Article | Published:

Competitive binding of antagonistic peptides fine-tunes stomatal patterning

Nature volume 522, pages 439443 (25 June 2015) | Download Citation


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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    & FGF signalling: diverse roles during early vertebrate embryogenesis. Development 137, 3731–3742 (2010)

  2. 2.

    & Diverse functions of plant peptides: entering a new phase. Plant Cell Physiol. 52, 1–4 (2011)

  3. 3.

    , , , & The secretory peptide gene EPF1 enforces the stomatal one-cell-spacing rule. Genes Dev. 21, 1720–1725 (2007)

  4. 4.

    et al. Epidermal cell density is auto-regulated via a secretory peptide, EPIDERMAL PATTERNING FACTOR 2 in Arabidopsis leaves. Plant Cell Physiol. 50, 1019–1031 (2009)

  5. 5.

    & The signaling peptide EPF2 controls asymmetric cell divisions during stomatal development. Curr. Biol. 19, 864–869 (2009)

  6. 6.

    et al. Direct interaction of ligand-receptor pairs specifying stomatal patterning. Genes Dev. 26, 126–136 (2012)

  7. 7.

    , , & Stomatal patterning and differentiation by synergistic interactions of receptor kinases. Science 309, 290–293 (2005)

  8. 8.

    & Control of stomatal distribution on the Arabidopsis leaf surface. Science 296, 1697–1700 (2002)

  9. 9.

    , , , & Stomatal development and patterning are regulated by environmentally responsive mitogen-activated protein kinases in Arabidopsis. Plant Cell 19, 63–73 (2007)

  10. 10.

    , & Arabidopsis stomatal initiation is controlled by MAPK-mediated regulation of the bHLH SPEECHLESS. Science 322, 1113–1116 (2008)

  11. 11.

    , , & Regulation of floral patterning and organ identity by Arabidopsis ERECTA-family receptor kinase genes. J. Exp. Bot. 64, 5323–5333 (2013)

  12. 12.

    , & Stomatal development and pattern controlled by a MAPKK kinase. Science 304, 1494–1497 (2004)

  13. 13.

    et al. Stomagen positively regulates stomatal density in Arabidopsis. Nature 463, 241–244 (2010)

  14. 14.

    et al. Stomatal density is controlled by a mesophyll-derived signaling molecule. Plant Cell Physiol. 51, 1–8 (2010)

  15. 15.

    , & The signalling peptide EPFL9 is a positive regulator of stomatal development. New Phytol. 186, 609–614 (2010)

  16. 16.

    , & The NMR structure of stomagen reveals the basis of stomatal density regulation by plant peptide hormones. Nat. Commun. 2, 512 (2011)

  17. 17.

    et al. Differential effects of the peptides Stomagen, EPF1 and EPF2 on activation of MAP kinase MPK6 and the SPCH protein level. Plant Cell Physiol. 54, 1253–1262 (2013)

  18. 18.

    , & Divergent regulation of stomatal initiation and patterning in organ and suborgan regions of the Arabidopsis mutants too many mouths and four lips. Planta 205, 522–530 (1998)

  19. 19.

    , & The bHLH protein, MUTE, controls differentiation of stomata and the hydathode pore in Arabidopsis. Plant Cell Physiol. 49, 934–943 (2008)

  20. 20.

    et al. Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells. Nature 458, 357–361 (2009)

  21. 21.

    & FLS2: An LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Mol. Cell 5, 1003–1011 (2000)

  22. 22.

    et al. Bacterial disease resistance in Arabidopsis through flagellin perception. Nature 428, 764–767 (2004)

  23. 23.

    et al. Small open reading frames associated with morphogenesis are hidden in plant genomes. Proc. Natl Acad. Sci. USA 110, 2395–2400 (2013)

  24. 24.

    & Regional specification of stomatal production by the putative ligand CHALLAH. Development 137, 447–455 (2010)

  25. 25.

    , & Generation of signaling specificity in Arabidopsis by spatially restricted buffering of ligand-receptor interactions. Plant Cell 23, 2864–2879 (2011)

  26. 26.

    et al. Regulation of inflorescence architecture by intertissue layer ligand-receptor communication between. Proc. Natl Acad. Sci. USA 109, 6337–6342 (2012)

  27. 27.

    , & Molecular mechanism for plant steroid receptor activation by somatic embryogenesis co-receptor kinases. Science 341, 889–892 (2013)

  28. 28.

    et al. Structural basis for flg22-induced activation of the Arabidopsis FLS2–BAK1 immune complex. Science 342, 624–628 (2013)

  29. 29.

    , & Dominant-negative receptor uncovers redundancy in the Arabidopsis ERECTA leucin-rich repeat receptor-like kinase signaling pathway that regulates organ shape. Plant Cell 15, 1095–1110 (2003)

  30. 30.

    & Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998)

  31. 31.

    , , & Molecular profiling of stomatal meristemoids reveals new component of asymmetric cell division and commonalities among stem cell populations in Arabidopsis. Plant Cell 23, 3260–3275 (2011)

  32. 32.

    , , & Termination of asymmetric cell division and differentiation of stomata. Nature 445, 501–505 (2007)

  33. 33.

    , , & An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J. 33, 949–956 (2003)

Download references


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.

Author information


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

    • Jin Suk Lee
    • , Ya-Chen Lisa Lin
    • , Soon-Ki Han
    • , Julian Avila
    •  & Keiko U. Torii
  2. Department of Biology, University of Washington, Seattle, Washington 98195, 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, Washington 98195, USA

    • Marketa Hnilova


  1. Search for Jin Suk Lee in:

  2. Search for Marketa Hnilova in:

  3. Search for Michal Maes in:

  4. Search for Ya-Chen Lisa Lin in:

  5. Search for Aarthi Putarjunan in:

  6. Search for Soon-Ki Han in:

  7. Search for Julian Avila in:

  8. Search for Keiko U. Torii in:


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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Keiko U. Torii.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains original uncropped scanned images of blots and gels.

About this article

Publication history





Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.