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

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.

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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.

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.

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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.

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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.

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Correspondence to Ning Zheng or Keiko U. Torii.

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The authors declare no competing interests.

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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.

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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.

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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

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