Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Stomagen positively regulates stomatal density in Arabidopsis

Nature volume 463, pages 241–244 (2010)Cite this article

Abstract

Stomata in the epidermal tissues of leaves are valves through which passes CO2, and as such they influence the global carbon cycle1. The two-dimensional pattern and density of stomata in the leaf epidermis are genetically and environmentally regulated to optimize gas exchange2. Two putative intercellular signalling factors, EPF1 and EPF2, function as negative regulators of stomatal development in Arabidopsis, possibly by interacting with the receptor-like protein TMM3,4,5,6. One or more positive intercellular signalling factors are assumed to be involved in stomatal development, but their identities are unknown7. Here we show that a novel secretory peptide, which we designate as stomagen, is a positive intercellular signalling factor that is conserved among vascular plants. Stomagen is a 45-amino--rich peptide that is generated from a 102-amino-acid precursor protein designated as STOMAGEN. Both an in planta analysis and a semi-in-vitro analysis with recombinant and chemically synthesized stomagen peptides showed that stomagen has stomata-inducing activity in a dose-dependent manner. A genetic analysis showed that TMM is epistatic to STOMAGEN (At4g12970), suggesting that stomatal development is finely regulated by competitive binding of positive and negative regulators to the same receptor. Notably, STOMAGEN is expressed in inner tissues (the mesophyll) of immature leaves but not in the epidermal tissues where stomata develop. This study provides evidence of a mesophyll-derived positive regulator of stomatal density. Our findings provide a conceptual advancement in understanding stomatal development: inner photosynthetic tissues optimize their function by regulating stomatal density in the epidermis for efficient uptake of CO2.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: STOMAGEN positively regulates stomatal development.
Figure 2: Stomagen is expressed in inner tissues of immature organs and secreted out of the cells to mediate inter-tissue signalling.
Figure 3: Stomagen, a 45-amino-acid cysteine-rich region of the STOMAGEN protein, is a mobile stomata-inducing factor.
Figure 4: STOMAGEN regulates stomatal density TMM -dependently in combination with the negative regulators EPF1 and EPF2.

Similar content being viewed by others

References

  1. Hetherington, A. M. & Woodward, F. I. The role of stomata in sensing and driving environmental change. Nature 424, 901–908 (2003)

    Article  ADS  CAS  PubMed  Google Scholar 

  2. Nadeau, J. A. & Sack, F. D. Stomatal development in Arabidopsis . In The Arabidopsis Book (eds Someville, C. & Meyerowitz, E) 10.1199/tab.0066 (American Society of Plant Biologists, 2002)

  3. Nadeau, J. A. & Sack, F. D. Control of stomatal distribution on the Arabidopsis leaf surface. Science 296, 1697–1700 (2002)

    Article  ADS  CAS  PubMed  Google Scholar 

  4. Hara, K., Kajita, R., Torii, K. U., Bergmann, D. C. & Kakimoto, T. The secretory peptide gene EPF1 enforces the stomatal one-cell-spacing rules. Genes Dev. 21, 1720–1725 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Hunt, L. & Gray, J. E. The signaling peptide EPF2 controls asymmetric cell divisions during stomatal development. Curr. Biol. 19, 864–869 (2009)

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  7. Bergmann, D. C. Integrating signals in stomatal development. Curr. Opin. Plant Biol. 7, 26–32 (2004)

    Article  CAS  PubMed  Google Scholar 

  8. Shpak, E. D., McAbee, J. M., Pillitteri, L. J. & Torii, K. U. Stomatal patterning and differentiation by synergistic interactions of receptor kinases. Science 309, 290–293 (2005)

    Article  ADS  CAS  PubMed  Google Scholar 

  9. Berger, D. & Altmann, T. A subtilisin-like serine protease involved in the regulation of stomatal density and distribution in Arabidopsis thaliana . Genes Dev. 14, 1119–1131 (2000)

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Nadeau, J. A. Stomatal development: new signals and fate determinants. Curr. Opin. Plant Biol. 12, 29–35 (2009)

    Article  CAS  PubMed  Google Scholar 

  11. Obayashi, T., Hayashi, S., Saeki, M., Ohta, H. & Kinoshita, K. ATTED-II provides coexpressed gene networks for Arabidopsis . Nucleic Acids Res. 37, D987–D991 (2009)

    Article  CAS  PubMed  Google Scholar 

  12. Sachs, T. The development of spacing patterns in the leaf epidermis. In Clonal Basis of Development 161–182 (Academic Press, 1987)

    Google Scholar 

  13. MacAlister, C. A., Ohashi-Ito, K. & Bergmann, D. C. Transcription factor control of asymmetric cell divisions that establish the stomatal lineage. Nature 445, 537–540 (2007)

    Article  ADS  CAS  PubMed  Google Scholar 

  14. Pillitteri, L. J., Sloan, D. B., Bogenschutz, N. L. & Torii, K. U. Termination of asymmetric cell division and differentiation of stomata. Nature 445, 501–505 (2007)

    Article  ADS  CAS  PubMed  Google Scholar 

  15. Serna, L., Torres-Contreras, J. & Fenoll, C. Specification of stomatal fate in Arabidopsis: evidences for cellular interactions. New Phytol. 153, 399–404 (2002)

