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Competitive binding of antagonistic peptides fine-tunes stomatal patterning

Nature volume 522, pages 439–443 (2015)Cite this article

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Abstract

During development, cells interpret complex and often conflicting signals to make optimal decisions. Plant stomata, the cellular interface between a plant and the atmosphere, develop according to positional cues, which include a family of secreted peptides called epidermal patterning factors (EPFs). How these signalling peptides orchestrate pattern formation at a molecular level remains unclear. Here we report in Arabidopsis that Stomagen (also called EPF-LIKE9) peptide, which promotes stomatal development, requires ERECTA (ER)-family receptor kinases and interferes with the inhibition of stomatal development by the EPIDERMAL PATTERNING FACTOR 2 (EPF2)–ER module. Both EPF2 and Stomagen directly bind to ER and its co-receptor TOO MANY MOUTHS. Stomagen peptide competitively replaced EPF2 binding to ER. Furthermore, application of EPF2, but not Stomagen, elicited rapid phosphorylation of downstream signalling components in vivo. Our findings demonstrate how a plant receptor agonist and antagonist define inhibitory and inductive cues to fine-tune tissue patterning on the plant epidermis.

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Figure 1: Complete loss of ER-family genes confers insensitivity to STOMAGEN overexpression and co-suppression.
Figure 2: STOMAGEN overexpression on stomatal development in tmm hypocotyl epidermis with combinatorial loss-of-function in ER-family genes.
Figure 3: Direct and competitive binding of Stomagen and EPF2 peptides to ER.
Figure 4: EPF2, but not Stomagen, triggers downstream MAPK activation in Arabidopsis seedlings.

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Acknowledgements

We thank I. Hara-Nishimura for STOMAGEN-ami lines and anti-Stomagen antibody; K. Peterson for iSTOMAGEN construct and transgenic lines; M. Kanaoka and N. Kamiya for LURE2 peptides; D. Baulcombe for p19 plasmid; C. Tamerler and M. Sarikaya for use of the HPLC, QCM and MALDI-ToF equipment; A. Hofstetter for technical assistance; and J. McAbee, K. Peterson, T. Imaizumi, B. Wakimoto, S. Di Rubbo and R. Horst for comments. K.U.T. is an HHMI-GBMF Investigator and an Endowed Distinguished Professor of Biology; J.S.L. was an NSERC Postdoctoral Fellow. Y.-C.L.L. was a Mary Gates Undergraduate Research Fellow of the University of Washington.

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Authors and Affiliations

  1. Howard Hughes Medical Institute, University of Washington, Seattle, 98195, Washington, USA

    Jin Suk Lee, Ya-Chen Lisa Lin, Soon-Ki Han, Julian Avila & Keiko U. Torii

  2. Department of Biology, University of Washington, Seattle, 98195, Washington, USA

    Jin Suk Lee, Michal Maes, Ya-Chen Lisa Lin, Aarthi Putarjunan, Soon-Ki Han, Julian Avila & Keiko U. Torii

  3. Department of Materials Science and Engineering, University of Washington, Seattle, 98195, Washington, USA

    Marketa Hnilova

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Contributions

J.S.L. and K.U.T. conceived the project. J.S.L., M.H., M.M., J.A. and Y.-C.L.L. purified peptides and performed ligand−receptor binding and bioassays. J.S.L and S.-K.H. performed RT–PCR. J.S.L. and A.P. performed MAPK assays. J.S.L. and Y.-C.L.L. performed quantitative analysis of stomatal phenotypes. K.U.T. constructed STOMAGEN cDNA plasmid. K.U.T., J.S.L., M.H., M.M., Y.-C.L.L., A.P. and S.-K.H. analysed the data. K.U.T. wrote the manuscript with inputs from all co-authors.

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Correspondence to Keiko U. Torii.

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Extended data figures and tables

Extended Data Figure 1 Stomatal clustering phenotype of induced STOMAGEN overexpression in multiple independent transgenic lines.

Shown are confocal microscopy images of abaxial cotyledon epidermis from 10-day-old light-grown seedlings of four independent transgenic lines carrying an oestradiol-inducible STOMAGEN overexpression construct (iSTOMAGEN). Left panels, no induction (control); right panels, oestradiol induction; each row shows representative images from individual lines. Yellow brackets indicate stomatal clusters. Images are taken under the same magnification. Scale bar, 40 μm. n = 3 for each panel.

