Letter

A small peptide modulates stomatal control via abscisic acid in long-distance signalling

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Abstract

Mammalian peptide hormones propagate extracellular stimuli from sensing tissues to appropriate targets to achieve optimal growth maintenance1. In land plants, root-to-shoot signalling is important to prevent water loss by transpiration and to adapt to water-deficient conditions2, 3. The phytohormone abscisic acid has a role in the regulation of stomatal movement to prevent water loss4. However, no mobile signalling molecules have yet been identified that can trigger abscisic acid accumulation in leaves. Here we show that the CLAVATA3/EMBRYO-SURROUNDING REGION-RELATED 25 (CLE25) peptide transmits water-deficiency signals through vascular tissues in Arabidopsis, and affects abscisic acid biosynthesis and stomatal control of transpiration in association with BARELY ANY MERISTEM (BAM) receptors in leaves. The CLE25 gene is expressed in vascular tissues and enhanced in roots in response to dehydration stress. The root-derived CLE25 peptide moves from the roots to the leaves, where it induces stomatal closure by modulating abscisic acid accumulation and thereby enhances resistance to dehydration stress. BAM receptors are required for the CLE25 peptide-induced dehydration stress response in leaves, and the CLE25–BAM module therefore probably functions as one of the signalling molecules for long-distance signalling in the dehydration response.

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Change history

  • Update 13 April 2018

    The Source Data files originally published with this article were missing for Figures 1-4 and Extended Data Figures 1-5, 7, 9 and 10. This has now been corrected.

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Acknowledgements

We thank S. Sawa, T. Ishida (Kumamoto University) and Y. Matsubayashi (Nagoya University) for their discussions, and S. Mizukado and H. Kobayashi for their technical assistance. This research was supported by the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (BRAIN; to K.S.); by the Ministry of Agriculture, Forestry, and Fisheries (MAFF); by JSPS KAKENHI Grant Numbers JP15K18563 (F.T.) and JP16H01475 (F.T.); and by Program on Open Innovation Platform with Enterprises, Research Institute and Academia (OPERA) (Y.O.).

Reviewer information

Nature thanks T. Kakimoto and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

    • Shigeyuki Betsuyaku

    Present address: Faculty of Life and Environmental Science, University of Tsukuba, Tsukuba, Japan

Affiliations

  1. Gene Discovery Research Group, RIKEN Center for Sustainable Resource Science, Tsukuba, Japan

    • Fuminori Takahashi
    • , Yuriko Osakabe
    •  & Kazuo Shinozaki
  2. Biomass Research Platform Team, RIKEN Center for Sustainable Resource Science, Tsukuba, Japan

    • Fuminori Takahashi
    •  & Kazuo Shinozaki
  3. Biomolecular Characterization Unit, RIKEN Center for Sustainable Resource Science, Wako, Japan

    • Takehiro Suzuki
    •  & Naoshi Dohmae
  4. Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima, Japan

    • Yuriko Osakabe
  5. Japan Science and Technology Agency (JST), PRESTO, Kawaguchi, Japan

    • Shigeyuki Betsuyaku
  6. Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan

    • Shigeyuki Betsuyaku
    • , Yuki Kondo
    •  & Hiroo Fukuda
  7. Graduate School of Agricultural and Life Science, The University of Tokyo, Tokyo, Japan

    • Kazuko Yamaguchi-Shinozaki

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Contributions

F.T. and K.S. designed the study. S.B., H.F. and F.T. prepared synthetic peptides and performed peptide screening. F.T., S.B. and K.S. performed stomatal aperture analyses. F.T., T.S. and N.D. conducted nLC–MS/MS analyses. S.B. helped with peptide labelling experiments. Y.O. and F.T. generated knockout mutants. F.T. and Y.O. performed drought stress sensitivity and stomatal conductance analyses. F.T. performed gene expression analyses and ABA measurement. Y.K., H.F. and F.T. performed structural analyses of vasculature. F.T., T.S., Y.O., S.B. and N.D. analysed the data. F.T., K.Y.-S. and K.S. wrote the manuscript with substantial input from S.B. and H.F. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Fuminori Takahashi or Kazuo Shinozaki.

