Understanding the role of abiotic stress in azadirachtin accumulation in callus cultures of neem (Azadirachta indica) through proteomics approach / Siti Ainnsyah Omar

Siti Ainnsyah , Omar (2023) Understanding the role of abiotic stress in azadirachtin accumulation in callus cultures of neem (Azadirachta indica) through proteomics approach / Siti Ainnsyah Omar. Masters thesis, Universiti Malaya.

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Abstract

Abiotic stress such as salinity and jasmonic acid have the potential to impact plant growth and synthesis of secondary compounds. Abiotic stresses might also act as elicitors which may stimulate the production of secondary metabolites as a part of defense system. In this research, in vitro callus culture of neem, a highly valuable medicinal tree was developed and induced with salinity and jasmonic acid stress to explore their effects on the production of azadirachtin, an essential neem secondary metabolite. Further investigation was carried out to evaluate the impact of these stresses on the expression and regulation of neem proteins via comparative proteomics. In this study, callus cultures were subjected to various concentrations of salinity stress (0.05 - 1.5% of NaCl) and jasmonic acid (1 - 6 mg/L) for 4 weeks. The outcomes showed that the callus was able to survive at increasing NaCl and jasmonic acid concentrations. However, overall fresh weight reduction was observed as the stress concentration increased. Remarkably, callus grown under salt and jasmonic stress showed an increase in the level of azadirachtin production compared to control. The highest total azadirachtin contents were recorded from callus supplemented with 6 mg/L jasmonic acid and 1.5% NaCl. In order to acquire some molecular insights of stress adaptations undergone by the callus cultures, the samples were subjected to shotgun proteomics analysis. From the analysis, a total of 129 (36 upregulated and 93 downregulated) proteins were differentially regulated in salinity stress; meanwhile, 320 (34 upregulated and 286 downregulated) proteins were associated with jasmonic acid elicitation. Overall, altered proteins in salinity stress were related to several functional groups such as cellular process, metabolic process, response to stimulus (stress, abiotic stimulus, and chemical), biological regulation, and biological process involved in interspecies interaction between organisms. Meanwhile, in jasmonic stress, differentially abundant proteins were related to several functional groups, for instance, cellular process, metabolite process, response to stimulus, localization, and development process involved in reproduction. The findings suggest that elicitation with jasmonic acid and salinity stress significantly improved the content of azadirachtin in the sample compared to control media. Thus, this information on stress-induced improvement of secondary metabolites synthesis can be used as a tool in manipulation of biosynthesis pathways for large scale production to produce natural product-like compounds under controlled conditions.

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