A Pair of Tabersonine 16-Hydroxylases Initiates the Synthesis of Vindoline in an Organ-Dependent Manner in Catharanthus roseus 1[C][W] Sébastien Besseau 2 , Franziska Kellner 2 , Arnaud Lanoue, Antje M.K. Thamm, Vonny Salim, Bernd Schneider, Fernando Geu-Flores, René Höfer, Grégory Guirimand, Anthony Guihur, Audrey Oudin, Gaëlle Glevarec, Emilien Foureau, Nicolas Papon, Marc Clastre, Nathalie Giglioli-Guivarc’h, Benoit St-Pierre, Danièle Werck-Reichhart, Vincent Burlat, Vincenzo De Luca, Sarah E. O’Connor, and Vincent Courdavault* Université François Rabelais de Tours, EA2106 Biomolécules et Biotechnologies Végétales, 37200 Tours, France (S.B., A.L., G.Gu., A.G., A.O., G.Gl., E.F., N.P., M.C., N.G.-G., B.S.-P., V.C.); Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom (F.K., F.G.-F., S.O.); Department of Biological Sciences, Brock University, St. Catharines, Ontario L2S 3A1, Canada (A.M.K.T., V.S., V.D.L.); Max-Planck-Institute for Chemical Ecology, Beutenberg Campus, D–07745 Jena, Germany (B.S.); Institut de Biologie Moléculaire des Plantes, Unité Propre de Recherche 2357 du CNRS, University of Strasbourg, F–67083 Strasbourg cedex, France (R.H., D.W.-R.); Université de Toulouse, Université Paul Sabatier, UMR 5546, Laboratoire de Recherche en Sciences Végétales, BP 42617 Auzeville, F–31326 Castanet-Tolosan, France (V.B.); and CNRS, UMR 5546, BP 42617, F–31326 Castanet-Tolosan, France (V.B.) Hydroxylation of tabersonine at the C-16 position, catalyzed by tabersonine 16-hydroxylase (T16H), initiates the synthesis of vindoline that constitutes the main alkaloid accumulated in leaves of Catharanthus roseus. Over the last decade, this reaction has been associated with CYP71D12 cloned from undifferentiated C. roseus cells. In this study, we isolated a second cytochrome P450 (CYP71D351) displaying T16H activity. Biochemical characterization demonstrated that CYP71D12 and CYP71D351 both exhibit high affinity for tabersonine and narrow substrate specificity, making of T16H, to our knowledge, the first alkaloid biosynthetic enzyme displaying two isoforms encoded by distinct genes characterized to date in C. roseus. However, both genes dramatically diverge in transcript distribution in planta. While CYP71D12 (T16H1) expression is restricted to flowers and undifferentiated cells, the CYP71D351 (T16H2) expression profile is similar to the other vindoline biosynthetic genes reaching a maximum in young leaves. Moreover, transcript localization by carborundum abrasion and RNA in situ hybridization demonstrated that CYP71D351 messenger RNAs are specifically located to leaf epidermis, which also hosts the next step of vindoline biosynthesis. Comparison of high- and low-vindoline-accumulating C. roseus cultivars also highlights the direct correlation between CYP71D351 transcript and vindoline levels. In addition, CYP71D351 down-regulation mediated by virus-induced gene silencing reduces vindoline accumulation in leaves and redirects the biosynthetic flux toward the production of unmodified alkaloids at the C-16 position. All these data demonstrate that tabersonine 16-hydroxylation is orchestrated in an organ-dependent manner by two genes including CYP71D351, which encodes the specific T16H isoform acting in the foliar vindoline biosynthesis. Catharanthus roseus displays an active specialized metabolism producing more than 100 monoterpene indole alkaloids (MIAs). MIAs are a major component of the chemical arsenal that likely protects the plant by acting against herbivores (Guirimand et al., 2010a; Roepke et al., 2010). These MIAs include highly valu- able molecules that exhibit interesting pharmacological properties for human health. For instance, vinblastine, vincristine, and their derivatives are widely used in cancer chemotherapy, where they act as microtubule disruptors (Gigant et al., 2005). Due to highly complex structures, these therapeutic molecules are prepared by semisynthesis using natural precursors extracted from C. roseus leaves. Their extremely low level in planta explains their high cost of production and justifies the extensive study of the MIA biosynthetic pathway that has been carried out over the last three decades, making 1 This work was supported by the Ministère de l’Enseignement Supérieur et de la Recherche, by a grant from the University of Tours (to S.B., A.L., G.Gu., A.G., A.O., G.Gl., E.F., N.P., M.C., N.G.-G., B.S.-P., and V.C.), by the Biotechnology and Biological Sciences Research Council (grant nos. BB/J004561/1 and BB/J009091/1 to F.K., F.G.-F., and S.O.), and by the Natural Sciences and Engineering Research Council of Canada (Discovery grants to A.T., V.S., and V.D.L.). 2 These authors contributed equally to the article. * Address correspondence to vincent.courdavault@univ-tours.fr. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy de- scribed in the Instructions for Authors (www.plantphysiol.org) is: Vincent Courdavault (vincent.courdavault@univ-tours.fr). [C] Some figures in this article are displayed in color online but in black and white in the print edition. [W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.113.222828 1792 Plant Physiology Ò , December 2013, Vol. 163, pp. 1792–1803, www.plantphysiol.org Ó 2013 American Society of Plant Biologists. All Rights Reserved. Downloaded from https://academic.oup.com/plphys/article/163/4/1792/6111124 by guest on 14 November 2022