Extended data figures and tables

  1. Extended Data Fig. 1 Effects of synthetic CLE peptide application on NCED3 expression and stomatal closure in leaves, and movement of CLE25 peptide from roots to leaves.

    a, NCED3 expression after application of 5 μM of each synthetic CLE peptide to roots for 3 h, or in response to dehydration stress for 3 h in leaves of wild-type plants (n = 4 biological replicates). **P < 0.01, no significant difference (NS) among treatment conditions as analysed by two-tailed Student’s t-test (see Methods for exact P values). b, NCED3 expression in leaves of wild-type plants after application of 1 μM peptide to roots for 3 h (n = 6 biological replicates). c, ABA content in leaves after application of 1 μM peptide to roots for 3 h (n = 8 biological replicates). d, Comparison of peptide sequences of CLE25 and CLE26. e, Typical images of wild-type (n = 6 biological replicates), 1 μM-ABA- or 1 μM-CLE25-induced stomatal closure (n = 4 and 6 biological replicates, respectively). Scale bars, 20 μm. f, Roots of whole plants were incubated without (0 h on x axis, n = 547) or with 0.01% acetonitrile (mock, n = 519), ABA (n = 562) or each CLE peptide (n = 546, CLE26; n = 578, CLV3; n = 762, CLE46; n = 561, TDIF) for 3 h. Data are from three experiments. **P < 0.01, ***P < 0.001 as analysed by one-way ANOVA followed by a Tukey’s (bc) or a Tukey–Kramer (f) post hoc test. g, h, Detection of non-labelled (g) and isotope-labelled (h) CLE25 peptide by nLC–MS/MS. These experiments were repeated two times independently with similar results. Hyp, hydroxyproline. Source data

  2. Extended Data Fig. 2 The cle25 mutants are generated using CRISPR–Cas9 method, and clv3-8, cle46-1 and tdr-1 mutants do not exhibit repression of NCED3 expression in response to dehydration stress.

    a, CEL-I analysis of T1 cle25 mutants. The CLE25 locus was amplified in wild-type and cle25 mutants, then digested with CEL-I. The asterisk indicates mutated bands digested with CEL-I nuclease. These experiments were repeated four times independently with similar results. For gel source data, see Supplementary Fig. 1. b, CRISPR–Cas9-induced mutation detected by amplicon sequencing in T3 plants. The exons (boxes) and intron (line) indicate the schematic arrangement of the CLE25 gene. Selected target sequences (18 base pairs) are shown in red boxes and protospacer adjacent motif sequences are shown as green characters. A base deletion (guanosine at position 22) was detected in the genomic DNA of the mutants. The red triangle shows the position of the base deletion site. This mutation created a stop codon after the mutation site (asterisk in the amino acid sequence). (ce, Dehydration-induced NCED3 expression in clv3-8 (c), cle46-1 (d) and tdr-1 (e) mutants was not repressed compared with that in wild-type (Cont.) plants in response to dehydration stress (n = 3 pooled biological replicates). CLV3, CLE46 and TDIF peptides do not have a primary function in the dehydration stress response that mediates ABA signalling. *P < 0.05, **P < 0.01 as analysed by one-way ANOVA followed by a Tukey’s post hoc test ((ce). The clv3-8 mutant was a point mutant of CLV3. The cle46-1 and tdr-1 mutants were transfer DNA mutants of CLE46 and TDIF RECEPTOR, respectively. Source data

  3. Extended Data Fig. 3 nced3-2 and aba2-1 mutants treated with CLE25 peptide do not exhibit stomatal closure.

    Detached rosette leaves were incubated without (labelled ‘0’ on x axis: n = 505, wild type (Cont.); n = 647, nced3-2; n = 564, aba2-1) or with ABA (n = 617, wild type; n = 591, nced3-2; n = 467, aba2-1) or the CLE25 peptide (n = 505, wild type; n = 517, nced3-2; n = 570, aba2-1) for 3 h. Data are from three experiments. ***P < 0.001 as analysed by one-way ANOVA followed by a Tukey–Kramer post hoc test. Source data