Extended Data Figure 2 RT–PCR analysis of STOMAGEN transcripts in transgenic lines used in this study.

a, Expression of oestradiol-inducible STOMAGEN transgene (iSTOMAGEN) in transgenic lines expressing oestradiol-inducible STOMAGEN overexpression (Est::STOMAGEN) lines from wild-type (wt), tmm and er erl1 erl2 triple mutant background with or without oestradiol induction. b, Expression of the endogenous STOMAGEN transcripts in each genotype carrying STOMAGEN-ami construct. tmm or er erl1 erl2 mutation does not seem to affect STOMAGEN transcript levels. c, Expression of EPF1, EPF2, total STOMAGEN and STOMAGEN transgene (iSTOMAGEN) transcripts in transgenic Est::STOMAGEN lines (in two different T1 populations (s1 and s2) and a representative T3 line (s3)) with or without oestradiol induction. STOMAGEN overexpression by oestradiol causes modest increase in EPF1 and EPF2 transcripts, which accords with increased stomatal differentiation by iSTOMAGEN. d, EPF1, EPF2 and STOMAGEN transcript accumulation in wild-type (wt) and single- and higher-order loss-of-function mutants of epf1, epf2 and stomagen (STOMAGEN-ami). For epf1 STOMAGEN-ami and epf2 STOMAGEN-ami lines, two different F3 populations derived from the same genetic crosses were used to test the reproducibility. STOMAGEN expression is not influenced by epf1 and epf2 mutations, consistent with the proto-mesophyll expression of STOMAGEN. However, EPF2 expression is reduced by STOMAGEN-ami, consistent with reduced stomatal cell lineages by STOMAGEN co-suppression. As reported, epf1 has a T-DNA insertion within the 5′ UTR3, which results in accumulation of aberrant transcripts. For all experiments, elF4A was used as a control. For primer sequences see Extended Data Table 1.

Extended Data Figure 3 STOMAGEN overexpression promotes stomatal differentiation in genetic backgrounds missing/blocking EPF2–ER and EPF1–ERL1 signalling components.

a–j, Representative confocal images of cotyledon abaxial epidermis from 10-day-old light-grown transgenic seedlings of the following genotypes, each carrying Est::STOMAGEN construct: epf2 (a, b); dominant-negative ER (ER(ΔK)) in er (c, d); epf1 (e, f); dominant-negative ERL1 (ERL1(ΔK)) in erl1 (g, h); er erl1 erl2 (i, j). For each genotype, a control uninduced phenotype (a, c, e, g, i) and induced STOMAGEN overexpression (iSTOMAGEN) (b, d, f, h, j) are shown. Blocking ER or lacking EPF2 produces small stomatal-lineage cells due to excessive entry divisions (a, c; yellow brackets). iSTOMAGEN confers stomatal clusters and small stomatal-lineage cells are no longer present (b and d). Blocking ERL1 or lacking EPF1 causes a stomatal pairing due to a violation of one-cell-spacing rule (e, g; dots). iSTOMAGEN enhances stomatal cluster phenotype in these genotypes (f, h). iSTOMAGEN does not enhance stomatal clustering defects in er erl1 erl2 (i, j). Images were taken under the same magnification. Scale bars, 30 µm. n = 29 (a); n = 24 (b); n = 16 (c); n = 17 (d); n = 22 (e); n = 23 (f); n = 17 (g); n = 20 (h); n = 24 (i); n = 24 (j). k–m, Stomatal density (number of stomata per mm2) (k); stomatal index (% of number of stomata per stomata + non-stomatal epidermal cells) (l); and stomatal cluster distribution (in %) (m) from 10-day-old abaxial cotyledons of transgenic lines of each genotype carrying Est::STOMAGEN construct. −, no induction; +, induced by 10 µM oestradiol. Stomagen overexpression significantly increases stomatal density in all genotypes except for er erl1 erl2 and tmm. Error bars indicate s.e.m. ***P < 0.001; **P < 0.01; NS, not significant; Welch 2-sample t-test. Number of seedlings subjected to analysis, n = 14–16. Total numbers of stomata counted: wt, no induction 1,277, induction 2,639; epf1, no induction 1,390, induction 3,485; ERL1(ΔK) erl1, no induction 1,573, induction 3,991; epf2, no induction 2,502, induction 3,317; ER(ΔK) er, no induction 2,899, induction 4,397; tmm, no induction 2,948, induction 3,212; er erl1 erl2, no induction 4,454, induction 4,464. All genotypes carry Est::STOMAGEN. wt, no induction n = 16, induction n = 14; epf1, no induction n = 16, induction n = 17; ERL1(ΔK) erl1, no induction n = 15, induction n = 15; epf2, no induction n = 15, induction n = 15; ER(ΔK) er, no induction n = 15, induction n = 15; tmm, no induction n = 15, induction n = 15; er erl1 erl2, no induction n = 15, induction n = 15.

