Late embryogenesis abundant (LEA) proteins are typical stress-related proteins widely distributed across various organisms. Their anti-stress functions in higher plants have garnered significant attention and have been extensively studied
however, no such studies have been reported on the entire protein family in Medicago sativa. In this study, we identified a total of 83 MsLEA proteins in M. sativa and conducted a comprehensive analysis to elucidate their functions in response to abiotic stresses. The results indicated that these proteins could be classified into seven groups and were distributed across eight chromosomes. Collineation analysis revealed that segmental duplication primarily drove the expansion of MsLEA genes. Furthermore, the promoters of MsLEA genes were found to be enriched with cis-elements associated with various stress responses. Through transcriptome and qRT-PCR analysis, nine MsLEA genes related to drought, salinity, and cold stress were identified, with MsLEA69 selected for further validation. The ectopic expression of MsLEA69 improves osmotic and extreme temperature tolerance by increasing the activity of stress-related enzymes in both prokaryotic and eukaryotic cells. These comprehensive analyses and identifications lay the groundwork for future research into the functional mechanisms of MsLEA proteins and offer potential candidate genes for enhancing resistance breeding in M. sativa.