Synergistic effects of chitin and nitric oxide on phytochemical and molecular defense mechanisms in Andrographis paniculata under nickel stress

Nickel (Ni) contamination is an increasing environmental concern that negatively affects plant growth, physiological performance, and the biosynthesis of medicinally important secondary metabolites. The use of natural biostimulants such as chitin and nitric oxide (NO) has emerged as a promising strategy to enhance plant tolerance against heavy metal stress. Therefore, this study investigated the potential of chitin and NO to enhance the physiological and phytochemical responses of Andrographis paniculata under Ni stress. The study was designed to assess the effects of varying concentrations of chitin (0, 15, and 30 µM) and NO (0, 0.5, and 1 g/L) on several growth parameters, including photosynthetic pigments, total phenolic content, total flavonoid content, protein accumulation, key secondary metabolites (andrographolide, neoandrographolide, and 14-deoxy-11,12-didehydroandrographolide), and the expression of isoprenoid biosynthesis-related genes (HMGR, HMGS, DXR, and DXS) in A. paniculata under different levels of Ni stress (0, 1.5, and 3 mM). The results showed that Ni stress significantly reduced chlorophyll, carotenoid, phenolic, and protein contents, whereas it altered secondary metabolite profiles and gene expression patterns. Application of NO and chitin significantly improved chlorophyll a, chlorophyll b, carotenoids, total phenols, and protein content under Ni stress conditions. In addition, NO and chitin treatments enhanced the accumulation of key bioactive compounds and positively regulated the expression of genes involved in terpenoid biosynthesis pathways. Overall, the findings indicate that NO and chitin alleviate Ni-induced stress in A. paniculata primarily through improving physiological performance and enhancing the accumulation of non-enzymatic antioxidant compounds such as phenolics and flavonoids, thereby contributing to improved metabolic stability and secondary metabolite production under heavy metal stress.