Searching Steroid C2-hydroxylase in Brassinosteroid Biosynthetic Pathways in Arabidopsis thaliana

Brassinosteroids (BRs) modulate virtually all the processes of plant growth and development. Brassinolide (BL) which is the most active form among known BRs is synthesized using campesterol derived from squalene 2, 3-oxide in sterol-specific biosynthetic pathway as precursor. Most of the BR-specific biosynthetic enzymes are coded by the genes belonging to cytochrome P450s (CYPs) family. CYPs catalyze the monooxygenation/hydroxylation reactions in primary and secondary metabolisms. Arabidopsis BR biosynthetic CYPs belong to the CYP85 clan, which includes CYP90A1 (cpd), CYP90B1 (dwarf4), CYP90C1 (rotundifolia), CYP85A1 (BR6ox1), and CYP85A2 (BR6ox2). But it is yet to be explored, which enzyme catalyzes the C2-hydroxylation step in Arabidopsis BR biosynthetic pathways. We aimed to find the candidates of C2-hydroxylase using diverse approaches. First, Arabidopsis CYP85A1 amino acid sequence was blasted against embryophyta database in Phytozome. Archived sequences with high similarity were used for generating a phylogenetic tree. Of the orphan P450s, we tentatively identified candidates to study further. Second, we explored microarray data and applied a selection category to screen the probes which were up- or down-regulated by BR related chemicals. We also examined to find Arabidopsis homologs of mammalian steroid C2-hydroxylases. Because many of known BR biosynthetic genes are down-regulated by exogenous treatment of BL, we prepared RNA from seedlings of a BR deficient mutant (dwf7-1) after treatment with mock or BL. Semi-real time PCR of the candidate genes will reveal if they are involved in BR biosynthesis. Furthermore, transgenic overexpression lines of the candidate genes will be used to determine the phenotypes, molecular and biochemical identities. Our studies would reveal long-sought Arabidopsis C2-hydroxylase and contribute to complete identification of the enzymes catalyzing every step of the BR biosynthetic pathways.