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Destabilization of interaction between cytokinin signaling intermediates AHP1 and ARR4 modulates Arabidopsis development

Verma, V.; Sivaraman, J.; Srivastava, A.K.; Sadanandom, A.; Kumar, P.P.

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Authors

V. Verma

J. Sivaraman

A.K. Srivastava

P.P. Kumar



Abstract

Eukaryotic two-component signaling involves the His–Asp–His–Asp multistep phosphorelay (MSP). In Arabidopsis thaliana, cytokinin-mediated MSP signaling intermediates include histidine kinases (HKs), histidine phosphotransfer proteins (Hpts) and response regulators (RRs). The structure–function relationship of interaction between Hpt (e.g. AHP1) and RR (e.g. ARR4) is poorly understood. Using a homology model and yeast two-hybrid analysis, we identified key amino acids of ARR4 at the AHP1–ΔARR4(16–175) interaction interface. Mutating them in Arabidopsis (arr3,4,5,6,8,9 hextuple mutant background) and performing root length assays provided functional relevance, and coimmunoprecipitation (coIP) assay provided biochemical evidence for the interaction. The homology model mimics crystal structures of Hpt–RR complexes. Mutating selected interface residues of ARR4 either abolished or destabilized the interaction. D45A and Y96A mutations weakened interaction with AHP1, and exhibited weaker rescue of root elongation in the hextuple mutants. CoIP analysis using cytokinin-treated transgenic Arabidopsis seedlings provided biochemical evidence for weakened AHP1–ARR4 interaction. The relevance of the selected residues for the interaction was further validated in two independent pairs of Hpt–RR proteins from Arabidopsis and rice (Oryza sativa). Our data provide evidence of a link between Hpt–RR interaction affinity and regulation of downstream functions of RRs. This establishes a structure–function relationship for the final step of a eukaryotic MSP signal cascade.

Citation

Verma, V., Sivaraman, J., Srivastava, A., Sadanandom, A., & Kumar, P. (2015). Destabilization of interaction between cytokinin signaling intermediates AHP1 and ARR4 modulates Arabidopsis development. New Phytologist, 206(2), 726-737. https://doi.org/10.1111/nph.13297

Journal Article Type Article
Acceptance Date Nov 26, 2014
Publication Date Apr 1, 2015
Deposit Date Sep 9, 2015
Publicly Available Date Mar 28, 2024
Journal New Phytologist
Print ISSN 0028-646X
Electronic ISSN 1469-8137
Publisher Wiley
Peer Reviewed Peer Reviewed
Volume 206
Issue 2
Pages 726-737
DOI https://doi.org/10.1111/nph.13297

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Copyright Statement
This is the accepted version of the following article: Verma, V., Sivaraman, J., Srivastava, A.K., Sadanandom, A. & Kumar, P.P. (2015). Destabilization of interaction between cytokinin signaling intermediates AHP1 and ARR4 modulates Arabidopsis development. New Phytologist 206(2): 726-737., which has been published in final form at https://doi.org/10.1111/nph.13297. This article may be used for non-commercial purposes in accordance With Wiley Terms and Conditions for self-archiving.





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