Localization of tabersonine 16‐hydroxylase and 16‐OH tabersonine‐16‐O‐methyltransferase to leaf epidermal cells defines them as a major site of precursor biosynthesis in the vindoline pathway in Catharanthus roseus

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Abstract
The Madagascar periwinkle (Catharanthus roseus) produces the well known and remarkably complex anticancer dimeric alkaloids vinblastine and vincristine, which are derived by the coupling of vindoline and catharanthine monomers. Recent data from in situ RNA hybridization and immunolocalization suggest that combinatorial cell factories within the leaf are involved in vindoline biosynthesis. In this study, the cell types responsible for vindoline biosynthesis were identified by laser‐capture microdissection/RNA isolation/RT–PCR to show that geraniol hydroxylase, secologanin synthase, tryptophan decarboxylase, strictosidine synthase, strictosidine ß‐glucosidase and tabersonine 16‐hydroxylase can be detected preferentially in epidermal cells. A new and complementary application of the carborundum abrasion (CA) technique was developed to obtain epidermis‐enriched leaf extracts that can be used to measure alkaloid metabolite levels, enzyme activities and gene expression. The CA technique showed that tabersonine and 16‐methoxytabersonine, together with 16‐hydroxytabersonine‐16‐O‐methyltransferase, are found predominantly in Catharanthus leaf epidermis, in contrast to vindoline, catharanthine and later enzymatic steps in vindoline biosynthesis. The results show that leaf epidermal cells are biosynthetically competent to produce tryptamine and secologanin precursors that are converted via many enzymatic transformations to make 16‐methoxytabersonine. This alkaloid or its 2,3 dihydro‐derivative is then transported to cells (mesophyll/idioblast/laticifer) within Catharanthus leaves to complete the last three or four enzymatic transformations to make vindoline.

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