Source data

Extended Data Figure 4 STOMAGEN co-suppression results in reduced stomatal development in genetic backgrounds missing or blocked in EPF2–ER and EPF1–ERL1 signalling pathways.

a–j, Representative confocal images of cotyledon abaxial epidermis from 10-day-old light-grown transgenic seedlings of the following genotypes: wild type (a); STOMAGEN-ami (b); epf2 (c); epf2 STOMAGEN-ami (d); dominant-negative ER (ER(ΔK)) in er (e); ER(ΔK) er STOMAGEN-ami (f); epf1 (g); epf1 STOMAGEN-ami (h); dominant-negative ERL1 (ERL1(ΔK)) in erl1 (i); ERL1(ΔK) erl1 STOMAGEN-ami (j). STOMAGEN-ami markedly reduces stomatal differentiation in wild type (a, b). Blocking ER or lacking EPF2 produces small stomatal-lineage cells due to excessive entry divisions (c, e; yellow brackets). STOMAGEN-ami exaggerates the small stomatal-lineage cells of epf2 (d; yellow brackets). STOMAGEN-ami ER(ΔK) er shows excessive asymmetric entry as well as amplifying divisions (f; yellow and pink brackets, respectively). Blocking ERL1 or lacking EPF1 causes a stomatal pairing due to a violation of one-cell-spacing rule (g, i; dots). STOMAGEN-ami suppresses these mild stomatal pairing phenotypes and reduces stomatal differentiation (h, j). Images were taken under the same magnification. Scale bars, 30 μm. n = 13 (a); n = 26 (b); n = 15 (c); n = 23 (d); n = 11 (e); n = 17 (f); n = 12 (g); n = 22 (h); n = 18 (i); n = 13 (j). k–n, Stomatal density (k), stomatal index (l), stomatal cluster distribution (in %; m), and non-stomatal epidermal cell density (n) from 10-day-old abaxial cotyledons of each genotype with or without carrying STOMAGEN-ami construct. Error bars, s.e.m. ***P < 0.001; *P ≤ 0.05; NS, not significant; Welch 2-sample t-test. n = 9–16. Total numbers of stomata counted: wt, 719; STOMAGEN-ami, 204; epf1, 1,004; epf1 STOMAGEN-ami, 383; ERL1(ΔK) erl1, 1,558; ERL1(ΔK) erl1 STOMAGEN-ami, 504; epf2, 1,505; epf2 STOMAGEN-ami, 1,165; ER(ΔK) er, 1,361; ER(ΔK) er STOMAGEN-ami, 782; tmm, 2,495; tmm STOMAGEN-ami, 2,688; er erl1 erl2, 1,853; er erl1 erl2 STOMAGEN-ami, 2,028. Total numbers of non-stomatal epidermal cells counted: wt, 1,494; STOMAGEN-ami, 1,299; epf1, 1,584, epf1 STOMAGEN-ami, 2,711; ERL1(ΔK) erl1, 871; ERL1(ΔK) erl1 STOMAGEN-ami, 1,348; epf2, 3,980; epf2 STOMAGEN-ami, 8,808; ER(ΔK) er, 5,739; ER(ΔK) er STOMAGEN-ami, 6,939; tmm, 790; tmm STOMAGEN-ami, 962; er erl1 erl2, 479; er erl1 erl2 STOMAGEN-ami, 391. wt, n = 8; STOMAGEN-ami, n = 8; epf1, n = 9, epf1 STOMAGEN-ami, n = 17; ERL1(ΔK) erl1, n = 13; ERL1(ΔK) erl1 STOMAGEN-ami, n = 9; epf2, n = 11; epf2 STOMAGEN-ami, n = 15; ER(ΔK) er, n = 9; ER(ΔK) er STOMAGEN-ami, n = 11; tmm, n = 8; tmm STOMAGEN-ami, n = 8; er erl1 erl2, n = 8; er erl1 erl2 STOMAGEN-ami, n = 8.