    Article  CAS  PubMed  Google Scholar 

  16. Geisler, M., Yang, M. & Sack, F. D. 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)

    Article  CAS  PubMed  Google Scholar 

  17. Geisler, M., Nadeau, J. & Sack, F. D. Oriented asymmetric divisions that generate the stomatal spacing pattern in Arabidopsis are disrupted by the too many mouths mutation. Plant Cell 12, 2075–2086 (2000)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. von Grolla, U., Bergera, D. & Altmann, T. The subtilisin-like serine protease SDD1 mediates cell-to-cell signaling during Arabidopsis stomatal development. Plant Cell. 14, 1527–1539 (2002)

    Article  Google Scholar 

  19. Schwab, R., Ossowski, S., Riester, M., Warthmann, N. & Weigel, D. Highly specific gene silencing by artificial microRNAs in Arabidopsis . Plant Cell 18, 1121–1133 (2006)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Dohi, K., Tamai, A. & Mori, M. Insertion in the coding region of the movement protein improves stability of the plasmid encoding a tomato mosaic virus-based expression vector. Arch. Virol. 153, 1667–1675 (2008)

    Article  CAS  PubMed  Google Scholar 

  21. Kono, A. et al. The Arabidopsis D-type cyclin CYCD4 controls cell division in the stomatal lineage of the hypocotyl epidermis. Plant Cell 19, 1265–1277 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Hara-Nishimura, I., Takeuchi, Y., Inoue, K. & Nishimura, M. Vesicle transport and processing of the precursor to 2S albumin in pumpkin. Plant J. 4, 793–800 (1993)

    Article  Google Scholar 

  23. Shimada, T. et al. AtVPS29, a putative component of a retromer complex, is required for the efficient sorting of seed storage proteins. Plant Cell Physiol. 47, 1187–1194 (2006)

    Article  CAS  PubMed  Google Scholar 

  24. Kunieda, T. et al. NAC family proteins NARS1/NAC2 and NARS2/NAM in the outer integument regulate embryogenesis in Arabidopsis . Plant Cell 20, 2631–2642 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔC(T) method. Methods 25, 402–408 (2001)

    Article  CAS  PubMed  Google Scholar 

  26. Kinoshita, T. et al. Vacuolar processing enzyme is up-regulated in the lytic vacuoles of vegetative tissues during senescence and under various stressed conditions. Plant J. 19, 43–53 (1999)

    Article  CAS  PubMed  Google Scholar 

  27. Jensen, O. N., Podtelejnikov, A. & Mann, M. Delayed extraction improves specificity in database searches by matrix-assisted laser desorption/ionization peptide maps. Rapid Commun. Mass Spectrom. 10, 1371–1378 (1996)

    Article  ADS  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank T. Nakagawa for donating the TMM::GFP seeds; K. Yamada for his technical support of quantitative PCR; and Y. Nakata for her technical support of in situ hybridization. We thank J. Raymond for reading and editing the manuscript. We thank the ABRC for providing seeds of Arabidopsis T-DNA insertion lines. This work was supported by Grants-in-Aid for Scientific Research (no. 16085203 and 17107002) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, by the Global Center of Excellence Program ‘Formation of a Strategic Base for Biodiversity and Evolutionary Research: from Genome to Ecosystem’ of MEXT, and by Japan Science and Technology Agency (JST).

Author Contributions T.S. performed in silico screening to identify STOMAGEN. S.S.S. established the STOMAGEN-RNAi lines, performed genetic interaction experiments and other experiments including antibody production and in situ hybridization. S.S.S. also purified recombinant stomagen, refolded chemically synthesized stomagen, and evaluated their stomata inducing activities. Y.I. developed the STOMAGEN-OX and pSTOMAGEN::GUS lines. A.T. and M.M. produced stomagen in tobacco-cultured cells by using their ToMV-expression system. K.O. conducted MS analysis of STOMAGEN–Venus. I.H.-N. and T.S. designed this project and supervised. S.S.S., T.S. and I.H.-N. wrote the paper with input from co-authors.

Author information

Author notes

  1. Shigeo S. Sugano and Tomoo Shimada: These authors contributed equally to this work.

Authors and Affiliations

  1. Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan ,

    Shigeo S. Sugano, Tomoo Shimada, Yu Imai & Ikuko Hara-Nishimura

  2. Innovative Drug Research Laboratories, Kyowa Hakko Kirin Co., Ltd., Taksasaki 370-1295, Japan

    Katsuya Okawa

  3. Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi-machi, Ishikawa 921-8836, Japan ,

    Atsushi Tamai & Masashi Mori

Authors
  1. Shigeo S. Sugano
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tomoo Shimada
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yu Imai
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Katsuya Okawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Atsushi Tamai
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Masashi Mori
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ikuko Hara-Nishimura
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ikuko Hara-Nishimura.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-10 with Legends and Supplementary Tables 1-2. (PDF 4673 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sugano, S., Shimada, T., Imai, Y. et al. Stomagen positively regulates stomatal density in Arabidopsis. Nature 463, 241–244 (2010). https://doi.org/10.1038/nature08682

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature08682

This article is cited by

Comments

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.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing