Physiological and molecular mechanisms of cadmium stress tolerance in timothy (Phleum pratense L.) seedlings mediated by exogenous melatonin

Melatonin (M) plays a key role in plant growth and stress tolerance; however, its role in mitigating cadmium (Cd) stress in timothy (Phleum pratense L.) remains poorly understood. In this study, timothy seedlings were exposed to four treatments: control (CK), melatonin (M), cadmium (Cd), and combined melatonin and cadmium (M + Cd) under hydroponic conditions. The results showed that M alleviated Cd-induced toxicity, as evidenced by improved biomass, photosynthetic performance, and enhanced antioxidant enzyme activities under M + Cd treatment compared with Cd alone. Transcriptomic analysis revealed that M-responsive differentially expressed genes (DEGs) under Cd stress were significantly enriched in oxidoreductase activity, catalytic activity, and phenylpropanoid biosynthesis pathways. In addition, genes related to metal transport, including heavy metal-associated (HMA) and natural resistance-associated macrophage protein (Nramp), along with antioxidant enzyme and phytohormone-related genes, displayed distinct expression patterns across treatments. Functional validation in yeast demonstrated that overexpression of the HMA gene Cluster-13128.5368 significantly enhanced Cd tolerance. Overall, M may enhance Cd tolerance in timothy by coordinating root retention and shoot detoxification. These findings provide new insights into the physiological and molecular mechanisms underlying M-mediated Cd tolerance in timothy seedlings.