Abstract
BACKGROUND: Late embryogenesis abundant (LEA) proteins are pivotal mediators of plant abiotic stress adaptation, but their functional roles and regulatory mechanisms in forage crops like Medicago sativa remain unclear. Through a genome-wide analysis of the LEA_3 subgroup in alfalfa, we identified MsLEA_3–6, whose expression is significantly induced by drought. To elucidate its role in drought tolerance, we conducted a comprehensive molecular and physiological characterization of MsLEA_3–6. RESULTS: Phylogenetic analysis placed MsLEA_3–6 within a conserved clade of drought-responsive LEA proteins. Its promoter harbored stress-related cis-elements, and its expression was upregulated under drought and salinity. Subcellular localization revealed plasma membrane association, while β-glucuronidase GUS staining confirmed ubiquitous expression in the roots, stems, leaves, flowers, and seeds of transgenic Arabidopsis. Overexpression of MsLEA_3–6 enhanced drought tolerance in Arabidopsis (improved root growth and germination under mannitol) and alfalfa (reduced H(2)O(2) and malondialdehyde accumulation, elevated proline/soluble sugars, and attenuated oxidative stress via ROS scavenging). A soil moisture monitoring system demonstrated that MsLEA_3–6 overexpression led to improved photosynthetic performance under drought conditions. Furthermore, the expression of stress/ABA-responsive genes (ABI5, ABF3, NCED5, and NCED9) was altered in the transgenic lines compared with those in the wild-type plants under both normal and drought conditions. Dual-luciferase assays identified MsWRKY71 (MS.gene023126) as a direct binder of the MsLEA_3–6 promoter, forming a stress-responsive regulatory module. CONCLUSIONS: Our findings identify MsLEA_3–6 as a functionally conserved, drought-responsive LEA protein that enhances osmotic adjustment and oxidative stress mitigation in transgenic plants, likely through coordinated regulation of ABA signaling and MsWRKY71-mediated transcriptional activation. While the findings support the functional relevance of MsLEA_3–6 under controlled conditions, further studies are needed to elucidate the underlying molecular mechanisms and to validate its utility in improving drought tolerance under field environments. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12870-025-07101-9.