Physiological and Metabolite Responses of Alfalfa to Cold Stress under Saline-Alkali Conditions

Alfalfa (Medicago sativa L.), a perennial leguminous herb, exhibits robust cold and saline-alkali tolerance. In this study, two alfalfa cultivars, LJ and 218TR, were treated with saline-alkali stress, cold stress, and combined saline-alkali-cold stress, and phenotype, physiology, key metabolite and stress-responsive genes were analyzed. The results showed malondialdehyde, soluble sugar, proline content, and the activities of phenylalanine ammonia-lyase (PAL), superoxide dismutase, catalase, and peroxidase initially increased under individual stresses but declined after combined stress. The maximum photochemical efficiency of photosystem II and chlorophyll content declined under individual and combined stresses. The staining of leaves revealed that combined stress induced significantly higher cell mortality and accumulation of superoxide anion compared to individual stresses. LJ exhibited superior resistant to saline-alkali, cold, and combined stress compared to 218TR. Metabolite analysis showed salicylic acid (SA) in two alfalfa was the most responsive metabolite to combined stress. The isochorismate synthase (ICS) and PAL as critical genes for SA biosynthesis were up-regulated in expression under single or combined stress, and promoted SA accumulation, thereby improving alfalfa resilience to combined saline-alkali-cold stress. This study elucidates the physiological and molecular mechanisms underlying alfalfa’s response to combined saline-alkali and cold stress, providing a theoretical basis for breeding stress-tolerant cultivars.