SUMMARYIn plants, geranylgeranyl diphosphate (GGPP, C20) synthesized by GGPP synthase (GGPPS) serves as precursor for vital metabolic branches including specialized metabolites. Here, we report the characterization of a GGPPS (CrGGPPS2) from the Madagascar periwinkle (Catharanthus roseus) and demonstrate its role in monoterpene (C10)‐indole alkaloids (MIA) biosynthesis. The expression of CrGGPPS2 was not induced in response to methyl jasmonate (MeJA), and was similar to the gene encoding type‐I protein geranylgeranyltransferase_β subunit (CrPGGT‐I_β), which modulates MIA formation in C. roseus cell cultures. Recombinant CrGGPPS2 exhibited a bona fide GGPPS activity by catalyzing the formation of GGPP as the sole product. Co‐localization of fluorescent protein fusions clearly showed CrGGPPS2 was targeted to plastids. Downregulation of CrGGPPS2 by virus‐induced gene silencing (VIGS) significantly decreased the expression of transcription factors and pathway genes related to MIA biosynthesis, resulting in reduced MIA. Chemical complementation of CrGGPPS2‐vigs leaves with geranylgeraniol (GGol, alcoholic form of GGPP) restored the negative effects of CrGGPPS2 silencing on MIA biosynthesis. In contrast to VIGS, transient and stable overexpression of CrGGPPS2 enhanced the MIA biosynthesis. Interestingly, VIGS and transgenic‐overexpression of CrGGPPS2 had no effect on the main GGPP‐derived metabolites, cholorophylls and carotenoids in C. roseus leaves. Moreover, silencing of CrPGGT‐I_β, similar to CrGGPPS2‐vigs, negatively affected the genes related to MIA biosynthesis resulting in reduced MIA. Overall, this study demonstrated that plastidial CrGGPPS2 plays an indirect but necessary role in MIA biosynthesis. We propose that CrGGPPS2 might be involved in providing GGPP for modifying proteins of the signaling pathway involved in MIA biosynthesis.
The plant Nothapodytes nimmoniana is a source of important anticancer compound camptothecin (CPT) which is being used in the treatment of colorectal, lung and ovarian cancers. Camptothecin is biosynthesized by the combination of seco-iridoid and indole pathway in plants. Majority of the biosynthetic steps and associated genes still remains mysterious. Few reactions in the seco-iridoid pathway are catalysed by cytochrome P450 enzymes. Hence, identifying transcriptionally active cytochrome P450 genes becomes essential in the elucidation of CPT biosynthetic pathway. Here, we report the identification of 94 cytochrome P450s from the assembled transcriptomic data of leaf and root of N. nimmoniana. The identified cytochrome P450s were full length and possessing all four conserved characteristic signature motifs of cytochrome P450 genes. Phylogenetic analysis of protein sequences revealed evolution, diversification and further categorized them into A type (52.12%) and non-A type (47.87%) cytochrome P450s. These 94 sequences represent 38 families and 63 subfamilies of cytochrome P450s. We also compared transcriptional activity of identified cytochrome P450s with the expression of their homologs in CPT producing plant Ophiorrhiza pumila. Based on expression profile and validation using quantitative PCR, we propose NnCYP81CB1 and NnCYP89R1 as the candidate cytochrome P450 genes involved in camptothecin biosynthesis in N. nimmoniana.
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