Sweet clover (Melilotus albus) is a high-quality leguminous forage grass with salinity tolerance, drought tolerance, and cold hardiness. We selected four varieties of sweet clover with different sensitivities (061898, 061930, No. 55 white flower, and Ningxia white flower) and analyzed the effects of different concentrations of three sodium salts (Na₂CO₃, NaHCO₃, and NaCl) on their physiology and biochemistry responses. Growth and development indexes (such as germination rate, root length, shoot length), chlorophyll content, osmotic regulators (proline, soluble sugar), malondialdehyde (MDA), superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were determined under saline–alkali stress. Seed germination and seedling growth of all four clover species were significantly inhibited under saline–alkali stress. During germination, seed germination rate, root length, and shoot length decreased with increasing saline and alkaline concentration. Under saline–alkali stress, chlorophyll content tended to increase and then decrease, cell damage and death increased, and malondialdehyde, soluble sugar, and proline content tended to increase and then decrease. Moreover, the activities of SOD, POD, and CAT all increased and then decreased. Under Na₂CO₃ stress, the decrease in chlorophyll content of the resistant variety 061898 was less than in the sensitive Ningxia white flower variety. As the concentrations of Na₂CO₃, NaHCO₃, and NaCl increased, the maximum photochemical efficiency of PSII was significantly affected. The resistant 061898 is capable of maintaining higher photosynthetic efficiency. Furthermore, under treatments with the three kinds of saline–alkali solutions, cell damage and death for Ningxia white flower were greater than in 061898. For 061898, the increases in soluble sugar and proline content were greater and the increase in malondialdehyde content was less, while the antioxidant enzyme activities were higher than those in Ningxia white flower. All four sweet clover varieties had higher stress resistance with neutral than with alkaline salts. When stressed by medium to high saline–alkali concentrations, sweet clover seedlings had increased osmotic substance content, enhanced antioxidant enzyme activity, and regulated physiological metabolism. Additionally, sweet clovers regulated the expression of WRKY33, GH3, CYCD3, OXI1, MKK2, MYC2, JAZ, COI1, PYL, PP2C, TGA, and MPK3 to adapt to the saline–alkali environment and improve saline–alkali tolerance. Our analysis of the sweet clover salinity tolerance mechanism contributes to its further use and is of significant importance for addressing land salinization and promoting sustainable agricultural and pastoral practices in China.

Alfalfa (Medicago sativa 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 isochorismate synthase (ICS) and PAL 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.

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 p henotype, physiology , key metaboli te and stress-responsive genes were analyzed . The results showed m alondialdehyde, 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 . T he 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 anio n 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 wa s 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 promote d 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.