling is also necessary for auxin-dependent floral primordia generation and that this function is partially masked by both inflorescence fasciation and heat-induced auxin biosynthesis. Stem cell regulation by CLAVATA signalling is separable from primordia formation but is also sensitized to temperature and auxin levels. In addition, we uncover a novel role for the CLV3 receptor CLAVATA1 in auxin-dependent meristem maintenance in cooler environments. As such, CLV3 signalling buffers multiple auxin-dependent shoot processes across divergent thermal environments, with opposing effects on cell proliferation in different meristem regions.

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Fig. 1: Dual CLV3p receptor systems regulate auxin-mediated primordia outgrowth.
Fig. 2: CLV3p is required for floral primordia formation across thermal environments.
Fig. 3: CLV3 functions with CLE25 in floral primordia and meristem regulation.
Fig. 4: CLV1 is required for auxin-dependent IM maintenance across thermal environments.

Data availability

Identifiers for published or publicly available lines are provided in Methods. RNA-seq data have been deposited in the NCBI SRA database under BioProject PRJNA661065. All other relevant data are available from the corresponding author upon request.

Code availability

All code used to analyse the data is published in the Nimchuk Lab Github (https://github.com/NimchukLab).

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Acknowledgements

We thank T. D. Perdue, director of the University of North Carolina-Chapel Hill Genome Sciences Microscopy Core, for assistance with confocal imaging and A. Willoughby for assistance with SEM and photography; J. Winshell and J. Garzoni for lab and plant growth facility support; UNC’s High-Throughput Sequencing Facility for sequencing services; and members of the Nimchuk Lab for critical feedback on this project. This research was supported by a National Institute of General Medical Sciences—Maximizing Investigators’ Research Award from the NIH (R35GM119614, Z.L.N.), a National Science Foundation (NSF) Plant Genome Research Program (PGRP) grant (IOS-1546837, Z.L.N.), a National Science Foundation Postdoctoral Research Fellowship in Biology through the PGRP (NSF 1906389, D.S.J.) and a National Science Foundation Graduate Research Fellowship (DGE-2040435, E.S.S.). Any opinions, findings and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.

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Author notes

  1. Daniel S. Jones

    Present address: Department of Biological Sciences, Auburn University, Auburn, AL, USA

  2. Cara L. Soyars

    Present address: Thermo Fisher Scientific, Raleigh, NC, USA

Authors and Affiliations

  1. Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Amala John, Elizabeth Sarkel Smith, Daniel S. Jones, Cara L. Soyars & Zachary L. Nimchuk

  2. Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Zachary L. Nimchuk

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Contributions

A.J., E.S.S., D.S.J. and Z.L.N. conceptualized the project. A.J., E.S.S., D.S.J., C.L.S. and Z.L.N. developed the methodology. A.J., E.S.S., D.S.J. and C.L.S. performed validation. A.J., E.S.S., D.S.J. and C.L.S. conducted formal analysis. A.J., E.S.S., D.S.J. and C.L.S. conducted investigations. A.J., E.S.S. and D.S.J. curated the data. A.J., E.S.S., D.S.J. and Z.L.N. wrote the original draft. A.J., E.S.S., D.S.J. and Z.L.N. reviewed and edited the manuscript. A.J., E.S.S. and D.S.J. performed visualization. Z.L.N. acquired funding, supervised and administered the project.

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Correspondence to Zachary L. Nimchuk.

