Abstract
Catabolic inactivation of (+)-abscisic acid (ABA) proceeds via hydroxylation at the 8' position to form the unstable intermediate, 8'-hydroxy ABA (8'-OH ABA), which subsequently cyclizes spontaneously to form (-)-phaseic acid (PA). ABA 8'-hydroxylase has been known as P450,but not isolated. Arabidopsis genome contains 246 cytochrome P450 (CYP or P450) genes, which are classified in two main clades : A-type and non-A-type. Several P450 genes involved in brassinosteroids and gibberellins biosynthesis are clustered in the 85 clan of non-A-type. CYP707A family is also in the 85 clan, but its function has not been identified yet. In this study, we have characterized biochemical properties of CYP707A. RT-PCR analysis of expression profiles in response to the phytohormone treatment revealed that expressions of CYP707A genes significantly increased in response to ABA. In addition, expressions of CYP707A genes also induced by various stress conditions such as high salinity, osmotic and drought stresses. These results suggest the involvement of CYP707A in ABA catabolism. To confirm the hypothesis, CYP707As were expressed in insect cells. (+)-ABA was added to the suspension-cultured cells. An EtOAc extract was analyzed by HPLC. PA and 8'-OH ABA were detected. For biochemical characterization, the microsomal fractions were prepared from the insect cells expressing CYP707A3. The recombinant CYP707A3 catalyzed the hydroxylation of ABA to form PA in the presence of NADPH, but not NADH. The recombinant CYP707A3 was solubilized and partially purified by column chromatography. Type I spectra were detected by adding (+)-ABA to the purified CYP707A3 in a dose-dependent manner. Thus, these results demonstrate that CYP707A genes encode ABA 8'-hydoxylase.
