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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