yang yue, Yuhan Liu, Peiqin Miao, Dan Yang, Kuiju Niu
L.), yet the metabolic determinants underlying cultivar-specific cold tolerance remain insufficiently understood. In this study, we compared a cold-tolerant cultivar ('WL343') and a cold-sensitive cultivar ('Gannong No. 4') using an integrated framework combining physiological analyses, non-targeted metabolomics, and functional validation with exogenous melatonin. Cold stress caused pronounced membrane damage, oxidative stress, and growth inhibition in 'Gannong No. 4', whereas 'WL343' maintained greater membrane integrity, stronger antioxidant capacity, and enhanced osmotic adjustment. Metabolomic profiling revealed extensive cold-induced metabolic reprogramming in both cultivars, but with distinct pathway prioritization. Notably, the tryptophan-melatonin metabolic pathway was specifically and strongly activated in 'WL343', leading to significant endogenous melatonin accumulation, while 'Gannong No. 4' exhibited a weaker response and preferential regulation of secondary metabolism, particularly isoflavonoid-related pathways. Functional experiments further demonstrated that exogenous melatonin markedly alleviated cold-induced injury in 'Gannong No. 4' by reducing reactive oxygen species accumulation, stabilizing membranes, and enhancing antioxidant enzyme activities and osmolyte accumulation. Among the tested concentrations, 100 μM melatonin conferred the greatest protective effect. Collectively, these results identify melatonin as a key functional metabolite underlying cold tolerance in alfalfa and highlight the tryptophan-melatonin pathway as a central metabolic hub conferring cultivar-specific cold resilience. This study provides a metabolic basis for improving alfalfa cold tolerance through breeding and targeted cultivation strategies.