Nitrogen application enhances thermotolerance in Pinellia ternata via modulation of antioxidant capacity

• 10 mM ammonium nitrate significantly improved heat tolerance in P. ternata. • Nitrogen application enhances the enzymatic activities of CAT, SOD, and POD. • Nitrogen application induced the accumulation of quercetin, myricetin, and nicotine. • Nitrogen application substantially suppressed ROS accumulation and MDA content. Pinellia ternata (Thunb.) Breit. is a commonly used medicinal herb in China. Heat stress (HS) inhibits the growth of P. ternata and reduces both yield and tuber quality. The application of exogenous substances is an effective strategy to enhance plant thermotolerance. Nitrogen has been reported to participate in the regulation of heat resistance in plants, but its role in P. ternata remains unclear. This study found that 10 mM ammonium nitrate significantly increased the survival rate and photosynthetic efficiency of P. ternata under HS. Additionally, ammonium nitrate application induced the activities of antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD), while suppressing the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA). Integrated transcriptomic and metabolomic analyses revealed that differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) were significantly enriched in the 'flavonoid biosynthesis pathway' and 'tropane, piperidine, and pyridine alkaloid biosynthesis pathway'. Notably, the relative contents of quercetin, myricetin, and nicotine were markedly induced by HS and ammonium nitrate, with key biosynthetic genes exhibiting expression patterns consistent with metabolite accumulation. In conclusion, moderate nitrogen application simultaneously enhances both enzymatic and non-enzymatic antioxidant systems in P. ternata under HS, effectively mitigating oxidative damage caused by ROS and thereby improving thermotolerance. Our results provide new insights into nitrogen-mediated heat resistance mechanisms and suggest practical applications for crop protection in warming climates.