Cytochrome P450 enzymes of the closely related CYP90 and CYP85 families catalyze essential oxidative reactions in the biosynthesis of brassinosteroid (BR) hormones. Arabidopsis CYP90B1/DWF4 and CYP90A1/CPD are responsible for respective C-22 and C-23 hydroxylation of the steroid side chain and CYP85A1 catalyzes C-6 oxidation of 6-deoxo intermediates, whereas the functions of CYP90C1/ROT3, CYP90D1, and CYP85A2 are still unknown. Semiquantitative reverse transcriptase-polymerase chain reaction analyses show that transcript levels of CYP85 and CYP90 genes are down-regulated by brassinolide, the end product of the BR biosynthesis pathway. Feedback control of the CYP90C1, CYP90D1, and CYP85A2 genes by brassinolide suggests that the corresponding enzymes might also participate in BR synthesis. CYP85 and CYP90 mRNAs show strong and transient accumulation during the 1st week of seedling development, as well as characteristic organ-specific distribution. Transcripts of CYP90A1 and CYP85A2 are preferentially represented in shoots and CYP90C1, CYP90D1, and CYP85A1 mRNAs are more abundant in roots, whereas CYP90B1 is ubiquitously expressed. Remarkably, the spatial pattern of CYP90A1 expression is maintained in the BR-insensitive cbb2 mutant, indicating the independence of organ-specific and BR-dependent regulation. Quantitative gas chromatography-mass spectrometry analysis of endogenous BRs in shoots and roots of Arabidopsis, pea (Pisum sativum), and tomato (Lycopersicon esculentum) reveal similar partitioning patterns of BR intermediates in these species. Inverse correlation between CYP90A1/ CPD transcript levels and the amounts of the CYP90A1 substrate 6-deoxocathasterone in shoots and roots suggests that transcriptional regulation plays an important role in controlling BR biosynthesis.Brassinosteroids (BRs) are plant steroid hormones that influence a wide range of important developmental processes, including germination, cell elongation, differentiation of vascular elements, photomorphogenesis, and pollen fertility (Clouse and Sasse, 1998; Steber and McCourt, 2001). The maintenance of optimal local BR concentrations by coordinated biosynthetic and inactivation mechanisms, in combination with the differential responsiveness of target cells and tissues, enables the proper regulation of these physiological functions during plant development. The pathways of BR biosynthesis have been elucidated by a series of detailed biochemical studies. Brassinolide (BL), the biologically most active BR, is synthesized from campesterol via either early or late C-6 oxidation routes (Fig. 1). The BR biosynthesis pathways are conserved between Catharanthus roseus, Arabidopsis, pea (Pisum sativum), tomato (Lycopersicon esculentum), and rice (Oryza sativa), although a limitation of early C-6 oxidation has been observed in some of these species (Fujioka and Sakurai, 1997; Fujioka et al., 2000; Noguchi et al., 2000; Nomura et al., 2001).Molecular genetic analysis of BR-deficient mutants has identified several BR biosynthesis genes that, with t...

Brassinosteroids (BRs) are biosynthesized from campesterol via several cytochrome P450 (P450)-catalyzed oxidative reactions. We report the functional characterization of two BR-biosynthetic P450s from Arabidopsis thaliana: CYP90C1/ ROTUNDIFOLIA3 and CYP90D1. The cyp90c1 cyp90d1 double mutant exhibits the characteristic BR-deficient dwarf phenotype, although the individual mutants do not display this phenotype. These data suggest redundant roles for these P450s. In vitro biochemical assays using insect cell-expressed proteins revealed that both CYP90C1 and CYP90D1 catalyze C-23 hydroxylation of various 22-hydroxylated BRs with markedly different catalytic efficiencies. Both enzymes preferentially convert 3-epi-6-deoxocathasterone, (22S,24R)-22-hydroxy-5a-ergostan-3-one, and (22S,24R)-22-hydroxyergost-4-en-3-one to 23-hydroxylated products, whereas they are less active on 6-deoxocathasterone. Likewise, cyp90c1 cyp90d1 plants were deficient in 23-hydroxylated BRs, and in feeding experiments using exogenously supplied intermediates, only 23-hydroxylated BRs rescued the growth deficiency of the cyp90c1 cyp90d1 mutant. Thus, CYP90C1 and CYP90D1 are redundant BR C-23 hydroxylases. Moreover, their preferential substrates are present in the endogenous Arabidopsis BR pool. Based on these results, we propose C-23 hydroxylation shortcuts that bypass campestanol, 6-deoxocathasterone, and 6-deoxoteasterone and lead directly from (22S,24R)-22-hydroxy-5a-ergostan-3-one and 3-epi-6-deoxocathasterone to 3-dehydro-6-deoxoteasterone and 6-deoxotyphasterol.