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Extended data

Extended Data Fig. 1 Temperature and CLV3p receptors intersect to buffer auxin mediated floral primordia formation.

a-e, CLE receptors and temperature intersect to buffer floral primordia formation. a, Quantification of flower primordia termination at 16 °C (n = 9–21), 23 °C (n = 10–24) and 30 °C (n = 12–15) for Col-0, pol, crn, crn pol, clv1-101, clv1-101 crn, clv1-101 pol, clv1-101 crn pol, cik124, cik124 crn, cik124 pol, cik124 crn pol. b, Inflorescence images at 16 °C and 30 °C of clv1-101, clv1-101 crn pol, cik124, and cik124 crn pol. All cik alleles in this figure are cik1-3, cik2-3, and cik4-3 CRISPR generated alleles. Same magnification within temperatures. c, Quantification of flower primordia termination at 16 °C (n = 8–9), 23 °C (n = 8–13), and 30 °C (n = 12) for Col-0, pol, crn, crn pol, clv1-8 crn, clv1-8 pol, clv1-8 crn pol. d, Inflorescence images at 16 °C, 23 °C and 30 °C of clv1-8 crn and clv1-8 crn pol. Same magnification within temperatures. e, Maximum intensity projection of DR5::GFP (green fire blue LUT) and PI (grey) and DR5::GFP alone in the IMs pol (n = 7), clv1-101 pol (n = 6), and clv1-101 crn (n = 8). f, Maximum intensity projection of Col-0 CLV1pro::2xYPET-N7 (green) inflorescence meristem. Expression patterns of CLV1pro::2xYPET-N7 in Col-0 (n = 4) and crn pol (n = 5) showing no expression changes. g, Expression patterns of POLpro::YPET-H2AX (green, n = 6) in Col-0 and WUSpro::POL-YPET (green, n = 3) and DR5::GFP (purple) in crn pol showing L5 and Z axis (PI, red). h, Diagram of flower/fruit production on primary inflorescence in wild-type and terminating and recovering crn plants. n = X biologically independent samples and statistical groupings based on significant differences using Kruskal-Wallis and Dunn’s multiple comparison test correction (a, c) where significance is defined as p-value < 0.05. Scale bars 10 mm (b, d) and 20 µm (e, f, g).

Extended Data Fig. 2 CIK co-receptors are required for temperature-dependent floral primordia outgrowth.

a, b, CIK receptors act redundantly in temperature sensitive floral primordia formation. a, Quantification of flower primordia termination at 16 °C (n = 7–14), 23 °C (n = 8–9) and 30 °C (n = 7–9) for Col-0, crn, cik1, cik2, cik3, cik4, cik12, cik13, cik14, cik234, cik124, cik134, cik123 and cik1234. b, Inflorescence images at 16 °C of cik1, cik12, cik13, cik14, cik124, cik134, cik123, cik1234 with arrows pointing to termination. Same magnification within single and double mutants as well as within triple and quadruple mutants. All cik alleles in this figure are cik1-1, cik2-1, cik3-1, and cik4-1 T-DNA lines. c. CIK genes are expressed in the IM. Expression patterns of YPET-H2AX (green fire blue LUT) reporter lines in the IM with XY view of L5 and axial view of same stack indicated with dotted line of CIK1pro (n = 10), CIK2pro (n = 15), CIK3pro (n = 8) and CIK4pro (n = 14) in Col-0 showing representative images (PI, magenta). n = X biologically independent samples and statistical groupings based on significant differences using Kruskal-Wallis and Dunn’s multiple comparison test correction (a) where significance is defined as p-value < 0.05. Scale bars 10 mm (b) and 20 µm (c).

Extended Data Fig. 3 CLV3p dependent stem fasciation and floral primordia outgrowth are temperature dependent.

a, Primordia outgrowth defects in clv3 plants are buffered by temperature. Quantification of termination at 16 °C (n = 8–21), 23 °C (n = 9–22), and 30 °C (n = 12–16) for Col-0, pol, crn, crn pol, clv3-20, clv3-20 crn, clv3-20 pol, clv3-20 crn pol. b, Stem fasciation in clv3 plants is buffered by temperature. Quantification of maximum stem width at 16 °C (n = 9), 23 °C (n = 9), and 30 °C (n = 15). c, Inflorescences at 16 °C and 30 °C of clv3-20, crn, clv3-20 pol and clv3-20 crn pol. Varying magnification is used at 16 °C to show clv3 fasciation and flower termination (arrows). All same magnification at 30 °C. n = X biologically independent samples and statistical groupings based on significant differences using a one-way ANOVA (a, 16 °C and 23 °C) and Kruskal-Wallis and Dunn’s multiple comparison test correction (a 30 °C and b) where significance is defined as p-value < 0.05. In box plots, box indicates interquartile zone (25–75th percentile) with median line at center, whiskers indicate minimum and maximum values with ‘+’ indicating mean (b). Scale bars 10 mm (c).

