title: The subcellular organization of strictosidine biosynthesis in Catharanthus roseus epidermis highlights several trans-tonoplast translocations of intermediate metabolites.
abstract: Catharanthus roseus synthesizes a wide range of valuable monoterpene indole alkaloids, some of which have recently been recognized as functioning in plant defence mechanisms. More specifically, in aerial organ epidermal cells, vacuole-accumulated strictosidine displays a dual fate, being either the precursor of all monoterpene indole alkaloids after export from the vacuole, or the substrate for a defence mechanism based on the massive protein cross-linking, which occurs subsequent to organelle membrane disruption during biotic attacks. Such a mechanism relies on a physical separation between the vacuolar strictosidine-synthesizing enzyme and the nucleus-targeted enzyme catalyzing its activation through deglucosylation. In the present study, we carried out the spatial characterization of this mechanism by a cellular and subcellular study of three enzymes catalyzing the synthesis of the two strictosidine precursors (i.e. tryptamine and secologanin). Using RNA in situ hybridization, we demonstrated that loganic acid O-methyltransferase transcript, catalysing the penultimate step of secologanin synthesis, is specifically localized in the epidermis. A combination of green fluorescent protein imaging, bimolecular fluorescence complementation assays and yeast two-hybrid analysis enabled us to establish that both loganic acid O-methyltransferase and the tryptamine-producing enzyme, tryptophan decarboxylase, form homodimers in the cytosol, thereby preventing their passive diffusion to the nucleus. We also showed that the cytochrome P450 secologanin synthase is anchored to the endoplasmic reticulum via a N-terminal helix, thus allowing the production of secologanin on the cytosolic side of the endoplasmic reticulum membrane. Consequently, secologanin and tryptamine must be transported to the vacuole to achieve strictosidine biosynthesis, demonstrating the importance of trans-tonoplast translocation events during these metabolic processes.
authors.affiliation: Université François Rabelais de Tours, EA2106 Biomolécules et Biotechnologies Végétales, IFR 135 Imagerie fonctionnelle, Tours, France.
authors.forename: Grégory
authors.initials: G
authors.lastname: Guirimand
authors.forename: Anthony
authors.initials: A
authors.lastname: Guihur
authors.forename: Olivia
authors.initials: O
authors.lastname: Ginis
authors.forename: Pierre
authors.initials: P
authors.lastname: Poutrain
authors.forename: François
authors.initials: F
authors.lastname: Héricourt
authors.forename: Audrey
authors.initials: A
authors.lastname: Oudin
authors.forename: Arnaud
authors.initials: A
authors.lastname: Lanoue
authors.forename: Benoit
authors.initials: B
authors.lastname: St-Pierre
authors.forename: Vincent
authors.initials: V
authors.lastname: Burlat
authors.forename: Vincent
authors.initials: V
authors.lastname: Courdavault
mesh_terms: D001692:Biological Transport / Q000502:physiology; D030008:Catharanthus / Q000378:metabolism*; D008780:Methyltransferases / Q000378:metabolism; D008954:Models, Biological; D019441:Plant Epidermis / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism*; D014748:Vinca Alkaloids / Q000096:biosynthesis*
chemical_list: D010940:Plant Proteins; D014748:Vinca Alkaloids; C021093:strictosidine; D008780:Methyltransferases