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-teminal 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.Abbreviations BiFC, bimolecular fluorescence complementation; CFP, cyan fluorescent protein; ER, endoplasmic reticulum; G10H, geraniol 10-hydroxylase; GFP, green fluorescent protein; GUS, b-glucuronidase; IPAP, internal phloem-associated parenchyma; LAMT, loganic acid O-methyltransferase; -LW, leucine-trytophan lacking medium; -LWH, leucine-trytophan-histidine lacking medium; MEP, 2-C-methyl-D-erythritol 4-phosphate; MIA, monoterpene indole alkaloid(s); pGAD, GAL4 activation domain; pLex, LexA DNA-binding domain; SLS, secologanin synthase; SGD, strictosidine b-D-glucosidase; STR, strictosidine synthase; TDC, tryptophan decarboxylase; YFP, yellow fluorescent protein.

A 7-Deoxyloganetic Acid Glucosyltransferase Contributes a Key Step in Secologanin Biosynthesis in Madagascar Periwinkle

“…RT-PCR analysis of leaf epidermis enriched transcripts showed that the levels of UGT6 and UGT8 were at least 10-fold lower than those found in whole leaves, while UGT7 transcripts were more equally distributed ( Figure 4A). By contrast, transcripts for LAMT and SLS, well known to be preferentially expressed in the leaf epidermis Guirimand et al, 2011), were two-to fourfold more enriched in these cells. Together, these results suggested that both UGT-6 and -8 are preferentially expressed within periwinkle leaves rather than in leaf epidermal cells, where the last two steps in secologanin assembly are expressed.…”

“…However, the preference of native and recombinant loganic acid O-methyltransferase (LAMT) from periwinkle for loganic acid (5, Figure 1G) but not 7-deoxyloganic acid (4, Figure 1I) makes the alternative pathway less likely in periwinkle plants. The last two steps to form secologanin involve LAMT and secologanin synthase (SLS) ( Figure 1H), which are preferentially expressed in the leaf epidermis of periwinkle plants, as determined by carborundum abrasion extraction and in situ hybridization methods (Guirimand et al, 2011). The formation of secologanin in the leaf epidermis suggests that an undefined iridoid, possibly loganic acid or some earlier intermediate, must be transported to the leaf epidermis for its elaboration, and this cell type is also the site of expression of enzymes such as Trp decarboxylase and strictosidine synthase, which are required to elaborate the formation of strictosidine from which all of the MIAs of periwinkle are derived (Facchini and De Luca, 2008;Guirimand et al, 2011).…”

A Pair of Tabersonine 16-Hydroxylases Initiates the Synthesis of Vindoline in an Organ-Dependent Manner inCatharanthus roseus

“…Most plant P450s characterized to date function as endoplasmic reticulum (ER)-anchored enzymes, as exemplified in C. roseus for CYP71D12, CYP72A1, and CYP76B6 (Guirimand et al, 2009(Guirimand et al, , 2011a(Guirimand et al, , 2011b. However, the recent localization of two carotenoid-hydroxylating P450s to plastids (Quinlan et al, 2012) prompted us to analyze the subcellular localization of CYP71D351 (T16H2).…”

A Novel Lactone-Forming Carboxylesterase: Molecular Identification of a Tuliposide A-Converting Enzyme in Tulip

“…Recently, glucosidal intermediates for alkaloid biosynthesis have also been suggested to serve as a defense system (Nomura et al, 2008;Guirimand et al, 2010Guirimand et al, , 2011. In these systems, toxic aglycons are released from inactive glucosides by corresponding b-glucosidases and myrosinases existing in distinct subcellular compartments.…”