Cloning and Characterization of a Candidate Gene from the Medicinal Plant Catharanthus roseus Through Transient Expression in Mesophyll Protoplasts

Catharanthus roseus (L.) G. Don. accumulates in the leaves the dimeric terpenoid indole alkaloids (TIAs) vinblastine and vincristine, which were the first natural anticancer products to be clinically used, and are still among the most valuable agents used in cancer chemotherapy. The great pharmacological importance of the TIAs, associated with the low abundance of the anticancer alkaloids in the plant, stimulated intense research on the TIA pathway, and C. roseus has now become one of the most extensively studied medicinal plants. About 130 TIAs have already been isolated from C. roseus, and it has been shown that the biosynthesis of vinblastine is highly complex, involving at least 30 steps from the amino-acid tryptophan and the monoterpenoid geraniol (Loyola-Vargas et al., 2007; van der Heijden et al., 2004). All the TIAs of C. roseus derive from the common precursor strictosidine, after which the TIA pathway splits into several branches including a short one leading to ajmalicine and serpentine (used as an antihypertensive and as a sedative respectively), and two long branches leading to vindoline and catharanthine the leaf abundant monomeric precursors of vinblastine and vincristine (Loyola-Vargas et al., 2007; van der Heijden et al., 2004). In our lab, we have performed the characterization of a key biosynthetic step of the anticancer TIAs the biosynthesis of the first dimeric TIA, α-3’,4’-anhydrovinblastine (AVLB), from vindoline and catharanthine. We identified a leaf class III peroxidase (Prx) with AVLB synthase activity and we purified and characterized this enzyme, which was named Catharanthus roseus peroxidase 1, CroPrx1 (Bakalovic et al., 2006; Peroxibase, http://peroxibase.toulouse.inra.fr index.php). We have further shown the localization of CroPrx1 in the same subcellular compartment where alkaloids accumulate, the vacuole, through biochemical and molecular methodologies,