The ORCA-ome as a key to understanding alkaloid biosynthesis in Catharanthus roseus

Abstract

Catharanthus roseus produces an important class of secondary metabolites known as terpenoid indole alkaloids (TIA). The dimeric TIA vincristine and vinblastine are effective anticancer drugs. Two transcription factors called ORCA2 and ORCA3 have been reported to regulate the MeJA-responsive expression of several biosynthesis genes. We report here comprehensive transcript profiling analysis of Catharanthus cell lines overexpressing ORCA2 or ORCA3 in an inducible manner. By using cDNA-amplified fragment-length polymorphism technology the quantitative accumulation patterns of 11,277 transcript tags were determined and analyzed. Thirty six transcripts were upregulated in response to overexpression of either by both ORCA2 or ORCA3 while 22 tags and 16 tags were upregulated specifically in ORCA2 overexpressing lines and ORCA3 over expression lines, respectively. Accumulation of downstream TIAs was also differentially affected by ORCA2 and ORCA3 overexpression. TIA accumulation patterns allowed us to predict in which set of cDNA-AFLP tags genes encoding the corresponding enzymes should be present. Metabolite analysis showed that overexpression of either ORCA2 or ORCA3 resulted in increased ajmalicine levels, indicating that the gene encoding Cathenamine Reductase (CR) must be regulated by both ORCAs. Since the CR-75 tag gave a TBLASTX hit to aldo/keto oxidoreductase enzymes we investigated the possibility that CR-75 corresponds to CR. Recombinant CR-75 protein produced in E. coli was able convert cathenamine to ajmalicine using NADPH as a cofactor, thereby identifying CR-75 as Cathenamine Reductase. We also report the isolation and characterization of alcohol dehydrogenase genes from C. roseus which may function as 10-hydroxygeraniol oxidoreductases (10HGO). Two ORCA regulated genes were also isolated and characterized from C. roseus cell suspension culture which may have roles in TIA biosynthesis