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Abstract  In Catharanthus roseus, the first step of monoterpenoid indole alkaloids (MIA) biosynthesis results from the condensation of the indole precursor tryptamine with the terpenoid precursor secologanin. Secologanin biosynthesis requires two successive biosynthetic pathways, the plastidial methyl-d-erythritol 4-phosphate (MEP) pathway and the monoterpene secoiridoid pathway. In C. roseus cell culture, the expression of several genes encoding enzymes of these two pathways is dramatically down-regulated by auxin, while strongly enhanced by cytokinin and methyl-jasmonate. Furthermore, our previous studies have shown that protein prenylation events are also involved in the transcriptional activation of some of these genes. In the present work, we investigate the involvement of protein prenylation in the jasmonate signalling pathway leading to MIA biosynthesis. Inhibition of protein prenyltransferase down-regulates the methyl-jasmonate-induced expression of MEP and monoterpene secoiridoid pathway genes and thus abolishes MIA biosynthesis. Jointly, it also inhibits the methyl-jasmonate-induced expression of the AP2/ERF transcription factor ORCA3 that acts as a central regulator of MIA biosynthesis. Finally, a specific silencing of protein prenyltransferases mediated by RNA interference in C. roseus cells shows that inhibition of type I protein geranylgeranyltransferase (PGGT-I) down-regulates the methyl-jasmonate-induced expression of ORCA3, suggesting that PGGT-I prenylated proteins are part of the early steps of jasmonate signalling leading to MIA biosynthesis. [ABSTRACT FROM AUTHOR]

Abstract  Precursor feeding experiments have shown that the terpene moiety biosynthesis is the most rate-limiting step of terpenoid indole alkaloids (TIA) produced by Catharanthus roseus suspension cells. The biosynthesis of TIA terpene moiety is strictly regulated by hormones: auxin inhibits, jasmonate stimulates and cytokinin and ethylene enhance their biosynthesis. Biochemical analyses coupled to molecular approaches have outlined a regulatory cross-talk between two metabolite pathways leading to the biosynthesis of terpene precursors. Terpenes derivatives produced through the mevalonic acid (MVA) pathway regulate the activity of the 2-C-methyl-d-erythritol 4-phosphate (MEP) non-mevalonate pathway producing the precursor of the TIA terpene moiety. This cross-talk involves regulatory prenylated proteins and acts on the regulation of the expression of early steps of monoterpenoid biosynthesis (ESMB) genes. The expression of the genes involved in the TIA terpene moiety biosynthesis is regulated in part by a general TIA transcription factor, ORCA3, and other unidentified transcription factors. This review sums up the essential information obtained at the metabolic, physiological, biochemical and molecular levels of regulation of TIA terpene moiety biosynthesis in C. roseus. We propose a synthetic scheme of the general regulatory network emerging from these experimental data, and discuss the related hypothesis and prospect in elucidating other key events involved in the regulation of TIA terpene moiety biosynthesis. [ABSTRACT FROM AUTHOR]

Abstract CaaX-prenyltransferases (CaaX-PTases) catalyse the covalent attachment of isoprenyl groups to conserved cysteine residues located at the C-terminal CaaX motif of a protein substrate. This post-translational modification is required for the function and/or subcellular localization of some transcription factors and components of signal transduction and membrane trafficking machinery. CaaX-PTases, including protein farnesyltransferase (PFT) and type-I protein geranylgeranyltransferase (PGGT-I), are heterodimeric enzymes composed of a common a subunit and a specific subunit. We have established RNA interference cell lines targeting the subunits of PFT and PGGT-I, respectively, in the Catharanthus roseus C20D cell line, which synthesizes monoterpenoid indole alkaloids in response to auxin depletion from the culture medium. In both types of RNAi cell lines, expression of a subset of genes involved in the early stage of monoterpenoid biosynthetic pathway (ESMB genes), including the MEP pathway, is strongly decreased. The role of CaaX-PTases in ESMB gene regulation was confirmed by using the general prenyltransferase inhibitor s-perillyl alcohol (SP) and the specific PFT inhibitor Manumycin A on the wild type line. Furthermore, supplementation of SP inhibited cells with monoterpenoid intermediates downstream of the steps encoded by the ESMB genes restores monoterpenoid indole alkaloids biosynthesis. We conclude that protein targets for both PFT and PGGT-I are required for the expression of ESMB genes and monoterpenoid biosynthesis in C. roseus, this represents a non previously described role for protein prenyltransferase in plants. [ABSTRACT FROM AUTHOR]