Heteromeric and Homomeric Geranyl Diphosphate Synthases from Catharanthus roseus and Their Role in Monoterpene Indole Alkaloid Biosynthesis

Abstract

Catharanthus roseus is the sole source of two most important monoterpene indole alkaloid (MIA) anti-cancer agents: vinblastine and vincristine. MIAs possess a terpene and an indole moiety derived from terpenoid and shikimate pathways, respectively. Geranyl diphosphate (GPP), the entry point to the formation of terpene moiety, is a product of the condensation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) by GPP synthase (GPPS). Here, we report three genes encoding proteins with sequence similarity to large subunit (CrGPPS.LSU) and small subunit (CrGPPS.SSU) of heteromeric GPPSs, and a homomeric GPPSs. CrGPPS.LSU is a bifunctional enzyme producing both GPP and geranyl geranyl diphosphate (GGPP), CrGPPS.SSU is inactive, whereas CrGPPS is a homomeric enzyme forming GPP. Co-expression of both subunits in Escherichia coli resulted in heteromeric enzyme with enhanced activity producing only GPP. While CrGPPS.LSU and CrGPPS showed higher expression in older and younger leaves, respectively, CrGPPS.SSU showed an increasing trend and decreased gradually. Methyl jasmonate (MeJA) treatment of leaves significantly induced the expression of only CrGPPS.SSU. GFP localization indicated that CrGPPS.SSU is plastidial whereas CrGPPS is mitochondrial. Transient overexpression of AmGPPS.SSU in C. roseus leaves resulted in increased vindoline, immediate monomeric precursor of vinblastine and vincristine. Although C. roseus has both heteromeric and homomeric GPPS enzymes, our results implicate the involvement of only heteromeric GPPS with CrGPPS.SSU regulating the GPP flux for MIA biosynthesis.

Precursor feeding studies and molecular characterization of geraniol synthase establish the limiting role of geraniol in monoterpene indole alkaloid biosynthesis in Catharanthus roseus leaves

“…This was in agreement with RNA in situ hybridization studies, which has shown the distribution of CrGES in internal phloem associated parenchyma (IPAP) cells of leaves, as well as carpels and stamens of flowers [14]. The gene encoding the small subunit of GPPS (GPPS.SSU), which was reported to regulate the formation of GPP in C. roseus, also exhibited higher expression in leaf, shoot and flowers [33] indicating co-regulation of GPP and geraniol production. MeJA is known to induce the expression of many genes of secondary metabolic pathways, including the genes of MIA biosynthesis leading to the accumulation of the corresponding metabolites such as alkaloids, terpenoids, flavonoids, and glucosinolates in different plant species [44].…”

“…MeJA is known to induce the expression of many genes of secondary metabolic pathways, including the genes of MIA biosynthesis leading to the accumulation of the corresponding metabolites such as alkaloids, terpenoids, flavonoids, and glucosinolates in different plant species [44]. In C. roseus, it has been previously reported that several MIA pathway genes such as GPPS, G8O, TDC, STR, D4H, DAT, and MIA pathway regulators like ORCA2, ORCA3 are induced in response to MeJA resulting in increased alkaloid accumulation [4,33,44,45]. Induction of CrGES in response to MeJA in leaf tissue (Fig.…”

“…In C. roseus, transient over-expression strategy has been utilized to validate gene function [31][32][33]. Since VIGS of CrGES in C. roseus leaves resulted in reduced MIA content ( Fig.…”

Transcriptomic insight into terpenoid and carbazole alkaloid biosynthesis, and functional characterization of two terpene synthases in curry tree (Murraya koenigii)

“…Primers used for cloning into GFP vector are listed in Table S9. The fusion constructs were transformed into Arabidopsis protoplasts as described previously47. Transient expression of GFP fusion proteins was observed 16–20 h after transformation using confocal laser scanning microscope (Zeiss LSM 880) with a 40 × 1.55 numerical aperture lens.…”

Virus‐induced gene silencing of Withania somnifera squalene synthase negatively regulates sterol and defence‐related genes resulting in reduced withanolides and biotic stress tolerance

“…RNA isolation, cDNA synthesis and qPCR analysis were performed as reported previously (Rai et al, 2013;Rastogi et al, 2014). For semi-quantitative RT-PCR, 18S rRNA gene served as an endogenous control for RNA quantity.…”