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.

  • Article
  • Published:

Bipartite anchoring of SCREAM enforces stomatal initiation by coupling MAP kinases to SPEECHLESS

Nature Plants volume 5, pages 742–754 (2019)Cite this article

Subjects

Abstract

Cell fate in eukaryotes is controlled by mitogen-activated protein kinases (MAPKs) that translate external cues into cellular responses. In plants, two MAPKs—MPK3 and MPK6—regulate diverse processes of development, environmental response and immunity. However, the mechanism that bridges these shared signalling components with a specific target remains unresolved. Focusing on the development of stomata—epidermal valves that are essential for gas exchange and transpiration—here, we report that the basic helix-loop-helix protein SCREAM functions as a scaffold that recruits MPK3/6 to downregulate SPEECHLESS, a transcription factor that initiates stomatal cell lineages. SCREAM directly binds to MPK3/6 through an evolutionarily conserved, yet unconventional, bipartite motif. Mutations in this motif abrogate association, phosphorylation and degradation of SCREAM, unmask hidden non-redundancies between MPK3 and MPK6, and result in uncontrolled stomatal differentiation. Structural analyses of MPK6 with a resolution of 2.75 Å showed bipartite binding of SCREAM to MPK6 that is distinct from an upstream MAPKK. Our findings elucidate, at the atomic resolution, the mechanism that directly links extrinsic signals to transcriptional reprogramming during the establishment of stomatal cell fate, and highlight a unique substrate-binding mode adopted by plant MAPKs.

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

Fig. 1: SCRM functions as a scaffold to recruit MAPK to interact with SPCH.
Fig. 2: The evolutionarily conserved KiDoK motif of SCRM defines a direct MPK3/6 interaction surface.
Fig. 3: MPK3 and MPK6 exhibit different binding modes to the SCRM KiDoK motif to repress stomatal cell fate.
Fig. 4: Structure–function analyses of the SCRM KiDoK–MAPK interaction module.
Fig. 5: Direct MPK3/6 association is required for the phosphorylation and degradation of SCRM.
Fig. 6: A mechanism that enforces the initiation of stomatal cell lineages through the SCRM KiDoK–MAPK interaction module.

Similar content being viewed by others

Data availability

The PDB accession number for the MPK6ΔNt structure reported in this paper is 6DTL. All data generated and/or analysed during the current study are available from the corresponding authors on reasonable request.

References

  1. Pitzschke, A. Modes of MAPK substrate recognition and control. Trends Plant Sci. 20, 49–55 (2015).

    Article  CAS  Google Scholar 

  2. Meng, X. & Zhang, S. MAPK cascades in plant disease resistance signaling. Annu. Rev. Phytopathol. 51, 245–266 (2013).

    Article  CAS  Google Scholar 

  3. Tena, G., Boudsocq, M. & Sheen, J. Protein kinase signaling networks in plant innate immunity. Curr. Opin. Plant Biol. 14, 519–529 (2011).

    Article  CAS  Google Scholar 

  4. Xu, J. & Zhang, S. Mitogen-activated protein kinase cascades in signaling plant growth and development. Trends Plant Sci. 20, 56–64 (2015).

    Article  CAS  Google Scholar 

  5. Colcombet, J. & Hirt, H. Arabidopsis MAPKs: a complex signalling network involved in multiple biological processes. Biochem. J. 413, 217–226 (2008).

    Article  CAS  Google Scholar 

  6. Meng, X. et al. A MAPK cascade downstream of ERECTA receptor-like protein kinase regulates Arabidopsis inflorescence architecture by promoting localized cell proliferation. Plant Cell 24, 4948–4960 (2012).

    Article  CAS  Google Scholar 

  7. Wang, H. et al. Haplo-insufficiency of MPK3 in MPK6 mutant background uncovers a novel function of these two MAPKs in Arabidopsis ovule development. Plant Cell 20, 602–613 (2008).

    Article  CAS  Google Scholar 

  8. Hord, C. L. et al. Regulation of Arabidopsis early anther development by the mitogen-activated protein kinases, MPK3 and MPK6, and the ERECTA and related receptor-like kinases. Mol. Plant 1, 645–658 (2008).

