Genome-wide identification and validation of Na+/H+ antiporter (NHX) gene family in finger millet under salt stress

• Genome-wide identification of five NHX genes in finger millet ( Eleusine coracana ) • EcNHX genes classified into vacuolar and plasma membrane NHX subfamilies. • Conserved NHX domains and structural features identified across EcNHX proteins. • Predicted interaction networks suggest coordinated roles in ion homeostasis. • Distinct temporal expression patterns of EcNHXs under salt stress. Salt stress poses a serious challenge to agricultural productivity, so it’s important to identify stress resilient genes in underutilized cereals like finger millet ( Eleusine coracana (L.) Gaertn.). In this study, we identified and characterized five EcNHX genes that encode Na + /H + antiporters and examining their physicochemical properties, structural features and regulatory elements. Subcellular localization analysis revealed that EcNHX1 - EcNHX4 are located in the vacuolar (Vac), while EcNHX5 is located in the plasma membrane (PM). Phylogenetic tree classified them into vac and PM NHX groups, with no members present in the endosomal class. Structural analysis confirmed that all the identified NHXs contain NHX protein domains. Furthermore, three-dimensional structural modeling suggested that all EcNHXs share structural features characteristic of Na + /H + antiporters. Protein-Protein interaction networks suggest that EcNHXs interact with major ion transporters, which indicating coordinated roles in ion homeostasis. Gene expression analysis of identified NHXs under salt stress exhibits early upregulation of EcNHX1-3 and late upregulation of EcNHX4-5 . These results suggested that NHX genes were involved in both immediate and long-term stress responses. Overall, this study enhances our knowledge of the structural and functional variations within the NHX gene family in finger millet and highlights potential candidate genes for future functional validation aimed at enhancing salt tolerance through crop improvement programs.