Extended Data Fig. 4 CLV-dependent FM stem cell regulation is sensitized to heat and auxin levels.

a, CLV-dependent FM stem cell regulation is repressed in warmer growth environments. Carpels per flower at 16 °C (blue) and 30 °C (red) with sample numbers as follow (16 °C, 30 °C) for Col-0 (n = 18, n = 8), pol (n = 25, n = 9), crn (n = 9, n = 14), clv1 (n = 9, n = 13), crn pol (n = 14, n = 11), clv1 pol (n = 11, n = 16), clv1 crn (n = 10, n = 15), clv1 crn pol (n = 10, n = 18), cik124 (n = 12, n = 9), cik124crn (n = 8, n = 16), cik124 pol (n = 13, n = 16), cik124 crn pol (n = 14, n = 17). Middle chart shows Col-0 (n = 18, n = 9), pol (n = 25, n = 9), crn (n = 9, n = 15), crn pol (n = 14, n = 14), clv3-20 (n = 8, n = 16), clv3-20 crn (n = 8, n = 13), clv3-20 pol (n = 10, n = 15), and clv3-20 crn pol (n = 12, n = 15). Last chart shows Col-0 (n = 9, n = 12), pol (n = 12, n = 12), crn (n = 9, n = 12), clv1-8 (n = 12, n = 12), crn pol (n = 9, n = 12), clv1-8 pol (n = 12, n = 12), clv1-8 crn (n = 9, n = 12), and clv1-8 crn pol (n = 12, n = 12). b, elf3 enhances carpel numbers in clv1-101 mutants. Carpels per flower at 16 °C (blue) and 30 °C (red) with sample numbers as follows (16 °C, 30 °C) for Col-0 (n = 10, n = 16), crn (n = 14, n = 12), elf3 (n = 14, n = 9), crn elf3 (n = 13, n = 10), clv1 (n = 9, n = 11), clv1-101 elf3 (n = 13, n = 12), clv1 crn (n = 11, n = 14) and clv1-101 crn elf3 (n = 14, n = 12) with all single mutant control alleles. c, CLV-mediated FM stem cell regulation is sensitized to auxin levels. Carpels per flower at 23 °C for Col-0 (n = 9), yuc4 (n = 7), clv1-101 (n = 8), clv1-101 yuc4 (n = 8), clv3-9 (n = 11), clv3-9 yuc4 (n = 12). In box plots, box indicates interquartile zone (25-75th percentile) with median line at center, whiskers indicate minimum and maximum values with ‘+’ indicating mean (a-c). Statistical comparisons based on two-tailed Mann-Whitney test (a) where * indicate p-value < 0.0001 and ns indicates not significant. n = X biologically independent samples and statistical groupings based on significant differences using a Kruskal-Wallis and Dunn’s multiple comparison test correction (b, c). Significance is defined as p-value < 0.05 (a-c).

Extended Data Fig. 5 CLV3 acts redundantly with CLE25 and auxin in IM function.