    Article  CAS  Google Scholar 

  9. Bush, S. M. & Krysan, P. J. Mutational evidence that the Arabidopsis MAP kinase MPK6 is involved in anther, inflorescence, and embryo development. J. Exp. Bot. 58, 2181–2191 (2007).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  11. Wang, B. et al. Analysis of crystal structure of Arabidopsis MPK6 and generation of its mutants with higher activity. Sci. Rep. 6, e25646 (2016).

    Article  Google Scholar 

  12. Qi, X. & Torii, K. U. Hormonal and environmental signals guiding stomatal development. BMC Biol. 16, 21 (2018).

    Article  Google Scholar 

  13. Pillitteri, L. J. & Dong, J. Stomatal development in Arabidopsis. Arabid. Book 11, e0162 (2013).

    Article  Google Scholar 

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

    Article  Google Scholar 

  15. Lee, J. S. et al. Competitive binding of antagonistic peptides fine-tunes stomatal patterning. Nature 522, 439–443 (2015).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. 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  Google Scholar 

  18. Zhang, Y., Wang, P., Shao, W., Zhu, J. K. & Dong, J. The BASL polarity protein controls a MAPK signaling feedback loop in asymmetric cell division. Dev. Cell 33, 136–149 (2015).

    Article  Google Scholar 

  19. Zhang, Y., Guo, X. & Dong, J. Phosphorylation of the polarity protein BASL differentiates asymmetric cell fate through MAPKs and SPCH. Curr. Biol. 26, 2957–2965 (2016).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  21. Kanaoka, M. M. et al. SCREAM/ICE1 and SCREAM2 specify three cell-state transitional steps leading to Arabidopsis stomatal differentiation. Plant Cell 20, 1775–1785 (2008).

    Article  CAS  Google Scholar 

  22. 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  Google Scholar 

  23. Horst, R. J. et al. Molecular framework of a regulatory circuit initiating two-dimensional spatial patterning of stomatal lineage. PLoS Genet. 11, e1005374 (2015).

    Article  Google Scholar 

  24. Walter, M. et al. Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J. 40, 428–438 (2004).

    Article  CAS  Google Scholar 

  25. Qu, X., Peterson, K. M. & Torii, K. U. Stomatal development in time: the past and the future. Curr. Opin. Genet. Dev. 45, 1–9 (2017).

    Article  CAS  Google Scholar 

  26. Abdiche, Y., Malashock, D., Pinkerton, A. & Pons, J. Determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor, the Octet. Anal. Biochem. 377, 209–217 (2008).

    Article  CAS  Google Scholar 

  27. Tanoue, T., Adachi, M., Moriguchi, T. & Nishida, E. A conserved docking motif in MAP kinases common to substrates, activators and regulators. Nat. Cell Biol. 2, 110–116 (2000).

    Article  CAS  Google Scholar 

  28. Garai, A. et al. Specificity of linear motifs that bind to a common mitogen-activated protein kinase docking groove. Sci. Signal. 5, RA74 (2012).

    Article  Google Scholar 

  29. Zeke, A. et al. Systematic discovery of linear binding motifs targeting an ancient protein interaction surface on MAP kinases. Mol. Syst. Biol. 11, 837 (2015).

    Article  Google Scholar 

  30. Lee, J. S., Huh, K. W., Bhargava, A. & Ellis, B. E. Comprehensive analysis of protein-protein interactions between Arabidopsis MAPKs and MAPK kinases helps define potential MAPK signalling modules. Plant Signal. Behav. 3, 1037–1041 (2008).

    Article  Google Scholar 

  31. Li, H. et al. MPK3-and MPK6-mediated ICE1 phosphorylation negatively regulates ICE1 stability and freezing tolerance in Arabidopsis. Dev. Cell 43, 630–642 (2017).

    Article  CAS  Google Scholar 

  32. Zhao, C. Z. et al. MAP kinase cascades regulate the cold response by modulating ICE1 protein stability. Dev. Cell 43, 618–629 (2017).

    Article  CAS  Google Scholar 

  33. Yang, K. Y., Liu, Y. & Zhang, S. Activation of a mitogen-activated protein kinase pathway is involved in disease resistance in tobacco. Proc. Natl Acad. Sci. USA 98, 741–746 (2001).

