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Molecular evidence of the involvement of heat shock protein 90 in brassinosteroid signaling in Arabidopsis T87 cultured cells

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Abstract

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A closer association of HSP90s with brassinosteroid signaling is suggested by the brassinosteroid-triggered formation of an HSP90-containing macromolecular complex and the direct interaction between HSP90.3 and BES1.

Abstract

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone that is reportedly involved in the proper folding, stabilization, intracellular trafficking, maintenance and degradation of numerous proteins, as well as the facilitation of cellular signaling in various organisms including plants. Brassinosteroids (BRs), a class of unique steroidal hormones, play crucial roles in plant growth and development. The interaction between HSP90 proteins and BR action has been poorly understood. Here, we present molecular evidence suggesting that HSP90 proteins have a function(s) in BR signal transduction. First, blue native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis linked immunoblotting demonstrated that a bioactive BR, brassinolide (BL), promotes the formation of some HSP90-containing macromolecular complexes with molecular weight more than 480 kDa in Arabidopsis T87 cultured cells. Second, HSP90.3, one of seven Arabidopsis HSP90 family proteins, was observed to interact in vitro with BRI1-EMS-SUPPRESSOR 1 (BES1), a transcription factor acting in BR signaling. Geldanamycin, an inhibitor of ATPase activity in HSP90, not only diminished HSP90.3 interaction with BES1 in vitro, but also suppressed BL-induced down-regulation of two BR biosynthesis genes, CONSTITUTIVE PHOTHOMORPHOGENESIS AND DWARFISM and DWARF4 in vivo. The results suggest the involvement of the HSP90/BES1 heterocomplexes in BR signaling-mediated feedback control in BR contents. Together, our results provide important clues to elucidate HSP90s’ functions in the BR signaling pathway in Arabidopsis.

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Acknowledgments

We thank Dr. Riichiro Yoshida at the Faculty of Agriculture of Kagoshima University for their technical assistance and valuable advice on the generation of transgenic cells and the real-time PCR analysis. We are grateful to Dr. Keiichiro Nemoto and Dr. Hirotaka Takahashi at Ehime University and Dr. Yuki Yanagawa at RIKEN for their technical advice regarding the wheat germ cell-free protein expression system and AlphaScreen assay. We also thank Dr. Yasuo Niwa at the Graduate School of Integrated Pharmaceutical and Nutritional Sciences of the University of Shizuoka for providing the pUC18 plasmid containing CaMV 35S::sGFP(S65T)::Nos-T.

Conflict of interest

The authors declare that they have no conflicts of interest.

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Authors and Affiliations

  1. The United Graduate School of Agricultural Sciences, Kagoshima University, 1-21-24 Korimoto, Kagoshima, 890-0065, Japan

    Tomoaki Shigeta, Shigehisa Okamoto, Tomoaki Matsuo & Yasushi Sugimoto

  2. Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima, 890-0065, Japan

    Yuichi Zaizen

  3. Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan

    Tadao Asami

  4. Kihara Institute for Biological Research, Yokohama City University, 641-12 Maioka-cho, Totsuka-ku, Yokohama, 244-0813, Japan

    Shigeo Yoshida

  5. Department of Food Sciences and Nutritional Health, Kyoto Prefectural University, Shimogamo-Hangi-cho, Sakyo-ku, Kyoto, 606-8522, Japan

    Yasushi Nakamura

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  1. Tomoaki Shigeta
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  2. Yuichi Zaizen
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Correspondence to Yasushi Sugimoto.

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Communicated by F. Sato.

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Shigeta, T., Zaizen, Y., Asami, T. et al. Molecular evidence of the involvement of heat shock protein 90 in brassinosteroid signaling in Arabidopsis T87 cultured cells. Plant Cell Rep 33, 499–510 (2014). https://doi.org/10.1007/s00299-013-1550-y

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  • DOI: https://doi.org/10.1007/s00299-013-1550-y

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