A double mutant for the CYP85A1 and CYP85A2 genes of Arabidopsis exhibits a brassinosteroid dwarf phenotype

Brassinosteroid (BR)-6-oxidases mediate the bridge reactions that connect the late and early C-6 oxidation pathways by converting 6-deoxoBR to 6-oxoBRs. Two similar genes of Arabidopsis, CYP85A1 (At5g38970) and CYP85A2 (At3g30180), are proposed to encode BR-6-oxidases based on findings that heterologously expressed genes mediate BR-6-oxidation reactions in yeast. However, genetic evidence that both genes are critically involved in the BR-6-oxidation step in Arabidopsis has been limited. Here, we show that a double mutant for the two genes displays dwarfism similar to that of typical BR biosynthesis-deficient mutants, suggesting that they are the major BR-6-oxidases in Arabidopsis. Examination of endogenous BR levels and metabolism monitoring tests using this double mutant revealed a great reduction in the levels of 6-oxoBRs, e.g., TY and CS, due to a lack in the conversion reactions from 6-deoxoCS to CS, and from 6-deoxoTY to TY. Surprisingly, the double mutant accumulated a significant amount of 6-oxocampestanol, suggesting that the upstream C-6 oxidation of campestanol to 6-oxocampestanol is not catalyzed by the two BR-6-oxidases in Arabidopsis, rather, by another enzyme yet to be discovered.