Spatial organization of the vindoline biosynthetic pathway in Catharanthus roseus.
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.
Université François Rabelais de Tours, EA 2106 Biomolécules et Biotechnologies Végétales, IFR 135 Imagerie fonctionnelle 37200 Tours, France.
Grégory
G
Guirimand
Anthony
A
Guihur
Pierre
P
Poutrain
François
F
Héricourt
Samira
S
Mahroug
Benoit
B
St-Pierre
Vincent
V
Burlat
Vincent
V
Courdavault
D000123:Acetyltransferases / Q000235:genetics / Q000378:metabolism; D053898:Biosynthetic Pathways; D030008:Catharanthus / Q000737:chemistry / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism*; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D026121:Indole Alkaloids / Q000378:metabolism*; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D019441:Plant Epidermis / Q000201:enzymology / Q000378:metabolism; D018515:Plant Leaves / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011804:Quinolines / Q000378:metabolism*; D046948:Secologanin Tryptamine Alkaloids / Q000378:metabolism; D014747:Vinblastine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism
D026121:Indole Alkaloids; D010940:Plant Proteins; D011804:Quinolines; D046948:Secologanin Tryptamine Alkaloids; C009373:tabersonine; C009667:vindoline; D014747:Vinblastine; D006899:Mixed Function Oxygenases; C527828:16-methoxytabersonine methyltransferase, Catharanthus roseus; D008780:Methyltransferases; D000123:Acetyltransferases; C064442:acetylcoenzyme A-deacetylvindoline 4-O-acetyltransferase