Alfalfa MsSOS2 confers salinity tolerance by promoting lateral root growth and regulating Na⁺/K⁺ homeostasis

Salinity is a major abiotic stress limiting plant productivity. The Salt Overly Sensitive (SOS) pathway, comprising SOS1, SOS2, and SOS3, is central to ion homeostasis under salt stress. While the pathway has been extensively studied in model plants, its components remain uncharacterized in Medicago sativa (alfalfa), a moderately salt-tolerant forage crop of global economic importance. Here, we report the functional characterization of MsSOS2, a CIPK family gene and putative ortholog of AtSOS2. Sequence analysis revealed that MsSOS2 possesses conserved domains essential for kinase activity, including a kinase domain, activation loop, the phosphatidic acid (PA)-binding site, and NAF motif. Phylogenetic analysis placed MsSOS2 with AtSOS2 and OsSOS2. Yeast-two-hybrid assays confirmed the interaction between MsSOS2 and MsSOS3, mimicking the AtSOS2-AtSOS3 complex. Transgenic expression of MsSOS2 in the Arabidopsis sos2 mutant restored salinity tolerance during seed germination, seedling development, and late vegetative growth. Ion profiling showed reduced Na accumulation and enhanced K retention in MsSOS2-expressing lines under salinity. We propose that MsSOS2 activates AtSOS1 to facilitate Na⁺ efflux and disrupts AtCBL10-mediated inhibition of AKT1 to promote K⁺ uptake. This study provides the first functional validation of an SOS pathway component in alfalfa and highlights MsSOS2 as a promising candidate for improving salinity tolerance in legumes.