Physiological and biochemical traits positively modulate tissue-specific withanolides and untargeted metabolites in Withania somnifera (L.) dunal under salinity stress

Withania somnifera (L.) Dunal (Ashwagandha) has been used in herbal medicines worldwide and in the Indian traditional medicinal system for 3000 years. It is a member of the Solanaceae family distributed across Asia, Africa, Australia, and Europe. Its bioactive secondary metabolite (withanolide) biosynthesis is sensitive to salinity stress, though the mechanism remains unexplored. Therefore, we investigated the effect of Sodium chloride (NaCl) on growth, photosynthesis, biochemical traits, tissue-specific withanolide, and untargeted metabolites in W. somnifera. Ashwagandha plants were raised in pots containing soil mixture and treated with different NaCl concentrations (0 as control, 10, 30, and 50 mM) for one month inside the greenhouse. NaCl stress significantly enhanced withaferin A (WFA) (3.79 mg/g), withanolide A (WA) (0.51 mg/g), and withanone (WN) (0.022 mg/g) at 50 mM NaCl groups in the shoot. Similarly, in the root, a significant increase in WFA (0.19 mg/g) and WN (0.0016 mg/g) were observed at 10 mM, WA (0.059 mg/g) at 30 mM, and withanolide B (WB) (0.013 mg/g) at 50 mM NaCl groups compared to control. LC-MS-based untargeted metabolite profiling revealed 37 differentially accumulated metabolites in all groups. Maximum abundance of glycyl-hydroxyproline (8X) followed by tyrosyl-valine (2X) and 3-hydroxy-beta-ionone (2X) were recorded at 50 mM NaCl groups compared to the control. This study showed for the first time that low NaCl stress enhances the biosynthesis of tissue-specific withanolides through physio-biochemical and metabolites adjustment. Overall, we demonstrated a multifaceted approach for cultivating medicinal crops in salt-affected areas with enhanced bioactive metabolites for healthcare and pharmaceutical industries.