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Stomagen positively regulates stomatal density in Arabidopsis

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.

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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.

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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.

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Correspondence to Ikuko Hara-Nishimura.

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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

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