Physiological and molecular mechanisms of Medicago ruthenica in response to different saline-alkali stresses

Soil salinization is a global issue constraining agricultural production and ecological restoration. Medicago ruthenica, a stress-tolerant leguminous forage, holds significant potential for rehabilitating salinized grasslands. This study integrated physiological-biochemical, transcriptomic, and metabolomic analyses to elucidate the mechanisms underlying its response to saline-alkali stress. Results showed that alkaline salt (NaHCO₃) stress caused significantly greater damage than neutral salts, with plant death occurring at 1.2% NaHCO₃. Transcriptome analysis revealed over 3.4-fold more differentially expressed genes under NaHCO₃ stress compared to other treatments. Four core pathways were identified: biosynthesis of secondary metabolites, motor proteins, plant hormone signal transduction, and the MAPK signaling pathway. Metabolomic analysis highlighted the central role of amino acid metabolism, with 26 common differential metabolites being amino acids or derivatives. L-arginine and L-ornithine accumulated markedly under alkaline stress. Conjoint analysis identified two key pathways—D-amino acid metabolism and lysine degradation—with D-amino acid metabolism being uniquely enriched under alkali stress. This study systematically deciphers the multi-level regulatory network of M. ruthenica under saline-alkali stress, providing theoretical insights and genetic resources for breeding tolerant forage varieties.