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Proteome study of somatic embryogenesis in Nothapodytes nimmoniana (J. Graham) Mabberly.

ABSTRACT: Somatic embryogenesis (SE) is the most suitable biotechnological tool for the rapid clonal propagation of endangered woody plants, but many bottlenecks limit understanding its molecular and physiological processes in Nothapodytes nimmoniana. Combinations of two-dimensional electrophoresis (2-DE) and mass spectrometry (MaSp) were used to study proteomic expression changes during SE of the forest tree. Callus was induced from mature seed embryos, and embryogenic callus (EC) obtained at very low frequency after about 6 month culture. Globular embryos were induced from the seed embryo-derived EC and the subsequent stages of the SE. Analysis of the extracted proteins from globular, heart/torpedo-shaped, and maturing embryo stages resolved in the 2-DE gels showed increased protein expression across developmental stages of the somatic embryos. The mass spectrometric analysis with database search aided identification of 55 out of 100 and 54 selected protein spots. Identified proteins classified by the cellular role which they perform are involved in aspects of stress responses, energy metabolism, carbon fixation, secondary metabolism, and other metabolic functions, while three proteins are of unknown cellular role. The putative role of the expressed proteins during SE provided insight into the physiology of somatic embryo development in N. nimmoniana.

SUBMITTER: Isah T

PROVIDER: S-EPMC6399373 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Proteome study of somatic embryogenesis in <i>Nothapodytes nimmoniana</i> (J. Graham) Mabberly.

Isah Tasiu T

3 Biotech 20190304 4

Somatic embryogenesis (SE) is the most suitable biotechnological tool for the rapid clonal propagation of endangered woody plants, but many bottlenecks limit understanding its molecular and physiological processes in <i>Nothapodytes nimmoniana</i>. Combinations of two-dimensional electrophoresis (2-DE) and mass spectrometry (MaSp) were used to study proteomic expression changes during SE of the forest tree. Callus was induced from mature seed embryos, and embryogenic callus (EC) obtained at very ...[more]

PMID: 30854279

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Project description: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.

Dataset Information

Proteome study of somatic embryogenesis in Nothapodytes nimmoniana (J. Graham) Mabberly.

Publications

Proteome study of somatic embryogenesis in <i>Nothapodytes nimmoniana</i> (J. Graham) Mabberly.

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