Abstract
BackgroundNothapodytes nimmoniana, a plant of pivotal medicinal significance is a source of potent anticancer monoterpene indole alkaloid (MIA) camptothecin (CPT). This compound owes its potency due to topoisomerase-I inhibitory activity. However, biosynthetic and regulatory aspects of CPT biosynthesis so far remain elusive. Production of CPT is also constrained due to unavailability of suitable in vitro experimental system. Contextually, there are two routes for the biosynthesis of MIAs: the mevalonate (MVA) pathway operating in cytosol and the methylerythritol phosphate (MEP) pathway in the plastids. Determination of relative precursor flux through either of these pathways may provide a new vista for manipulating the enhanced CPT production.ResultsIn present study, specific enzyme inhibitors of MVA (lovastatin) and MEP pathways (fosmidomycin) were used to perturb the metabolic flux in N. nimmoniana. Interaction of both these pathways was investigated at transcriptional level by using qRT-PCR and at metabolite level by evaluating secologanin, tryptamine and CPT contents. In fosmidomycin treated plants, highly significant reduction was observed in both secologanin and CPT accumulation in the range 40–57% and 64–71.5% respectively, while 4.61–7.69% increase was observed in tryptamine content as compared to control. Lovastatin treatment showed reduction in CPT (7–11%) and secologanin (7.5%) accumulation while tryptamine registered slight increase (3.84%) in comparison to control. These inhibitor mediated changes were reflected at transcriptional level via altering expression levels of deoxy-xylulose-5-phosphate reductoisomerase (DXR) and hydroxymethylglutaryl-CoA reductase (HMG). Further, mRNA expression of four more genes downstream to DXR and HMG of MEP and MVA pathways respectively were also investigated. Expression analysis also included secologanin synthase (SLS) and strictosidine synthase (STR) of seco-iridoid pathway. Present investigation also entailed development of an efficient in vitro multiplication system as a precursor to pathway flux studies. Further, a robust Agrobacterium-mediated transformed hairy root protocol was also developed for its amenability for up-scaling as a future prospect.ConclusionsMetabolic and transcriptional changes reveal differential efficacy of cytosolic and plastidial inhibitors in context to pathway flux perturbations on seco-iridoid end-product camptothecin. MEP pathway plausibly is the major precursor contributor towards CPT production. These empirical findings allude towards developing suitable biotechnological interventions for enhanced CPT production.
Highlights
Nothapodytes nimmoniana, a plant of pivotal medicinal significance is a source of potent anticancer monoterpene indole alkaloid (MIA) camptothecin (CPT)
The chemoprofiling of inhibitor treated in vitro cultures revealed drastic reduction of CPT, secologanin accumulation in presence of fosmidomycin. These results suggest the pivotal role of methylerythritol phosphate (MEP) pathway in CPT biosynthesis
Experimental design and effects of the pathway inhibitors on CPT accumulation in Nothapodytes nimmoniana From the nodal segments of field grown N. nimmoniana, an efficient and highly reproducible in vitro regeneration system was established on Woody Plant Medium (WPM) fortified with different phytohormone concentrations and combinations (Fig. 2) and their morphogenetic response is summarized in the Table 1
Summary
Experimental design and effects of the pathway inhibitors on CPT accumulation in Nothapodytes nimmoniana From the nodal segments of field grown N. nimmoniana, an efficient and highly reproducible in vitro regeneration system was established on WPM fortified with different phytohormone concentrations and combinations (Fig. 2) and their morphogenetic response is summarized in the Table 1. Expression levels of three more downstream genes of MEP pathway (DPCMEK, MECDPS, HMBEDPR) and MVA pathway (MK, PMK, DPMD) were examined (Fig. 6a-d) These genes showed 0.87–0.10 fold reduction in their transcript levels. The inhibitors fosmidomycin and lovastatin strongly reduced the transcript levels of DXR and HMG respectively It was further obvious in terms of modulating the expression levels of other examined downstream genes viz. DPCMEK, MECDPS, HMBEDPR, MK, PMK, DPMD, SLS, STR. The amount of CPT on dry weight basis (DWB) in the fruit, Fig. 7 Agrobacterium rhizogenes induced hairy roots in Nothapodytes nimmoniana: Hairy roots grown on hormone free MS medium supplemented with 500 mg/L cefotaxime (bar = 10 mm) (a, b). Hairy roots grown in hormone free MS liquid medium supplemented with 3% sucrose (bar = 15 mm) (c); PCR amplification of rol b gene from genomic DNA of hairy roots (d)
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