Enhanced azadirachtin production in neem (Azadirachta indica) callus through NaCl elicitation: Insights into differential protein regulation via shotgun proteomics.

With their remarkable bioactivity and evolving commercial importance, plant secondary metabolites (PSMs) have gained significant research interest in recent years. Plant tissue culture serves as a credible tool to examine how abiotic stresses modulate the production of PSMs, enabling clear insights into plant stress responses and the prospects for controlled synthesis of bioactive compounds. Azadirachta indica, or neem has been recognized as a repository of secondary metabolites for centuries, particularly for the compound named azadirachtin, due to its bio-pesticidal and high antioxidant properties. Introducing salt stress as an elicitor makes it possible to enhance the synthesis of secondary metabolites, specifically azadirachtin. Thus, in this research, in vitro callus cultures of neem were micro-propagated and induced with salinity stress to explore their effects on the production of azadirachtin and identify potential proteins associated with salinity stress through comparative shotgun proteomics (LCMS/MS). To induce salinity stress, 2-month-old calli were subjected to various concentrations of NaCl (0.05-1.5%) for 4 weeks. The results showed that the callus cultures were able to adapt and survive in the salinity treatments, but displayed a reduction in fresh weight as the NaCl concentration increased. Notably, azadirachtin production was significantly enhanced in the salinity treatment compared to control, where 1.5% NaCl-treated calli produced the highest azadirachtin amount (10.847 ± 0.037 mg/g DW). The proteomics analysis showed that key proteins related to primary metabolism, such as defence, energy, cell structure, redox, transcriptional and photosynthesis, were predominantly differentially regulated (36 upregulated and 93 downregulated). While a few proteins were identified as being regulated in secondary metabolism, they were not directly involved in the synthesis of azadirachtin. In conjunction with azadirachtin elicitation, salinity stress treatment could therefore be successfully applied in commercial settings for the controlled synthesis of azadirachtin and other plant-based compounds. Further complementary omics approaches can be employed to enhance molecular-level modifications, to facilitate large-scale production of bioactive compounds in the future.