Identification of methyl jasmonate-inducible cytochrome P450s and diterpene cyclase involved in cyclic diterpene biosynthesis in Scoparia dulcis

Diterpenes including the phytoalexins and phytohormone gibberellin are one of biologically important pharmaceutical sources from natural origin. Scoparia dulcis L. (Scrophulariaceae), a tropical medicinal plant, produce tetracyclic diterpenes such as scopadulcic acid B (SDB) and scopadulciol (SDC). SDB exhibits various pharmacological activities including inhibitory effects on replication of herpes simplex virus type 1 and antitumor. Furthermore, SDB formation in S. dulcis leaf tissue is rapidly and transiently stimulated by addition of methyl jasmonate (MJ) as an elicitor. In order to gain insight into the molecular mechanisms underlying diterpene biosynthesis, we have focused on cytochrome P450 (P450) enzymes often appear to form key regulatory steps in plant secondary metabolism. As a first step, we performed differenzial display analysis of P450 genes induced during elicitation of SDB biosynthesis after MJ addition to S. dulcis leaf tissues. As a result, nine genes were found to be up-regulated and were highly homologous to the corresponding region of P450 cDNA. We further examined the change in these gene expressions in the course of induction of SDB synthesis by MJ or Yeast extract. In addition, we also isolated a gene encoding ent-kaurene synthase (KS) which catalyzes the cyclization of copalyl diphosphate to ent-kaurene from S. dulcis and analyzed its function. The KS gene has been duplicated in the S. dulcis genome and is highly expressed in mature leaves. Here, we discuss the physiological roles of these isolated P450s and KS in diterpene biosynthesis in S. dulcis.