Chitosan activates a sequential Ca 2+ -ROS-NO-jasmonate signaling cascade governing withanolide accumulation in Withania somnifera.

Chitosan promotes withanolide accumulation in W. somnifera; however, the temporal organization of the underlying signaling network remains unresolved. We examined whether calcium (Ca2⁺), reactive oxygen species (ROS), nitric oxide (NO), and jasmone form an ordered cascade linking elicitor perception to specialized metabolism. Sixty-day-old greenhouse-grown plants were treated with 100 mg L-1 foliar chitosan alone or after pretreatment with LaCl₃, diphenyleneiodonium, cPTIO, or diethyldithiocarbamate, with inhibitor-only controls. Cytosolic Ca2+ -dependent fluorescence, NADPH oxidase activity, hydrogen peroxide, superoxide, NO, jasmone acid (JA), and jasmonoyl-isoleucine (JA-Ile) were monitored from 0 minutes to 7 d. Chitosan induced a temporally ordered response: Ca2+ fluorescence increased 3.01-fold within 15 minutes and returned to baseline by 3 hours; NADPH oxidase activity and hydrogen peroxide peaked at 3 hours; NO peaked at 6 hours; and JA and JA-Ile peaked at 12 hours. LaCl₃ suppressed all downstream responses, diphenyleneiodonium largely eliminated the oxidative burst, cPTIO abolished NO accumulation without affecting ROS production, and diethyldithiocarbamate selectively suppressed jasmone accumulation. These interventions support the sequence Ca2+→ NADPH oxidase-dependent ROS → NO → jasmone. Total targeted withanolides increased by 84% at day 7, including a 113.5% increase in withaferin A, whereas pathway inhibitors reduced this gain by 36%-49%. Enhanced antioxidant activity and improved glutathione and ascorbate redox states occurred without appreciable changes in lipid peroxidation, electrolyte leakage, photosynthesis, or biomass, indicating regulated redox signaling rather than injury. Untargeted LC-MS/MS revealed enrichment of steroid, terpenoid-backbone, and α-linolenic-acid metabolism and a 61.7% increase in the withanolide-precursor pool. Early signaling integrals predicted day-7 withanolide accumulation (r = 0.88-0.92), while structural equation modeling identified jasmone as the strongest direct predictor. Computational analyzes prioritized withaferin A and withanolide A as candidate IκB kinase β ligands, although biochemical validation is required. These findings show that signal timing organizes chitosan-induced withanolide biosynthesis in W. somnifera, with signaling dynamics predicting metabolic output. This pharmacological asymmetry provides strong directional evidence for pathway ordering, but it does not constitute definitive molecular proof of causality because inhibitor specificity is incomplete and no genetic or rescue experiment was performed.