Extreme temperatures elicit the accumulation of cardiac glycoside and their genin units inCalotropis proceraby altering the expression of transcripts involved in its biosynthesis.

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Calotropis procera (Aiton) W.T. Aiton is a medicinal plant belonging to the family Apocynaceae as a core source of natural cardenolides. Cardiac glycosides (CGs) are steroid derivatives reported to have the ability to regulate cancer cell survival and death through multiple signaling pathways. Earlier stage-specific and wound-responsive accumulation of CGs and their genin units have already been reported. Recent cumulative evidences have implicated stress and defense response signaling in the production of secondary metabolite in plants. In this report, seasonal accumulation of CGs and its genin units have been explored along with their profiling under control vs stress conditions with a significant accumulation using LC-MS/MS. The study showed that Calotropis procera plants efficiently accumulate CGs and genin units in both winter and summer beside rainy season, as well as under thermal and salinity stress. Among the three cardenolides, the calotropagenin was accumulated more than coroglaucigenin and uzarigenin whereas CGs like calotropin, frugoside, uscharidin, uscharin, and asclepin were significantly accumulated in response to heat, cold and NaCl. Comparatively for hormonal treatments like methyl jasmonate and salicylic acid, targeted metabsolites showed upto twofold accumulation. Gene expression analysis of CG biosynthetic genes also validated the accumulation pattern of the targeted metabolite. This targeted metabolites accumulation enhances plant tolerance to adverse conditions. Gene expression analysis supports this strategy, emphasizing the plant's effective stress management. These findings significantly contribute to our understanding of how plants adapt to stress through the accumulation of metabolites, thereby enhancing their tolerance to challenging environmental conditions.