Genome-wide characterization of NHX gene family in Medicago sativa under abiotic stress

Soil salinization is one of the major abiotic stresses limiting global crop production. As key regulators of plant salt tolerance, the NHX gene family remains systematically uncharacterized in alfalfa (Medicago sativa). In this study, genome-wide analysis identified 35 NHX family genes (MsNHXs) in alfalfa, phylogenetically classified into three subfamilies. Subcellular localization prediction revealed that while six genes localized to the vacuole, the majority were distributed on plasma membranes, suggesting their primary contribution to salt tolerance through membrane-associated mechanisms. Tissue-specific expression analysis identified four genes (MsNHX7, MsNHX12, MsNHX21, and MsNHX24) with distinct tissue expression preferences. Analysis of abiotic stress responses demonstrated that MsNHX1 and MsNHX10 responded to salt, drought, and cold stress, while MsNHX1 and MsNHX19 were specifically induced by drought. cis-acting element analysis further revealed promoter enrichment in defense/stress-responsive and phytohormone-related elements, consistent with their roles in environmental adaptation. This study provides a theoretical foundation for elucidating salt tolerance mechanisms in alfalfa and supports its genetic improvement.