Characterization of cytochrome P450 monooxygenases isolated from trichome enriched fraction of Artemisia annua L. leaf.

CYPs have major role in the biosynthesis and modification of secondary metabolites. Predicting the possible involvement of CYPs in secondary metabolism, 20 partial sequences were amplified from the cDNA of trichome enriched tissue of Artemisia annua. Seven CYPs were converted to full length and assigned to different families based on sequence homology. These were co-expressed with CPR in Saccharomyces cerevisiae and microsome fractions were assayed for conversion of sesquiterpenes, phenols and fatty acid substrates. CIM_CYP02(c73) and CIM_CYP05(c81) converted trans-cinnamic acid to p-coumaric acid; and capric acid, lauric acid to their hydroxylated products, respectively. Higher expression of CIM_CYP71AV1, CIM_CYP03(c72a), CIM_CYP06(c72b), CIM_CYP02(c73) and CIM_CYP04(c83) was observed in the mature leaf, whereas expression of CIM_CYP05(c81) was more in the seedling. CIM_CYP71AV1, CIM_CYP02(c73) and CIM_CYP04(c83) expressed more in the flower bud compared to the leaf, with minor expression in stem. All CYPs' expression increased progressively with time after wounding except for CIM_CYP07(c92). These results relate involvement of CIM_CYP02(c73) to phenyl-propanoid metabolism in the leaf and CIM_CYP05(c81) to fatty acid metabolism in the seedling. Expression of CIM_CYP71AV1 and CIM_CYP02(c73) significantly increased when sprayed with trans-cinnamic acid indicating a relationship between phenylpropanoid and artemisinic acid pathways.