  4. Extended Data Fig. 4 Stomatal conductance, rosette diameter, fresh weight and dry weight of wild-type and cle25 mutants grown on soil under control conditions.

    a, The stomatal conductance of wild-type (Cont.) plants, and cle25 and nced3-2 mutants (n = 6 biological replicate) was measured. Data are plotted at 5-min intervals for 20 min under control conditions. b, Rosette size of wild-type plants and cle25 mutants (n = 6 biological replicates) grown on soil was scored. c, Fresh weight of wild-type plants and cle25 mutants (n = 6 biological replicates) grown on soil was measured. d, Dry weight of wild-type plants and cle25 mutants (n = 6 biological replicates) grown on soil was measured. **P < 0.01 as analysed by one-way ANOVA followed by a Tukey’s post hoc test ((ad). Source data

  5. Extended Data Fig. 5 Repression of CLE25 in transgenic plants affects expression of dehydration-induced genes and hypersensitivity to dehydration stress.

    ad, Dehydration-induced CLE25 (a), NCED3 (b), LEA (c) and RD29B (d) expression in wild-type (Cont.) and CLE25 RNAi plants in response to dehydration stress (n = 6 biological replicates). **P < 0.01 as analysed by one-way ANOVA followed by a Tukey’s post hoc test (ad). e, CLE25 RNAi plants and the nced3-2 mutant had a dehydration stress-sensitive phenotype (plants per group; n = 85, wild type (Cont.); n = 85, nced3-2; n = 85, CLE25 RNAi #10; n = 60, CLE25 RNAi #12; n = 60, CLE25 RNAi #13). Data are from three experiments. Scale bars, 2 cm. Source data

  6. Extended Data Fig. 6 Endogenous CLE25 peptide is secreted extracellularly.

    Arabidopsis T87 cells were cultured with or without 0.4 M mannitol for 4 h. Then, peptides in the liquid culture medium were purified and analysed by nLC–MS/MS. a, Selected MS/MS ion chromatograms of the y4-ion from triply charged CLE25 peptides treated with 0.4 M mannitol. b, MS/MS spectra of endogenous (upper, with 0.4 M mannitol treatment) and synthetic (lower) CLE25 peptide obtained by nLC–MS/MS. Endogenous CLE peptide with 0.4 M mannitol treatment was detected only in di-hydroxy form. These experiments were repeated two times independently with similar results (a, b). Hyp, hydroxyproline. c, List indicates top 10 proteins in T87 cells in response to mannitol treatment. Amounts of these top 10 proteins accumulated in the liquid culture medium were the same under control conditions and in response to mannitol treatment. Cell lysis did not occur in response to mannitol treatment.

  7. Extended Data Fig. 7 CLE25 peptide moves from roots to leaves and modulates NCED3 expression in leaves according to grafting experiments.

    a, b, CLE25 expression (a) and NCED3 expression (b) after application of CLE25 peptide to roots in leaves of wild-type (Cont.) and CLE25 RNAi plants (n = 6 biological replicates). c, Dehydration-induced CLE25 expression in grafted plants in which shoots and roots were grafted between wild-type and CLE25 RNAi plants (n = 6 biological replicates). *P < 0.05, **P < 0.01 as analysed by one-way ANOVA followed by a Tukey’s post hoc test (ac). d, Dehydration-induced NCED3 expression in grafted leaves in which shoots and roots were grafted between wild-type and CLE25 RNAi plants (n = 6 biological replicates). No significant difference (NS) among the three genotypes as analysed by two-tailed Student’s t-test (see Methods for exact P values). Source data

