Exogenous Melatonin Enhances Salt Tolerance in Alfalfa Through Dynamic Coordination of Molecular and Physiological Responses

Soil salinization severely constrains the productivity of Medicago sativa L. Although exogenous melatonin (MT) has been proven to effectively alleviate salt stress injury in plants, the molecular regulatory networks underlying its function during the early stages of stress response remain not fully elucidated. In this study, we systematically investigated the specific regulatory mechanisms of exogenous MT-mediated salt tolerance in alfalfa seedlings during the early phase (12–24 h) of salt stress by integrating physiological, biochemical, and transcriptomic analyses. The results showed that MT treatment significantly inhibited membrane lipid peroxidation (indicated by decreased MDA content) in leaves and upregulated the activities of antioxidant enzymes as well as the levels of osmoprotectants, such as soluble sugars. Transcriptomic (RNA-seq) analysis revealed that MT induced a precise strategy of temporal transcriptional reconfiguration. At the initial stage of stress (12 h), MT preferentially downregulated the expression of genes related to ribosome biogenesis and chromatin remodeling. This transcriptional suppression suggests that plants adopted an “energy saving strategy,” aiming to minimize basal metabolic consumption and potentially reallocate limited energy resources toward the antioxidant defense system. Subsequently, at 24 h, MT orchestrated the comprehensive activation of the ABA signaling cascade and secondary metabolic pathways, such as phenylpropanoid and flavonoid biosynthesis, thereby establishing a long-term chemical defense barrier. Furthermore, weighted gene co-expression network analysis (WGCNA) identified ABF2 and Susy as key hub genes mediating soluble sugar accumulation. This study elucidates the molecular basis by which melatonin enhances early salt tolerance in alfalfa through a temporal transition from an “energy-saving” strategy to “active defense,” providing new theoretical insights for the molecular breeding of stress resistance in leguminous forage crops.