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SPEECHLESS integrates brassinosteroid and stomata signalling pathways

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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Figure 1: BRs control stomatal development.
Figure 2: SPCH is required for the effect of BRs on stomatal development and epidermal cell division in the hypocotyl.
Figure 3: BIN2 phosphorylates SPCH.
Figure 4: Effect of BRs on stomatal development in leaves and cotyledons.
Figure 5: BRs and the MAPK signalling pathways concertedly control SPCH activity to regulate stomatal development.

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References

  1. Bergmann, D. C. & Sack, F. D. Stomatal development. Annu. Rev. Plant Biol. 58, 163–181 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. Lampard, G. R., MacAlister, C. A. & Bergmann, D. C. Arabidopsis stomatal initiation is controlled by MAPK-mediated regulation of the bHLH SPEECHLESS. Science 322, 1113–1116 (2008).

    Article  CAS  PubMed  Google Scholar 

  3. Robinson, S. et al. Generation of spatial patterns through cell polarity switching. Science 333, 1436–1440 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Pillitteri, L. J., Peterson, K. M., Horst, R. J. & Torii, K. U. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 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  CAS  PubMed  Google Scholar 

  6. Ohashi-Ito, K. & Bergmann, D. C. Arabidopsis FAMA controls the final proliferation/differentiation switch during stomatal development. Plant Cell 18, 2493–2505 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. 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  CAS  PubMed  Google Scholar 

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

    Article  PubMed  PubMed Central  Google Scholar 

  9. Lampard, G. R., Lukowitz, W., Ellis, B. E. & Bergmann, D. C. Novel and expanded roles for MAPK signaling in Arabidopsis stomatal cell fate revealed by cell type-specific manipulations. Plant Cell 21, 3506–3517 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Bergmann, D. C., Lukowitz, W. & Somerville, C. R. Stomatal development and pattern controlled by a MAPKK kinase. Science 304, 1494–1497 (2004).

    Article  CAS  PubMed  Google Scholar 

  11. Wang, H., Ngwenyama, N., Liu, Y., Walker, J. C. & Zhang, S. Stomatal development and patterning are regulated by environmentally responsive mitogen-activated protein kinases in Arabidopsis. Plant Cell 19, 63–73 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Gudesblat, G. E. & Russinova, E. Plants grow on brassinosteroids. Curr. Opin. Plant Biol. 14, 530–537 (2011).

    Article  CAS  PubMed  Google Scholar 

  13. González-Garcı´a, M-P. et al. Brassinosteroids control meristem size by promoting cell cycle progression in Arabidopsis roots. Development 138, 849–859 (2011).

    Article  Google Scholar 

  14. Gonzalez, N. et al. Increased leaf size: different means to an end. Plant Physiol. 153, 1261–1279 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Hacham, Y. et al. Brassinosteroid perception in the epidermis controls root meristem size. Development 138, 839–848 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Kim, T-W. & Wang, Z-Y. Brassinosteroid signal transduction from receptor kinases to transcription factors. Annu. Rev. Plant Biol. 61, 681–704 (2010).

    Article  CAS  PubMed  Google Scholar 

  17. Szekeres, M. et al. Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and de-etiolation in Arabidopsis. Cell 85, 171–182 (1996).

    Article  CAS  PubMed  Google Scholar 

  18. Friedrichsen, D. M., Joazeiro, C. A. P., Li, J., Hunter, T. & Chory, J. Brassinosteroid-insensitive-1 is a ubiquitously expressed leucine-rich repeat receptor serine/threonine kinase. Plant Physiol. 123, 1247–1256 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wang, Z. Y., Seto, H., Fujioka, S., Yoshida, S. & Chory, J. BRI1 is a critical component of a plasma-membrane receptor for plant steroids. Nature 410, 380–383 (2001).

    Article  CAS  PubMed  Google Scholar 

  20. Asami, T. et al. Characterization of brassinazole, a triazole-type brassinosteroid biosynthesis inhibitor. Plant Physiol. 123, 93–100 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kuppusamy, K. T., Chen, A. Y. & Nemhauser, J. L. Steroids are required for epidermal cell fate establishment in Arabidopsis roots. Proc. Natl Acad. Sci. USA 106, 8073–8076 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Serna, L. Epidermal cell patterning and differentiation throughout the apical-basal axis of the seedling. J. Exp. Bot. 56, 1983–1989 (2005).