  8. Extended Data Fig. 8 Root-derived endogenous CLE25 peptide accumulates in dehydrated leaves.

    Accumulation of CLE25 peptide in leaves of wild-type and cle25-mutant shoot scions grafted onto wild-type rootstocks was analysed by nLC–MS/MS. a, b, MS/MS ion chromatograms of triple-charged CLE25 peptides in dehydrated leaves of grafted wild-type/wild-type (a) or cle25 #10/wild-type (b) plants under 3-h dehydration-stress conditions. c, MS/MS spectra of endogenous CLE25 peptide in leaves of grafted cle25 #10/wild-type plants under 3-h dehydration-stress conditions. d, MS/MS spectra of synthetic CLE25 peptide. These experiments were repeated two times independently with similar results (ad). Hyp, hydroxyproline.

  9. Extended Data Fig. 9 cle25 and bam1-5 bam3-3 mutants show salinity stress-sensitive phenotype.

    a, Images represent 16-day-old seedlings for each genotype grown on germination medium agar plates containing 0 mM or 150 mM NaCl (n = 3 biological replicates). Scale bars, 0.5 cm. b, Measurements of relative chlorophyll contents in response to treatment with different NaCl concentrations were shown for 16-day-old seedlings after germination (n = 3 pooled biological replicates). *P < 0.05, **P < 0.01 as analysed by two-tailed Student’s t-test (see Methods for exact P values). Source data

  10. Extended Data Fig. 10 NCED3 expression in CLE25-treated leaves of bam1-5 bam3-3 mutants, vascular development of wild-type, cle25 and bam1-5 bam3-3 mutants under control conditions and root growth phenotypes of cle25, nced3-2 and aba2-1 mutants under control conditions or long-term application of CLE25 peptide.

    a, NCED3 expression in the leaves of wild-type (Cont.) and bam1-5 bam3-3 mutants (n = 6 biological replicates) after application of CLE25 peptide to leaves. **P < 0.01 as analysed by one-way ANOVA followed by a Tukey’s post hoc test. be, Microscopy images of the leaf vasculature of wild-type (b; n = 6 biological replicates), cle25 #6 (c; n = 12 biological replicates), cle25 #10 (d; n = 12 biological replicates) and bam1-5 bam3-3 mutants (e; n = 12 biological replicates). Scale bars, 1 mm. fi, Microscopy images of the protoxylem and metaxylem vessel formation of wild-type (f; n = 4 biological replicates), cle25 #6 (g; n = 3 biological replicates), cle25 #10 (hn = 4 biological replicates) and bam1-5 bam3-3 mutants (i; n = 4 biological replicates). Scale bars, 50 μm. jm, Cross section of primary root of wild-type (j; n = 9 biological replicates), cle25 #6 (kn = 10 biological replicates), cle25 #10 (l; n = 10 biological replicates) and bam1-5 bam3-3 mutants (m; n = 8 biological replicates). Scale bars, 20 μm. n, Root length of wild-type and cle25 mutants from six to eleven days after germination, on germination medium agar plates (n = 16 biological replicates). o, Images represent 8-day-old or 10-day-old seedlings after germination of each genotype on germination medium agar plates (n = 4 biological replicates). Scale bars, 2 cm. p, Relative root length of wild-type, and nced3-2 and aba2-1 mutants from seven to eleven days after germination, on germination medium agar plate (n = 12 biological replicates). q, Images represent 8-day-old or 10-day-old seedlings after germination of each genotype on germination medium agar plates (n = 3 biological replicates). Scale bars, 2 cm. r, Relative root length of wild-type, and nced3-2 and aba2-1 mutants from seven to eleven days after germination, on germination medium agar plates containing 1 μM CLE25 peptide (n = 12 biological replicates). s, Images represent 8-day-old or 10-day-old seedlings after germination of each genotype on germination medium agar plates containing 1 μM CLE25 peptide (n = 3 biological replicates). Scale bars, 2 cm. Two-way ANOVA followed by a Tukey’s post hoc test indicated that there were no differences among each genotype (np, r). Source data

Supplementary information

  1. Supplementary Information

    This file contains Supplementary Figure 1

  2. Reporting Summary

Source data

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