Abstract
Stomatal formation is regulated by multiple developmental and environmental signals, but how these signals are integrated to control this process is not fully understood1. In Arabidopsis thaliana, the basic helix-loop-helix transcription factor SPEECHLESS (SPCH) regulates the entry, amplifying and spacing divisions that occur during stomatal lineage development. SPCH activity is negatively regulated by mitogen-activated protein kinase (MAPK)-mediated phosphorylation2. Here, we show that in addition to MAPKs, SPCH activity is also modulated by brassinosteroid (BR) signalling. The GSK3/SHAGGY-like kinase BIN2 (BR INSENSITIVE2) phosphorylates residues overlapping those targeted by the MAPKs, as well as four residues in the amino-terminal region of the protein outside the MAPK target domain. These phosphorylation events antagonize SPCH activity and limit epidermal cell proliferation. Conversely, inhibition of BIN2 activity in vivo stabilizes SPCH and triggers excessive stomatal and non-stomatal cell formation. We demonstrate that through phosphorylation inputs from both MAPKs and BIN2, SPCH serves as an integration node for stomata and BR signalling pathways to control stomatal development in Arabidopsis.
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Acknowledgements
We thank D. Bergmann (Stanford University, USA), K. Torii (University of Washington, USA), F. Sack (University of British Columbia, Canada), G. Vert (CNRS, France), S. Mora-Garcı´a (FIL, Argentina), A. I. Caño-Delgado (CSIC-IRTA-UAB, Spain), H-Q. Yang (Chinese Academy of Sciences, China) and T. Kakimoto (Osaka University, Japan) for providing materials; K. Mechtler and N. Li for help in mass spectrometry; E. Mylle for technical assistance; and M. De Cock, A. Bleys, G. Van Isterdael and K. Van Lierde for help in preparing the manuscript. This work is supported by the Marie-Curie Initial Training Network ‘BRAVISSIMO’ (grant no. PITN-GA-2008-215118), the Research Foundation-Flanders (grant no. G.0065.08), the Agency for Innovation by Science and Technology (‘Strategisch Basisonderzoek’ grant no. 60839), the Centre for BioSystems Genomics Proteomics project CBSG2012-AA6 (S.d.V.) and the Austrian Academy of Sciences (J.M. and C.J.). G.E.G. and M.Z. are indebted to the Belgian Science Policy Office (BELSPO) and the Agency for Innovation by Science and Technology for a postdoctoral fellowship, respectively. G.E.G. is a Career Investigator of the Consejo Nacional de Investigaciones Cientı´ficas y Técnicas.
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G.E.G. and E.R. conceived the project and designed experiments. G.E.G., C.B. and I.V. performed microscopy experiments. G.E.G., J.S-P., C.B. and I.V. did DNA manipulations. J.S-P. expressed proteins in bacteria; J.S-P. and C.B performed SPCH immunoprecipitation experiments. M.Z. segregated and characterized the bin2-3, atsk22 and atsk23 mutants. C.J. and J.M. designed and performed in vitro phosphorylation assays and subsequent mass spectrometry analyses. J.S-P., S.B. W.v.D. and S.d.V. did in vivo mass spectrometry analysis. G.E.G. and E.R. wrote the manuscript and J.S-P., M.Z., S.d.V., C.B. and C.J. revised it.
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Gudesblat, G., Schneider-Pizoń, J., Betti, C. et al. SPEECHLESS integrates brassinosteroid and stomata signalling pathways. Nat Cell Biol 14, 548–554 (2012). https://doi.org/10.1038/ncb2471
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DOI: https://doi.org/10.1038/ncb2471
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