Multicellular compartmentation of Catharanthus roseus alkaloid biosynthesis predicts intercellular translocation of a pathway intermediate

In situ RNA hybridization and immunocytochemistry were used to establish th e cellular distribution of monoterpenoid indole alkaloid biosynthesis in Ma dagascar periwinkle (Catharanthus roseus). Tryptophan decarboxylase (TDC) a nd strictosidine synthase (STR1), which are involved in the biosynthesis of the central intermediate strictosidine, and desacetoxyvindoline 4-hydroxyl ase (D4H) and deacetylvindoline 4-O-acetyltransferase (DAT), which are invo lved in the terminal steps of vindoline biosynthesis, were localized. tdc a nd str1 mRNAs were present in the epidermis of stems, leaves, and flower bu ds, whereas they appeared in most protoderm and cortical cells around the a pical meristem of root tips. In marked contrast, d4h and dat mRNAs were ass ociated with the laticifer and idioblast cells of leaves, stems, and flower buds. Immunocytochemical localization for TDC, D4H, and DAT proteins confi rmed the differential localization of early and late stages of vindoline bi osynthesis. Therefore, we concluded that the elaboration of the major leaf alkaloids involves the participation of at least two cell types and require s the intercellular translocation of a pathway intermediate. A basipetal gr adient of expression in maturing leaves also was shown for all four genes b y in situ RNA hybridization studies and by complementary studies with disse cted leaves, suggesting that expression of the vindoline pathway occurs tra nsiently during early leaf development. These results partially explain why attempts to produce vindoline by cell culture technology have failed.