Physiological and Molecular Mechanisms of Medicago ruthenica in Response to Different Saline-Alkali Stresses

Soil salinization is a global issue that constrains agricultural production and ecological restoration. Melissitus ruthenica , a stress-tolerant leguminous forage, holds significant potential for the rehabilitation of salinized grasslands. This study systematically compared the effects of three single salts (NaCl, Na₂SO₄, NaHCO₃) and their mixed saline-alkali solutions at varying concentrations on M. ruthenica seedlings. Through integrated physiological-biochemical assays, as well as transcriptomic and metabolomic analyses, we elucidated the physiological and molecular mechanisms underlying the response of M. ruthenica to saline-alkali stress. The results indicated that alkaline salt (NaHCO₃) stress caused significantly greater damage to plants compared to neutral salt, with M. ruthenica being unable to survive under 1.2% NaHCO₃ stress. Osmotic adjustment substances increased significantly with rising stress concentrations and were notably higher under alkaline salt treatment than in other treatments ( P < 0.05 ). Transcriptome analysis revealed that the number of upregulated genes (4,835) and downregulated genes (7,286) in the NaHCO₃ versus CK groups was over 3.4 times higher than in other groups. The four core pathways identified were the biosynthesis of secondary metabolites, motor proteins, plant hormone signal transduction, and the MAPK signaling pathway in plants. Transcriptomic results demonstrated that amino acid metabolism plays a central role in the stress response, with 26 common differential metabolites identified as amino acids and their derivatives. L-arginine and L-ornithine exhibited significant accumulation under alkaline stress. Two pathways, D-amino acid metabolism and lysine degradation, were identified through conjoint analysis, with D-amino acid metabolism showing significantly greater enrichment under alkali stress compared to other treatments. This study systematically elucidates the multi-level regulatory mechanisms of M. ruthenica in response to saline-alkali stress, providing both theoretical foundations and candidate gene resources for the genetic improvement of saline-alkali tolerant forage varieties.