Functional analysis of arabidopsis CYP714A1 and CYP714A2 reveals that they are distinct gibberellin modification enzymes

Endogenous levels of bioactive gibberellins (GAs) are controlled by both biosynthetic and inactivation processes, and some cytochrome P450s are involved in this control mechanism. We have previously reported that CYP714B1 and CYP714B2 encode the enzyme GA 13-oxidase, which is required for GA₁ biosynthesis, and that CYP714D1 encodes GA 16a, 17-epoxidase, which inactivates the non-13-hydroxy GAs in rice. Arabidopsis has two CYP714 members, CYP714A1 and CYP714A2. To clarify the possible role of these genes in GA metabolism, enzymatic activities of their recombinant proteins were analyzed using a yeast expression system. We found that the recombinant CYP714A1 protein catalyzes the conversion of GA₁₂ to 16-carboxylated GA₁₂ (16-carboxy-16b, 17-dihydro GA₁₂), a previously unidentified GA metabolite. Bioassays of this GA product showed that CYP714A1 is an inactivation enzyme in Arabidopsis. This was confirmed by the extreme GA-deficient dwarf phenotype shown by CYP714A1-overexpres-sing plants. Intriguingly, the recombinant CYP714A2 protein catalyzed the conversion of ent-kaurenoic acid into steviol (ent-13-hydroxy kaurenoic acid). When GA₁₂ was used as a substrate for CYP714A2, 12a-hydroxy GA₁₂ (GA₁₁₁) was produced as a major product and 13-hydroxy GA₁₂ (GA₅₃) as a minor product. Transgenic Arabidopsis plants overexpres-sing the CYP714A2 gene showed semi-dwarfism. GA analysis showed that the levels of non-13-hydroxy GAs, including GA₄, were decreased, whereas those of 13-hydroxy GAs, including GA₁ (which is less active than GA₄), were increased in the transgenic plants. Our results suggest that the CYP714 family proteins contribute to the production of diverse GA compounds through various oxidations of C and D rings in both monocots and eudicots.