Journal of Steroid Biochemistry & Molecular Biology 91 (2004) 41–47 A mammalian steroid action inhibitor spironolactone retards plant growth by inhibition of brassinosteroid action and induces light-induced gene expression in the dark Tadao Asami a,∗ , Keimei Oh b , Yusuke Jikumaru a , Yukihisa Shimada c , Iriko Kaneko a , Takeshi Nakano a , Suguru Takatsuto d , Shozo Fujioka a,c , Shigeo Yoshida a,c a RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan b Department of Biotechnology, Akita Prefectural University, Akita, Akita 010-0195, Japan c RIKEN Plant Science Center, 1-7-22 Suehirocho, Tsurumi, Yokohama 230-0045, Japan d Department of Chemistry, Joetsu University of Education, Joetsu, Niigata 943-8512, Japan Received 1 August 2003; accepted 19 January 2004 Abstract We screened steroid derivatives and found that spironolactone, an inhibitor of both 17-hydroxysteroid dehydrogenase (17-HSD) and aldosterone receptor, is an inhibitor of phytohormone brassinosteroid (BR) action in plants. Under both dark and light growing conditions, spironolactone induced morphological changes in Arabidopsis, characteristic of brassinosteroid-deficient mutants. Spironolactone-treated plants were also nearly restored to the wild-type phenotype by treatment with additional BRs. In the spironolactone-treated Arabidop- sis, the CPD gene in the BR biosynthesis pathway was up-regulated, probably due to feedback regulation caused by BR-deficiency. Spironolactone-treated tobacco plants grown in the dark showed expression of light-regulated genes as was observed in the deficient mutant. These data suggest that spironolactone inhibits brassinosteroid action probably due to the blockage of biosynthesis and exerts its activity against plants. Thus, spironolactone, in conjunction with brassinosteroid-deficient mutants, can be used to clarify the function of BRs in plants and characterize mutants. The spironolactone action site was also investigated by feeding BR biosynthesis intermediates to Arabidopsis grown in the dark, and the results are discussed. © 2004 Elsevier Ltd. All rights reserved. Keywords: Brassinosteroid biosynthesis; Receptor; Inhibitor; Photomorphogenesis 1. Introduction The application of biologically active brassinosteroid (BR) homologues causes remarkable growth responses in plants, including stem elongation, pollen tube growth, leaf bending, leaf unrolling, root inhibition, proton pump acti- vation [1], promotion of ethylene production [2], tracheary element differentiation [3,4], and cell elongation [5]. The functions of endogenous BRs have been revealed by anal- ysis of several BR-deficient mutants in Arabidopsis, pea, tomato [6] and rice [7]. These mutants have been invaluable in determining the essential roles BRs have in plant growth and development; consequently, BRs have recently been recognized as a new class of phytohormones [6,8]. Abbreviations: BR, brassinosteroid; GA, gibberellin; BL, brassinolide; 3-HSD, 3-hydroxysteroid dehydrogenase; 17-HSD, 17-hydroxy- steroid dehydrogenase ∗ Corresponding author. Tel.: +81 48 467 9526; fax: +81 48 462 4674. E-mail address: tasami@postman.riken.go.jp (T. Asami). The use of specific inhibitors is an alternative way to de- termine the physiological functions of BRs. KM-01 is the first reported selective BR inhibitor, but it appears to be of limited use for investigating BR function in plants due to its very low activity when it is applied alone [9]. Re- cently, we reported the series of specific BR biosynthesis inhibitors and found that all of them target the conversions of 6-oxocampestanol to cathasterone catalyzed by the cy- tochrome P450 monooxygenases DWF4 [10–16]. Although BR biosynthesis inhibitors were proved to be very useful for investigating BR functions and gene expressions in plants [17–19] and isolating and characterizing mutants in which a component involved in BR signal transduction is altered [20–22], BR biosynthesis inhibitors, which target other en- zymes catalyzing reactions in BR biosynthesis pathway, are required. If we have two types of BR biosynthesis inhibitors targeting different enzymes, then we are readily able to iden- tify whether the mutant is altered in BR signal transduction or in inhibitor target enzymes. In this context, we started the 0960-0760/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2004.01.011