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

KEY MESSAGE: 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.

Heat shock protein 90 acts in brassinosteroid signaling through interaction with BES1/BZR1 transcription factor.

Brassinosteroids (BRs), a class of phytohormones, control various physiological and developmental processes in plants. Two highly homologous transcription factors, brassinosteroid insensitive 1-EMS-SUPRESSOR 1 (BES1) and brassinazole resistant 1 (BZR1), act downstream of BR signaling to control several thousands of putative target genes. We reported previously that BES1 forms a complex with a molecular chaperone: heat shock protein 90 (HSP90). This study demonstrates that the amino-terminal and central parts of BES1 are responsible for its physical interaction with HSP90.3 in vitro. Additionally, we present evidence that BZR1 is a novel HSP90 partner aside from two BR signaling components previously identified as its clients: BES1 and brassinosteroid insensitive 2 (BIN2). Furthermore, geldanamycin, an inhibitor of ATPase activity in HSP90, caused BES1 hyperphosphorylation and disrupted the expression of BR-responsive genes. Considered together, our results imply that HSP90 takes a part in BR-mediated gene expression through complex formation with two major transcription factors.