Melatonin and calcium synergistically improve salt tolerance in alfalfa (Medicago sativa. L)

Salt stress has become a significant constraint on agricultural yield and plant development. Melatonin (MT) and calcium (Ca^2 +) are well recognized as key elements in salt-stress resistance in plants. In spite of this, the underlying mechanisms governing the effects of MT and Ca²⁺ interplay on alfalfa (Medicago sativa L.) salt tolerance remain a mystery. This research sought to investigate the regulatory mechanisms of MT and Ca²⁺ in alfalfa salinity response through physiological and comparative transcriptome. Physiological results indicated that exogenous MT and CaCl₂ alleviated salinity stress-induced damage to alfalfa, which was reflected by increased plant growth parameters, Ca²⁺ in the cytosol ([Ca²⁺]_cyt), antioxidant enzyme activities, K⁺/Na⁺ ratio, endogenous MT content, and decreased of electrolyte leakage (EL) and superoxide anion (O₂^·−) levels, especially when they were applied simultaneously. Transcriptome analysis suggested that MT and Ca²⁺ mainly regulated genes related to Ca²⁺ signal transduction, hormone signal transduction, photosynthesis, reactive oxygen species (ROS) metabolism and ion transport to mediate salt stress in alfalfa. Additionally, transcription factor (TF) families like ERF, bHLH, WRKY, and NAC were also active in salt stress response mediated by MT and Ca²⁺. Moreover, nine hub genes were identified by weighted gene co-expression network analysis (WGCNA). Overall, this research revealed that MT and Ca²⁺ exert a synergistic influence on the regulation of salinity resistance, offering valuable insights for the development of salt-tolerant alfalfa varieties.