a-c, cle25 and yuc1/4 mutations enhance clv3 IM phenotypes. a, SEM micrographs of IMs of cle26-10 clv3-20, cle27-10 clv3-20, cle26-10 cle27-10 clv3-20, clv3-20, cle25-10 clv3-20, cle25-10 cle26-10 clv3-20, cle25-10 cle27-10 clv3-20, and cle25-10 cle26-10 cle27-10 clv3-20. clv3-9 yuc14 and clv3-9 inset at same magnification for size comparison. clv3-9 yuc1/4 side view. Scale bars 200 µm and representative images shown from 4 independent experiments. b, Flower primordia termination visualized with SEM side view of IMs in Col-0, crn, clv3-9, clv3-20, cle26-10 cle27-10 clv3-20, cle25-10 clv3-20, cle25-10 cle26-10 cle27-10 clv3-20. Scale bars 1 mm. Representative images from 2 independent experiments shown. c, Inflorescence of the cle25-11 allele with clv3-20 mutation showing characteristic disk-like IM phenotype. d, Quantification of disk meristem expansion using ratio of maximum width and maximum length of clv3-20 (n = 12), clv3-20 crn (n = 9), clv3-9 yuc1/4 (n = 9), cle25-10 clv3-20 (n = 14), cle26-10 cle27-10 clv3-20 (n = 8) and cle25-10 cle26-10 cle27-10 clv3-20 (n = 10). e, CRISPR guide and PAM sites for CLE genes indicating SNPs and indels in red or with dash. WT, wild-type; m, mutant. f, Gene diagrams of CLE ORFs with red line indicating CRISPR target positioned before the CLE domain indicated by the blue rectangle. g, carpels per flower of Col-0, cle25-10, clv3-20 and cle25-10 clv3-20 (n = 12). h-i, cle25 alone does not enhance crn. h, Inflorescence images (scale bars 10 mm) and i, quantification of termination of Col-0, cle25-10, crn and cle25-10 crn (n = 9). CLE25 is expressed in root phloem cells independent of clv3. j, Confocal images of CLE25pro::YPET-H2AX (green) of Col-0 and clv3-20 in roots. f, CLE25 is ectopically expressed in clv3-20 floral primordia. Col-0 (n = 4) and clv3-20 (n = 4) with same single transgene CLE25pro::YPET-H2AX (green) insertion line (#8) in IM and FMs (PI, magenta). Scale bar 20 µm (j, k). In box plots, box indicates interquartile zone (25–75th percentile) with median line at center, whiskers indicate minimum and maximum values with ‘+’ indicating mean (d, g). n = X biologically independent samples and statistical groupings based on significant differences using a Kruskal-Wallis and Dunn’s multiple comparison test correction (d, g, i) where significance is defined as p-value < 0.05.

Extended Data Fig. 6 CLV1 is required for IM maintenance.

a-e, CLV1 is required for primary IM maintenance. a, Side view of primary inflorescence with arrows pointing to primary inflorescence (white) with cauline leaf (green) subtending the secondary inflorescence (yellow) in both Col-0 and clv1-101 revealing primary inflorescence termination (PIT) in clv1-101. b, Categories: buried, hook, weak, WT-like, and fasciated. Range of flowers that emerge in each category before PIT. c, Population analysis of clv1-101 at 16 °C (n = 87). d, UBQ10::DII-Venus in L1 cells of the IM in clv1-101/+ and clv1-101 homozygous mutant undergoing PIT. The same transgenic insertion line was used in d. 4/4 WT UBQ10::DII-Venus and 4/5 clv1-101 UBQ10::DII-Venus (green) independent transgenic lines showed representative expression. clv1-20 allele inflorescence undergoing PIT at 23 °C but not at 30 °C (PI, magenta) (e) and quantification of PIT in Col-0, clv1-101, clv1-20 and clv1-8 plants (f, n = 15). g, Summary of interactions between CLEp signaling and heat on auxin dependent stem cell regulation, flower primordia outgrowth, and SAM maintenance. n = X biologically independent samples. Scale bars 10 mm (b, d, e) and 20 µm (d).

Extended Data Table 1 Protein sequences resulting from CRISPR CLE gene mutants used in this study
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Extended Data Table 2 Genotyping primers generated for this study
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John, A., Smith, E.S., Jones, D.S. et al. A network of CLAVATA receptors buffers auxin-dependent meristem maintenance. Nat. Plants 9, 1306–1317 (2023). https://doi.org/10.1038/s41477-023-01485-y

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