    Article  CAS  PubMed  Google Scholar 

  23. Berger, F., Linstead, P., Dolan, L. & Haseloff, J. Stomata patterning on the hypocotyl of Arabidopsis thaliana is controlled by genes involved in the control of root epidermis patterning. Dev. Biol. 194, 226–234 (1998).

    Article  CAS  PubMed  Google Scholar 

  24. Vert, G. & Chory, J. Downstream nuclear events in brassinosteroid signalling. Nature 441, 96–100 (2006).

    Article  CAS  PubMed  Google Scholar 

  25. Yan, Z., Zhao, J., Peng, P., Chihara, R. K. & Li, J. BIN2 functions redundantly with other Arabidopsis GSK3-like kinases to regulate brassinosteroid signaling. Plant Physiol. 150, 710–721 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Li, J. & Nam, K. H. Regulation of brassinosteroid signaling by a GSK3/SHAGGY-like kinase. Science 295, 1299–1301 (2002).

    CAS  PubMed  Google Scholar 

  27. Yin, Y. et al. BES1 accumulates in the nucleus in response to brassinosteroids to regulate gene expression and promote stem elongation. Cell 109, 181–191 (2002).

    Article  CAS  PubMed  Google Scholar 

  28. Wang, Z-Y. et al. Nuclear-localized BZR1 mediates brassinosteroid-induced growth and feedback suppression of brassinosteroid biosynthesis. Dev. Cell 2, 505–513 (2002).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 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 

  31. 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 

  32. 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  CAS  PubMed  Google Scholar 

  33. 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 

  34. Dong, J., MacAlister, C. A. & Bergmann, D. C. BASL controls asymmetric cell division in Arabidopsis. Cell 137, 1320–1330 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  36. Bhave, N. S. et al. TOO MANY MOUTHS promotes cell fate progression in stomatal development of Arabidopsis stems. Planta 229, 357–367 (2009).

    Article  CAS  PubMed  Google Scholar 

  37. De Rybel, B. et al. Chemical inhibition of a subset of Arabidopsis thaliana GSK3-like kinases activates brassinosteroid signaling. Chem. Biol. 16, 594–604 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Kim, T-W. et al. Brassinosteroid signal transduction from cell-surface receptor kinases to nuclear transcription factors. Nat. Cell Biol. 11, 1254–1260 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Rozhon, W., Mayerhofer, J., Petutschnig, E., Fujioka, S. & Jonak, C. ASKθ, a group-III Arabidopsis GSK3, functions in the brassinosteroid signalling pathway. Plant J. 62, 215–223 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Kim, T-W., Michniewicz, M., Bergmann, D. C. & Wang, Z-Y. Brassinosteroid regulates stomatal development by GSK3-mediated inhibition of a MAPK pathway. Nature 482, 419–422 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Karimi, M., Bleys, A., Vanderhaeghen, R. & Hilson, P. Building blocks for plant gene assembly. Plant Physiol. 145, 1183–1191 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Mazanek, M. et al. Titanium dioxide as a chemo-affinity solid phase in offline phosphopeptide chromatography prior to HPLC-MS/MS analysis. Nat. Protoc. 2, 1059–1069 (2007).

    Article  CAS  PubMed  Google Scholar 

  43. Mazanek, M. et al. A new acid mix enhances phosphopeptide enrichment on titanium- and zirconium dioxide for mapping of phosphorylation sites on protein complexes. J. Chromatogr. B 878, 515–524 (2010).

    Article  CAS  Google Scholar 

  44. Karlova, R. et al. Identification of in vitro phosphorylation sites in the Arabidopsis thaliana somatic embryogenesis receptor-like kinases. Proteomics 9, 368–379 (2009).

    Article  CAS  PubMed  Google Scholar 

  45. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotech. 26, 1367–1372 (2008).

    Article  CAS  Google Scholar 

  46. Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant Environment. J. Proteome Res. 10, 1794–1805 (2011).

    Article  CAS  PubMed  Google Scholar 

  47. Tingholm, T. E., Jørgensen, T. J., Jensen, O. N. & Larsen, M. R. Highly selective enrichment of phosphorylated peptides using titanium dioxide. Nat. Protoc. 1, 1929–1935 (2006).

    Article  Google Scholar 

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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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Correspondence to Gustavo E. Gudesblat or Eugenia Russinova.

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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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