In situ RNA hybridization and immunocytochemistry were used to establish the cellular distribution of monoterpenoid indole alkaloid biosynthesis in Madagascar periwinkle ( Catharanthus roseus ). Tryptophan decarboxylase (TDC) and strictosidine synthase (STR1), which are involved in the biosynthesis of the central intermediate strictosidine, and desacetoxyvindoline 4-hydroxylase (D4H) and deacetylvindoline 4-O -acetyltransferase (DAT), which are involved in the terminal steps of vindoline biosynthesis, were localized. tdc and str1 mRNAs were present in the epidermis of stems, leaves, and flower buds, whereas they appeared in most protoderm and cortical cells around the apical meristem of root tips. In marked contrast, d4h and dat mRNAs were associated with the laticifer and idioblast cells of leaves, stems, and flower buds. Immunocytochemical localization for TDC, D4H, and DAT proteins confirmed the differential localization of early and late stages of vindoline biosynthesis. Therefore, we concluded that the elaboration of the major leaf alkaloids involves the participation of at least two cell types and requires the intercellular translocation of a pathway intermediate. A basipetal gradient of expression in maturing leaves also was shown for all four genes by in situ RNA hybridization studies and by complementary studies with dissected leaves, suggesting that expression of the vindoline pathway occurs transiently during early leaf development. These results partially explain why attempts to produce vindoline by cell culture technology have failed. INTRODUCTIONThe organs forming the plant body consist of several different cell types that are organized in relation to each other and that confer specific functions to the resulting organ. Each cell type emerges from an undifferentiated meristem according to a sophisticated and partially understood developmental program (Sylvester et al., 1996;von Arnim and Deng, 1996). The commitment to differentiate into specialized structures involves the perception by cells in the meristem of a complex array of signals, which communicate cellular age, position in relation to other cells, and hormonal balance. Environmental factors, such as light and temperature, also play a critical role in modulating these signals throughout the process of organogenesis (Bernier, 1988;Dale, 1988;Sylvester et al., 1996).In addition to morphogenesis, developmental processes result in biochemical specialization of cells for the biosynthesis and/or accumulation of secondary metabolites, such as phenylpropanoids (Ibrahim et al., 1987;Reinold and Hahlbrock, 1997), monoterpenoids (Fahn, 1988;McCaskill et al., 1992), and alkaloids (Robinson, 1974(Robinson, , 1981Nessler and Mahlberg, 1977;Eilert et al., 1985;Hashimoto and Yamada, 1994;Facchini and De Luca, 1995). Studies with germinating seedlings have suggested that alkaloid biosynthesis and accumulation are associated with seedling development (Weeks and Bush, 1974;De Luca et al., 1986;Aerts et al., 1994). Studies with mature plants also reveal this ty...

SummaryThe gene encoding acetyl CoA:deacetylvindoline 4-Oacetyltransferase (DAT) (EC 2.3.1.107) which catalyzes the last step in vindoline biosynthesis was isolated and characterized. The genomic clone encoded a 50 kDa polypeptide containing the sequences of nine tryptic fragments derived from the purified DAT heterodimer. However, cleavage of DAT protein to yield a heterodimer appears to be an artifact of the protein purification procedure, since the size of the protein (50 kDa) crossreacting with anti-DAT antibody in seedlings and in leaves of various ages also corresponds to the size of the active recombinant enzyme. Studies with the intact plant and with developing seedlings showed that induction of DAT mRNA, protein accumulation and enzyme activity occurred preferentially in vindoline producing tissues such as leaves and cotyledons of light-treated etiolated seedlings. The ORF of DAT showed significant sequence identity to 19 other plant genes, whose biochemical functions were mostly unknown. The Mr of µ 50 kDa, a HXXXDG triad, and a DFGWGKP consensus sequence are highly conserved among the 20 plant genes and these criteria may be useful to identify this type of acyltransferase. The involvement of some of these genes in epicuticular wax biosynthesis, fruit-ripening and in benzoyltransfer reactions indicates that the plant kingdom contains a superfamily of multifunctional acyltransferases which operate by a reaction mechanism related to the ancient chloramphenicol Oacetyltransferase and dihydrolipoyl acetyltransferase class of enzymes.

SummaryIn higher plants, isopentenyl diphosphate (IPP) is synthesised both from the plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) and from the cytosolic mevalonate (MVA) pathways. Primary metabolites, such as phytol group of chlorophylls, carotenoids and the plant hormones abscisic acid (ABA) and gibberellins (GAs) are derived directly from the MEP pathway. Many secondary metabolites, such as monoterpene indole alkaloids (MIAs) in Catharanthus roseus, are also synthesised from this source of IPP. Using Northern blot and in situ hybridisation experiments, we show that three MEP pathway genes (1-deoxy-D-xylulose 5-phosphate synthase (DXS), 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR ) and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MECS)) and the gene encoding geraniol 10-hydroxylase (G10H ), a cytochrome P450 monooxygenase involved in the ®rst committed step in the formation of iridoid monoterpenoids display identical cell-speci®c expression patterns. The co-localisation of these four transcripts to internal phloem parenchyma of young aerial organs of C. roseus adds a new level of complexity to the multicellular nature of MIA biosynthesis. We predict the translocation of pathway intermediates from the internal phloem parenchyma to the epidermis and, ultimately, to laticifers and idioblasts during MIA biosynthesis. Similarly, the translocation of intermediates from the phloem parenchyma is probably also required during the biosynthesis of hormones and photosynthetic primary metabolites derived from the MEP pathway.

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BackgroundThe 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 β-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.ResultsIn 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.ConclusionsA 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.