Source data

Extended Data Figure 5 STOMAGEN overexpression on stomatal development in tmm hypocotyl epidermis with combinatorial loss-of-function in ER-family genes: a complete set.

a–r, Representative confocal microscopy images of hypocotyl epidermis from 10-day-old light-grown transgenic seedlings of the following genotypes, each carrying Est::STOMAGEN: wild-type (wt) (a, b); tmm (c, d); tmm er (e, f); tmm erl2 (g, h); tmm erl1 (i, j); tmm er erl2 (k, l); tmm erl1 erl2 (m, n); tmm er erl1 (o, p); and tmm er erl1 erl2 (q, r). A control, uninduced phenotype (a, c, e, g, i, k, m, o, q); iSTOMAGEN (b, d, f, h, j, l, n, p, r). iSTOMAGEN results in arrested stomatal precursor cells (asterisks) and stomatal-lineage ground cells (SLGCs; brackets) in tmm hypocotyls (d). iSTOMAGEN triggers entry divisions in tmm er and tmm erl2 (f, h; brackets), and exaggerate the SLGC clusters in tmm er erl2 (k, l; brackets). Images were taken under the same magnification. Scale bar, 30 μm. n = 19 (a); n = 19 (b); n = 20 (c); n = 20 (d); n = 19 (e); n = 22 (f); n = 20 (g); n = 17 (h); n = 18 (i); n = 19 (j); n = 19 (k), n = 21 (l); n = 17 (m); n = 20 (n); n = 19 (o); n = 21 (p); n = 20 (q); n = 20 (r). s, t, Stomatal index and SLGC index. s, ***P < 0.0001; **P < 0.01; *P < 0.5 (Wilcoxon rank sum test). NS, not significant. 0, no stomata or SLGC observed; n = 15. Total number of stomata and SLGCs counted; tmm non-induced, 0 and 0; induced, 0 and 211; tmm er non-induced, 0 and 0; induced, 0 and 308; tmm erl2 non-induced, 0 and 32; induced, 0 and 171; tmm erl1 non-induced, 58 and 116; induced, 142 and 138; tmm er erl2 non-induced, 0 and 270; induced, 10 and 676; tmm er erl1 non-induced, 422 and 283; induced, 817 and 422; tmm erl1 erl2 non-induced, 72 and 83; induced, 163 and 97; tmm er erl1 erl2 non-induced, 1,229 and 295; induced, 1,068 and 222. n = 15 for all genotypes (s, t).

Source data

Extended Data Figure 6 Association of Stomagen with ER-family receptors and TMM.

a, Shown are co-immunoprecipitation assays of ligand–receptor pairs expressed in N. benthamiana leaves. The ectodomains and membrane-spanning domains of ER, ERL1 and ERL2 fused with GFP were separately expressed in N. benthamiana, and microsomal fractions were incubated with 1 µM Stomagen peptides followed by immunoprecipitation using anti-GFP (anti-GFP) antibody. Inputs and immunoprecipitates were immunoblotted using anti-GFP (anti-GFP) or anti-Stomagen (anti-Stomagen) antibodies. Experiments were repeated three times (three biological replicates). b, Co-immunoprecipitation of LURE2 peptide fused with hexa-histidine tag (LURE2–His) with N. benthamiana microsomal fractions expressing the ectodomains and membrane-spanning domains of ER and FLS2 fused with GFP, a full-length TMM fused with GFP, or a control, uninoculated leaf sample. Immunoprecipitation was performed using anti-GFP and immunoblotted using anti-GFP (for detection of receptors) or anti-His (for detection of LURE2–His) antibodies. Experiments were repeated twice (two biological replicates). c, Co-immunoprecipitation of Stomagen peptide with N. benthamiana microsomal fractions expressing the ectodomains and membrane-spanning domains of ER and FLS2 fused with GFP or a control, uninoculated leaf sample. Immunoprecipitation was performed using anti-GFP and immunoblotted using anti-GFP (for detection of receptors) or anti-Stomagen antibodies. Experiments were repeated four times (four biological replicates).

