Regulation of Transcript Levels of the Arabidopsis Cytochrome P450 Genes Involved in Brassinosteroid Biosynthesis

Abstract

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...

Diurnal Regulation of the Brassinosteroid-Biosynthetic CPD Gene in Arabidopsis

“…Biosynthesis and inactivation are coordinated via BR-dependent transcriptional control of the genes involved in both processes. All BR-biosynthetic P450 genes of Arabidopsis are under negative feedback regulation by active BRs (Bancos et al, 2002;Tanaka et al, 2005), whereas of those encoding inactivating enzymes only BAS1 was found BR inducible (Choe et al, 2001;Takahashi et al, 2005). Feedback regulation of CPD is abolished in the BR-insensitive bin2 and brassinosteroid insensitive 1-2 (bri1-2)/cbb2 mutants (Li et al, 2001;Bancos et al, 2002), indicating a key role of BR signaling in the hormonal self-regulation of BR biosynthesis and perhaps also inactivation.…”

“…Recent studies indicated that in Arabidopsis the expression of all BR-biosynthetic P450 genes is under both developmental and organ-specific regulation that take place primarily at the level of transcription (Bancos et al, 2002;Shimada et al, 2003;Kim et al, 2006). This can influence the efficiency of biosynthesis because activities of certain genes encoding rate-limiting enzymes, such as CPD in Arabidopsis or DWARF/ CYP85A1 and CYP85A3 in tomato, show good correlation with the accumulation of active BRs (Bancos et al, 2002;Montoya et al, 2005;Nomura et al, 2005).…”

Plant P450s as versatile drivers for evolution of species-specific chemical diversity

“…Sterols are precursors of brassinosteroids/brassinolides, which are steroid hormones that promote plant growth and cell division, and of steroidal triterpenoids such as the glycoalkaloids in Solanum species [58]. Four P450 families act downstream of CYP51 and CYP710 in brassinosteroid metabolism: CYP85 catalyses C-6 oxidations, whereas CYP90A, CYP90B, CYP90C and CYP90D subfamily members catalyse C-22 and C-23 hydroxylations [59]. Two P450 families are involved in inactivation of brassinosteroids.…”

Brassinosteroids, de-etiolation and the re-emerging art of plant hormone quantification

“…2 This is consistent with previous studies conducted in Arabidopsis and tomato, where expression levels of selected BR-synthesis genes were also closely correlated with the actual endogenous BR levels. [11][12][13][14] However, this situation did not hold true when we examined the full complement of genes involved in the BR synthesis pathway. Indeed, there were just as many examples where the expression of individual genes did not reflect the endogenous BR levels.…”