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.

Figure 1

The pathway of BR biosynthesis. Black arrows represent conversion steps with confirmed or assumed involvement of cytochrome P450 monooxygenases. Identified Arabidopsis P450 enzymes of the pathway are indicated. Numbering of the carbon positions oxidized in BRs is given at the structural formula of campesterol.

Figure 2

Structural relationship between selected Arabidopsis cytochrome P450 proteins and their genes. A, Unrooted cladogram based on the primary structure of P450 families involved in BR biosynthesis (CYP85 and CYP90), BR catabolism (CYP72), and GA biosynthesis (CYP88). Amino acid identity values, as compared with CPD/CYP90A1, are given in brackets. B, Exon/intron structure of the genes encoding CYP85A1 (AB009048), CYP85A2 (AP002060), CPD/CYP90A1 (X87367), DWF4/CYP90B1 (AL132979), ROT3/CYP90C1 (Z99708), CYP90D1 (AP001307), CYP88A3 (AC000098), CYP88A4 (AC005700), BAS1/CYP72B1 (AC003105), and CHIBI2/CYP72C1 (AC007651). Exon sizes are given in bp.

Figure 3

Effect of BL on the steady-state mRNA levels of BR-biosynthetic P450s. Reverse transcriptase (RT)-PCR products obtained from total RNA of 7-d-old seedlings incubated for 4 h in the presence (BL) or absence (Ctr) of 100 nm BL. A, Wild type; B, BR-deficient cpd and cbb3 mutants; C, BR-insensitive cbb2 mutant. UBQ10 was used as internal control.

Figure 4

Changes in transcript levels of CYP85 and CYP90 genes during germination and seedling development. RT-PCR products prepared from total RNA of developing wild-type seedlings and young plants (1 through 8 and 14 d after imbibition). Quantitative data are plotted as percentage of the highest value measured during the experimental period.

Figure 5

Differential accumulation of BR-biosynthetic P450 mRNAs in shoots and roots. A, Transcript levels in shoots (S) and roots (R) of wild-type seedlings. B, CPD/CYP90A1 transcript levels in shoots (S) and roots (R) of BR-insensitive cbb2 seedlings. C, CPD/CYP90A1 transcript levels in roots (R) of wild-type seedlings incubated for 4 h in the presence (BL) or absence (Ctr) of 100 nm BL. RT-PCR products were obtained from total RNA of 7-d-old seedlings. UBQ10 was used as internal control.