Extended Data Figure 7 Purified mEPF2 and Stomagen recombinant peptides and separation of bioactive mEPF2 by reverse-phase chromatography.

a, SDS–PAGE gel of purified and refolded mEPF2–MYC–HIS and Stomagen recombinant peptides (asterisks). Left: molecular mass markers. b, HPLC chromatogram of purified, refolded mEPF2. Peaks 1 and 2 in UV chromatogram were collected and subjected to bioassays. c, Confocal image of cotyledon epidermis from wild-type seedling grown a solution with peak 1 for 5 days. No stoma is visible, indicating that peak 1 contains bioactive mEPF2. Scale bar, 20 μm. n = 19. d, Confocal image of cotyledon epidermis from wild-type seedling grown in a solution with peak 2 for 5 days, with normal stomatal differentiation, indicating that the peptide is not bioactive. Scale bar, 20 μm. n = 9.

Extended Data Figure 8 Separation of properly folded, bioactive Stomagen and mutant Stomagen peptides by reverse-phase chromatography followed by mass spectrometry and bioassays.

a, HPLC chromatogram of purified, refolded Stomagen. Peaks 1 and 2 in UV chromatogram were collected and subjected to MALDI-TOF mass spectrometry (b, d) as well as for bioassays (c, e). b, MALDI-TOF spectrum of peak 1 from a. A single-charged peptide corresponding to synthetic Stomagen peptide was observed at m/z = 5,118.5 ([M+H]+) and a double charged peptide at m/z = 2,559.8 ([M+2H]2+). c, Confocal image of cotyledon epidermis from wild-type seedling grown a solution with peak 1. Severe stomatal clustering and overproduction of stomata are observed. Scale bar, 20 μm. n = 8. d, MALDI-TOF spectrum of peak 2 from a. e, Confocal image of cotyledon epidermis from wild-type seedling grown in a solution with peak 2 from a, with no stomatal clustering, indicating that the fraction is not bioactive. Scale bar, 20 μm. n = 6. f, HPLC chromatogram and bioassays of an independent batch of Stomagen peptides used for QCM analysis in direct comparison with non-folding mutant Stomagen peptides in Fig. 3c. Peaks 1 and 2 in UV chromatogram were collected and subjected for bioassays. Insets: confocal microscopy images of cotyledon epidermis from wild-type seedling grown a solution with peak 1 (bioactive) and peak 2 (non-active) for 5 days. Scale bars, 50 μm. n = 8 (peak 1); n = 6 (peak 2). g, HPLC chromatogram of purified, mutant Stomagen peptide in which all cysteine residues were substituted to serine residues (Stomagen_6C→S). The mutant Stomagen peptide yielded a single peak, which was subjected for bioassays followed by confocal microscopy (inset). No stomatal clustering was observed, indicating that non-folding Stomagen peptide is not bioactive, confirming the previous results18. Scale bar, 50 μm. n = 8 for each peptide treatment.

Extended Data Figure 9 Raw QCM recording data.

Shown are raw recording data of frequency shifts for representative QCM analysis using biosensor chips immobilized with ER(ΔK)–GFP and GFP (a, b, inset) after sequential injection of active Stomagen (a, c), mEPF2 (b), non-folding, inactive mutant Stomagen (c, inset), or LURE2 (d) in increasing concentrations. Bioactive Stomagen and inactive Stomagen experiments in c were performed side by side. Arrows indicate time of additional peptide application. Numbers of experiments performed for each analysis: Stomagen–ER, n = 3; Stomagen–TMM, n = 2; Stomagen–GFP, n = 3; mEPF2–ER, n = 2; mEPF2–TMM, n = 3; mEPF2–GFP, n = 2; inactive Stomagen_C6→S–ER, n = 3; and LURE2–ER, n = 2.

Extended Data Table 1 List of plasmids and primers used in this study
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Lee, J., Hnilova, M., Maes, M. et al. Competitive binding of antagonistic peptides fine-tunes stomatal patterning. Nature 522, 439–443 (2015). https://doi.org/10.1038/nature14561

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