Identification and characterization of salt-, drought-, and combined salt-drought stress-responsive NHX gene family in Medicago sativa

Na⁺/H⁺ antiporters (NHXs) play essential roles in ion homeostasis and osmotic adjustment under abiotic stress; however, a systematic analysis of the NHX gene family in Medicago sativa remains limited. Here, eight MsNHX genes were identified and mapped to four chromosomes, all encoding multi-pass membrane proteins with conserved Na⁺/H⁺ exchanger domains. Phylogenetic analysis classified MsNHX proteins into vacuolar-type (Vac-type) and plasma membrane-type (PM-type) clades. Gene structure and promoter analyses revealed variation in exon-intron organization and enrichment of cis-acting elements related to hormone signaling, light responsiveness, and stress responses, indicating potential functional diversification. qRT-PCR analysis demonstrated distinct tissue-specific expression patterns, with several MsNHX genes showing relatively higher expression in roots. Under salt stress (200 mM NaCl), MsNHX1, MsNHX3, MsNHX4, MsNHX5, and MsNHX7 were significantly upregulated. Under PEG-induced drought stress (20% PEG6000), MsNHX1, MsNHX2, MsNHX3, MsNHX4, MsNHX5, and MsNHX7 showed increased expression. Under combined salt-drought stress (100 mM NaCl + 10% PEG6000), most MsNHX genes (MsNHX1-MsNHX7) were induced, including MsNHX6. These findings provide a comprehensive overview of the MsNHX gene family and identify candidate genes potentially involved in alfalfa responses to salt, drought, and combined stress conditions.