Tomato cytochrome P450 CYP734A7 functions in brassinosteroid catabolism Toshiyuki Ohnishi a , Takahito Nomura b,1 , Bunta Watanabe a,2 , Daisaku Ohta c , Takao Yokota b , Hisashi Miyagawa d , Kanzo Sakata a , Masaharu Mizutani a, * a Institute for Chemical Research, Kyoto University, Gokasyo, Uji, Kyoto 611-0011, Japan b Department of Bioscience, Teikyo University, Utsunomiya 320-8551, Japan c College of Agriculture, Osaka Prefecture University, Osaka 599-8531, Japan d Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan Received 27 January 2006; received in revised form 28 April 2006 Available online 25 July 2006 Abstract Several cytochrome P450 monooxygenases (P450s) catalyze essential oxidative reactions in brassinosteroid (BR) biosynthesis as well as in BR catabolism; however, only limited information exists on the P450s involved in the BR catabolic pathway. Here, we report the characterization of two P450 mRNAs, CYP734A7 and CYP734A8, from Lycopersicon esculentum. These P450s show high homology with Arabidopsis CYP734A1/BAS1 (formerly CYP72B1), which inactivates BRs via C-26 hydroxylation. Transgenic tobacco plants that constitutively overexpressed CYP734A7 showed an extreme dwarf phenotype similar to BR deficiency. Quantitative gas chromatogra- phy–mass spectrometry analysis of endogenous BRs in the transgenic plants showed that the levels of castasterone and 6-deoxocastas- terone significantly decreased in comparison with those in wild-type plants. By measuring the Type I substrate-binding spectra using recombinant CYP734A7, the dissociation constants for castasterone, brassinolide, and 6-deoxocastasterone were determined to be 6.7, 12, and 12 lM, respectively. In an in vitro assay, CYP734A7 was confirmed to metabolize castasterone to 26-hydroxycastasterone. In addition, 28-norcastasterone and brassinolide were converted to the hydroxylated products. The expression of CYP734A7 and CYP734A8 genes in tomato seedlings was upregulated by exogenous application of bioactive BRs. These results indicated that CYP734A7 is a C-26 hydroxylase of BRs and is likely involved in BR catabolism in tomato. The presence of the CYP734A subfamily in various plant species suggests that oxidative inactivation of BRs by these proteins is a widespread phenomenon in plants. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Lycopersicon esculentum; Solanaceae; Brassinosteroid; Brassinolide; Castasterone; Brassinosteroid catabolism; Cytochrome P450; CYP734A7; CYP734A8 1. Introduction Brassinosteroids (BR) are steroidal phytohormones that are ubiquitously distributed throughout the plant kingdom (Bishop and Yokota, 2001). BRs regulate many important physiological and developmental processes, such as cell elongation and division, pollen fertility, photomorphogen- esis, differentiation of vascular elements, and stress resis- tance (Mandava, 1988; Clouse and Sasse, 1998). Recent molecular genetic studies for BR-deficient mutants of Ara- bidopsis thaliana L., rice (Oryza sativa L.), tomato (Lycop- ersicon esculentum L.), and garden pea (Pisum sativum L.) have identified several genes in the pathway of BR biosyn- thesis from campesterol to brassinolide (BL) (Fujioka and Yokota, 2003). Cytochrome P450 monooxygenases (P450s) play crucial roles in BR biosynthesis as many oxygenations 0031-9422/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2006.05.042 Abbreviations: BL, brassinolide; BR, brassinosteroid; CS, castasterone; P450, cytochrome P450 monooxygenase. * Corresponding author. Tel.: +81 774 38 3232; fax: +81 774 38 3229. E-mail address: mizutani@scl.kyoto-u.ac.jp (M. Mizutani). 1 Present address: Plant Science Center, RIKEN, Yokohama 230-0045, Japan. 2 Present address: Sagami Chemical Research Center, Ayase 252-1193, Japan. www.elsevier.com/locate/phytochem Phytochemistry 67 (2006) 1895–1906 PHYTOCHEMISTRY