Spatial organization of the vindoline biosynthetic pathway in Catharanthus roseus

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• 作者：Gré ; gory Guirimand^a ; ¹ ; Anthony Guihur^a ; ¹ ; Pierre Poutrain^a ; Franç ; ois Hé ; ricourt^b ; Samira Mahroug^c ; Benoit St-Pierre^a ; Vincent Burlat^a ; ² ; Vincent Courdavault^a ; ^{vincent.courdavault@univ-tours.fr}
• 关键词：Alkaloid ; Bimolecular fluorescence complementation ; Catharanthus roseus ; O-methyltransferase ; Vindoline
• 刊名：Journal of Plant Physiology
• 出版年：2011
• 出版时间：15 April 2011
• 年：2011
• 卷：168
• 期：6
• 页码：549-557
• 全文大小：1998 K

文摘

Vindoline constitutes the main terpenoid indole alkaloid accumulated in leaves of Catharanthus roseus, and four genes involved in its biosynthesis have been identified. However, the spatial organization of the tabersonine-to-vindoline biosynthetic pathway is still incomplete. To pursue the characterization of this six-step conversion, we illustrated, with in situ hybridization, that the transcripts of the second biosynthetic enzyme, 16-hydroxytabersonine 16-O-methyltransferase (16OMT), are specifically localized to the aerial organ epidermis. At the subcellular level, by combining GFP imaging, bimolecular fluorescence complementation assays and yeast two-hybrid analysis, we established that the first biosynthetic enzyme, tabersonine 16-hydroxylase (T16H), is anchored to the ER as a monomer via a putative N-terminal helix that we cloned using a PCR approach. We also showed that 16OMT homodimerizes in the cytoplasm, allowing its exclusion from the nucleus and thus facilitating the uptake of T16H conversion product, although no T16H/16OMT interactions occur. Moreover, the two last biosynthetic enzymes, desacetoxyvindoline-4-hydroxylase (D4H) and deacetylvindoline-4-O-acetyltransferase (DAT), were shown to operate as monomers that reside in the nucleocytoplasmic compartment following passive diffusion to the nucleus allowed by the protein size. No D4H/DAT interactions were detected, suggesting the absence of metabolic channeling in the vindoline biosynthetic pathway. Finally, these results highlight the importance of the inter- and intracellular translocations of intermediates during the vindoline biosynthesis and their potential regulatory role.