    Article  CAS  Google Scholar 

  34. Raissig, M. T., Abrash, E., Bettadapur, A., Vogel, J. P. & Bergmann, D. C. Grasses use an alternatively wired bHLH transcription factor network to establish stomatal identity. Proc. Natl Acad. Sci. USA 113, 8326–8331 (2016).

    Article  CAS  Google Scholar 

  35. Dong, C. H., Agarwal, M., Zhang, Y., Xie, Q. & Zhu, J. K. The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1. Proc. Natl Acad. Sci. USA 103, 8281–8286 (2006).

    Article  CAS  Google Scholar 

  36. Miura, K. et al. SIZ1-mediated sumoylation of ICE1 controls CBF3/DREB1A expression and freezing tolerance in Arabidopsis. Plant Cell 19, 1403–1414 (2007).

    Article  CAS  Google Scholar 

  37. Ding, Y. et al. OST1 kinase modulates freezing tolerance by enhancing ICE1 stability in Arabidopsis. Dev. Cell 32, 278–289 (2015).

    Article  CAS  Google Scholar 

  38. Lee, J. H., Jung, J. H. & Park, C. M. Light inhibits COP1-mediated degradation of ICE transcription factors to induce stomatal development in Arabidopsis. Plant Cell 29, 2817–2830 (2017).

    Article  CAS  Google Scholar 

  39. Lee, J. H., Jung, J. H. & Park, C. M. Inducer of cbf expression 1 integrates cold signals into FLOWERING LOCUS C-mediated flowering pathways in Arabidopsis. Plant J. 84, 29–40 (2015).

    Article  CAS  Google Scholar 

  40. Chinnusamy, V. et al. ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis. Genes Dev. 17, 1043–1054 (2003).

    Article  CAS  Google Scholar 

  41. Denay, G. et al. Endosperm breakdown in Arabidopsis requires heterodimers of the basic helix-loop-helix proteins ZHOUPI and INDUCER OF CBP EXPRESSION 1. Development 141, 1222–1227 (2014).

    Article  CAS  Google Scholar 

  42. Tanoue, T. & Nishida, E. Molecular recognitions in the MAP kinase cascades. Cell. Signal. 15, 455–462 (2003).

    Article  CAS  Google Scholar 

  43. Schweighofer, A. et al. The PP2C-type phosphatase AP2C1, which negatively regulates MPK4 and MPK6, modulates innate immunity, jasmonic acid, and ethylene levels in Arabidopsis. Plant Cell 19, 2213–2224 (2007).

    Article  CAS  Google Scholar 

  44. 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  Google Scholar 

  45. Houbaert, A. et al. POLAR-guided signalling complex assembly and localization drive asymmetric cell division. Nature 563, 574–578 (2018).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  47. Vojtek, A. B. & Hollenberg, S. M. Ras-Raf interaction: two-hybrid analysis. Methods Enzymol. 255, 331–342 (1995).

    Article  CAS  Google Scholar 

  48. Bartel, P. L., Chien, C.-T., Sternglanz, R. & Fields, S. in Cellular Interactions in Development: A Practical Approach (Ed. Hartley, D. A.) 153–179 (Oxford Univ. Press, 1993).

  49. FromontRacine, M., Rain, J. C. & Legrain, P. Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens. Nat. Genet. 16, 277–282 (1997).

    Article  CAS  Google Scholar 

  50. Formstecher, E. et al. Protein interaction mapping: a Drosophila case study. Genome Res. 15, 376–384 (2005).

    Article  CAS  Google Scholar 

  51. Qi, X. Autocrine regulation of stomatal differentiation potential by EPF1 and ERECTA-LIKE1 ligand-receptor signaling. eLife 6, e24102 (2017).

    Article  Google Scholar 

  52. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  53. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  54. Emsley, P., Lohkamp, B., ScottW. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr 66, 486–501 (2010).

    CAS  Google Scholar 

  55. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  56. Sali, A. & Blundell, T. L. Comparative protein modeling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993).

    Article  CAS  Google Scholar 

  57. Raveh, B., London, N., Zimmerman, L. & Schueler-Furman, O. Rosetta FlexPepDock ab-initio: simultaneous folding, docking and refinement of peptides onto their receptors. PLoS ONE 6, e18934 (2011).

