Poster: Hormones

Abs # 577: Functional analysis of tomato cytochromes P450 involved in brassinosteroid catabolism

Presenter:	Mizutani, Masaharu , mizutani@scl.kyoto-u.ac.jp
Authors	Mizutani, Masaharu  (A)   Onishi, Toshiyuki  (A)   Ohta, Daisaku  (C)   Nomura, Takahito  (B)   Yokota, Takao  (B)   Sakata, Kanzo  (A)
Affiliations:	(A): Institute for Chemical Research, Kyoto University (B): Teikyo University (C): Osaka prefecture University
Web Site:	http://biofun.kuicr.kyoto-u.ac.jp/index-j.html

Brassinosteroids (BRs) are steroidal plant hormones that regulate growth and development. Biochemical analyses have suggested that several cytochromes P450 (P450s) are participated in the pathways of BR catabolism. Recently, Arabidopsis BAS1 gene (CYP72B1) has been suggested to function as a P450 catalyzing the inactivation of BRs. However, the pathways of BR catabolism are not yet clear in detail. We have obtained two tomato cDNAs encoding P450s (CYP72B2 and 72B3), which show 60-70% identity to BAS1. In BR treated tomato, the expression of CYP72B2 and 72B3 increased while the expression of P450 genes (CYP90A, 90B, and 90C from tomato) in BR biosynthesis decreased. Transgenic tobacco plants expressing the CYP72B2 cDNA under control of CaMV 35S promoter were dwarf remarkably and their leaves were dark-green, which are typical phenotypes of BR deficiency. Endogenous BR levels in transgenic tobacco were analyzed by GC-MS. BL was not detected in both wild type and transgenic tobacco. CS and 6-deoxocastasterone levels in transgenic tobacco were significantly decreased as compared to those in wild type. The recombinant CYP72B2 enzyme was expressed by using insect cell-baculovirus expression system. Analysis of type I spectral shift of CYP72B2 with BRs revealed that CS showed a higher binding affinity (7 mM) than BL and 6-deoxocastasterone (11 m)M). The CYP72B2 activity with CS as a substrate was estimated as 47 nmol/min/nmol P450 in a reconstituted system containing NADPH-P450 reductase. GC-MS analysis demonstrated that the metabolite was a hydroxylated CS and a hydroxy group was introduced to the side chain of CS. Thus this study revealed that CYP72B2 catalyzes the hydroxylation reaction of CS to an inactive BR.