Physiological and Metabolic Responses of Alfalfa to Cold Stress Under Saline–Alkaline Conditions

Alfalfa (<i>Medicago sativa</i> L.), a perennial leguminous herb, can tolerate cold and saline-alkaline conditions. In this study, alfalfa cultivars LJ and 218TR were exposed to saline-alkaline, cold, and saline-alkaline-cold conditions and compared in terms of phenotypes, physiological indices, key metabolite contents, and stress-responsive gene expression. Malondialdehyde, soluble sugar, proline contents and phenylalanine ammonia-lyase (PAL), superoxide dismutase, catalase, and peroxidase activities initially increased under individual stress conditions, but decreased when stresses were combined. Photosystem II maximum photochemical efficiency and chlorophyll contents decreased under individual and combined stress conditions. Nitroblue tetrazolium-stained leaves revealed that the combined stress treatment significantly increased cell mortality rates and superoxide anion levels. LJ was more tolerant to saline-alkaline, cold, and combined stress treatments than 218TR. Metabolite analyses indicated that for LJ and 218TR, salicylic acid (SA) was the most responsive metabolite to combined stress conditions. Additionally, the expression of <i>isochorismate synthase</i> (<i>ICS</i>) and <i>PAL</i> genes critical for SA biosynthesis was upregulated under single or combined stress conditions, leading to SA accumulation and improved tolerance to saline-alkaline-cold conditions. This study revealed the physiological indices and molecular changes underlying alfalfa responses to saline-alkaline stress combined with cold stress, providing a theoretical basis for breeding stress-tolerant cultivars.