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Abstract: 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. [Copyright &y& Elsevier]

Background: The first two enzymatic steps of monoterpene indole alkaloid (MIA) biosynthetic pathway are catalysed by strictosidine synthase (STR) that condensates tryptamine and secologanin to form strictosidine and by strictosidine b-D-glucosidase (SGD) that subsequently hydrolyses the glucose moiety of strictosidine. The resulting unstable aglycon is rapidly converted into a highly reactive dialdehyde, from which more than 2,000 MIAs are derived. Many studies were conducted to elucidate the biosynthesis and regulation of pharmacologically valuable MIAs such as vinblastine and vincristine in Catharanthus roseus or ajmaline in Rauvolfia serpentina. However, very few reports focused on the MIA physiological functions. Results: In this study we showed that a strictosidine pool existed in planta and that the strictosidine deglucosylation product(s) was (were) specifically responsible for in vitro protein cross-linking and precipitation suggesting a potential role for strictosidine activation in plant defence. The spatial feasibility of such an activation process was evaluated in planta. On the one hand, in situ hybridisation studies showed that CrSTR and CrSGD were coexpressed in the epidermal first barrier of C. roseus aerial organs. However, a combination of GFP-imaging, bimolecular fluorescence complementation and electromobility shift-zymogram experiments revealed that STR from both C. roseus and R. serpentina were localised to the vacuole whereas SGD from both species were shown to accumulate as highly stable supramolecular aggregates within the nucleus. Deletion and fusion studies allowed us to identify and to demonstrate the functionality of CrSTR and CrSGD targeting sequences. Conclusions: A spatial model was drawn to explain the role of the subcellular sequestration of STR and SGD to control the MIA metabolic flux under normal physiological conditions. The model also illustrates the possible mechanism of massive activation of the strictosidine vacuolar pool upon enzyme-substrate reunion occurring during potential herbivore feeding constituting a so-called "nuclear time bomb" in reference to the "mustard oil bomb" commonly used to describe the myrosinase-glucosinolate defence system in Brassicaceae. [ABSTRACT FROM AUTHOR]

The Madagascar periwinkle (Catharanthus roseus) synthesizes the highly valuable monoterpene indole alkaloids (MIAs) through a long metabolic route initiated by the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. In leaves, a complex compartmentation of the MIA biosynthetic pathway occurs at both the cellular and subcellular levels, notably for some gene products of the MEP pathway. To get a complete overview of the pathway organization, we cloned four genes encoding missing enzymes involved in the MEP pathway before conducting a systematic analysis of transcript distribution and protein subcellular localization. RNA in situ hybridization revealed that all MEP pathway genes were coordinately and mainly expressed in internal phloem-associated parenchyma of young leaves, reinforcing the role of this tissue in MIA biosynthesis. At the subcellular level, transient cell transformation and expression of fluorescent protein fusions showed that all MEP pathway enzymes were targeted to plastids. Surprisingly, two isoforms of 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose 5-phosphate reductoisomerase initially exhibited an artifactual aggregated pattern of localization due to high protein accumulation. Immunogold combined with transmission electron microscopy, transient transformations performed with a low amount of transforming DNA and fusion/deletion experiments established that both enzymes were rather diffuse in stroma and stromules of plastids as also observed for the last six enzymes of the pathway. Taken together, these results provide new insights into a potential role of stromules in enhancing MIA precursor exchange with other cell compartments to favor metabolic fluxes towards the MIA biosynthesis. [ABSTRACT FROM AUTHOR]