    Article  CAS  Google Scholar 

  58. Simons, K. T., Bonneau, R., Ruczinski, I. & Baker, D. Ab initio protein structure prediction of CASP III targets using ROSETTA. Proteins 3, 171–176 (1999).

    Article  Google Scholar 

  59. Leaver-Fay, A. et al. ROSETTA3: An object-oriented software suite for the simulation and design of macromolecules. Methods Enzymol. 487, 545–574 (2011).

    Article  CAS  Google Scholar 

  60. Pettersen, E. F. et al. UCSF Chimera—A visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  Google Scholar 

  61. Sanner, M. F., Olson, A. J. & Spehner, J. C. Reduced surface: an efficient way to compute molecular surfaces. Biopolymers 38, 305–320 (1996).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank S. Zhang for his gifts of Arabidopsis mpk3, mpk6 and inducible NtMEK2DD overexpression line, J. Dong for thoughtful discussions and T. Hinds for helping to set up the in vitro interaction assays using the Octet system. This work was supported by the US National Science Foundation (MCB-0855659) and the Gordon and Betty Moore Foundation (GBMF-3035) to K.U.T., and Grants in Aid for Scientific Research on Innovative Areas (17H06476), Japan Society for Promotion of Sciences (JSPS) (26119006 and 15K21711) to F.T. N.Z. and K.U.T. are Howard Hughes Medical Institute (HHMI) Investigators.

Author information

Authors and Affiliations

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

    Aarthi Putarjunan, Alex K. Hofstetter, Ning Zheng & Keiko U. Torii

  2. Department of Biology, University of Washington, Seattle, WA, USA

    Aarthi Putarjunan, Amanda L. Rychel & Keiko U. Torii

  3. Department of Pharmacology, University of Washington, Seattle, WA, USA

    Jim Ruble, Xiaobo Tang & Ning Zheng

  4. Institute of Transformative Bio-Molecules, Nagoya University, Nagoya, Japan

    Ashutosh Srivastava, Florence Tama & Keiko U. Torii

  5. Shanghai Center for Plant Stress Biology and Center of Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China

    Chunzhao Zhao & Jian-Kang Zhu

  6. Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN, USA

    Chunzhao Zhao & Jian-Kang Zhu

  7. Department of Physics, Graduate School of Science, Nagoya University, Nagoya, Japan

    Florence Tama

  8. Computational Structural Biology Team, Center for Computational Science, Kobe, Japan

    Florence Tama

Authors
  1. Aarthi Putarjunan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jim Ruble
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ashutosh Srivastava
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chunzhao Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Amanda L. Rychel
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Alex K. Hofstetter
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xiaobo Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jian-Kang Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Florence Tama
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Ning Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Keiko U. Torii
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.U.T. conceived the project; A.P. and K.U.T. conceptualized the study; A.P., A.L.R., N.Z. and K.U.T. designed the experiments; A.P., J.R., C.Z., A.L.R., A.K.H. and X.T. performed the experiments; J.R., A.S., F.T. and N.Z. performed the structural analysis and modelling; A.P., J.R., A.S., A.L.R., N.Z. and K.U.T. carried out the formal analysis; A.P., J.R., A.S. and K.U.T. visualized the data; A.P. and K.U.T wrote the original draft; A.P., J.R., A.S., C.Z., A.L.R., A.K.H, F.T., N.Z. and K.U.T reviewed and edited the paper; N.Z. and K.U.T. supervised the project; K.U.T. oversaw project administration; and J.-K.Z., F.T., N.Z. and K.U.T acquired funding.

Corresponding authors

Correspondence to Ning Zheng or Keiko U. Torii.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information: Nature Plants thanks Laszlo Bogre, Shuqun Zhang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–8 and Supplementary Table 2.

Reporting Summary

Supplementary Table 1

Y2H screen and interactors.

Supplementary Table 3

List of plasmids and primers.

Supplementary Table 4

Raw data of individual BLI experiments.

Supplementary Table 5

Exact P values for statistical analysis.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Putarjunan, A., Ruble, J., Srivastava, A. et al. Bipartite anchoring of SCREAM enforces stomatal initiation by coupling MAP kinases to SPEECHLESS. Nat. Plants 5, 742–754 (2019). https://doi.org/10.1038/s41477-019-0440-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41477-019-0440-x

This article is cited by

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