Genome-Wide Analysis of the MsRCI2 Gene Family in Medicago sativa and Functional Characterization of MsRCI2B in Salt Tolerance

The Rare Cold-Inducible 2 (<i>RCI2</i>) gene encodes a conserved hydrophobic peptide that plays a crucial role in ion homeostasis, membrane stability, and responses to abiotic stress. In this study, six members of the <i>MsRCI2</i> gene family were identified in <i>Medicago sativa</i> L., all of which contain highly conserved PMP₃ domains. Comparative collinearity analysis revealed syntenic relationships between <i>M. sativa</i> and <i>M. truncatula</i>, with each gene displaying distinct expression profiles under various stress conditions. Among them, <i>MsRCI2B</i> was significantly upregulated in response to salt stress. Alfalfa plants overexpressing <i>MsRCI2B</i> exhibited enhanced salt tolerance, as evidenced by increased antioxidant enzyme activities and reduced accumulation of malondialdehyde (MDA), hydrogen peroxide (H₂O₂), and superoxide anion (O₂^-) compared to wild-type plants. Furthermore, the transgenic lines maintained better Na⁺/K⁺ homeostasis under salt stress, reflected by a lower Na⁺/K⁺ ratio and significantly elevated expression of key ion transport genes, including <i>MsSOS1</i>, <i>MsAKT1</i>, and <i>MsNHX1</i>. To elucidate the molecular mechanisms underlying <i>MsRCI2B</i> function, a yeast two-hybrid (Y2H) screen identified 151 potential interacting proteins. Gene Ontology (GO) enrichment analysis revealed that these interactors are mainly involved in antioxidant defense and ion transport. Further validation confirmed direct interactions between <i>MsRCI2B</i> and both calmodulin (CaM) and vacuola H⁺-ATPase (V-H⁺-ATPase), suggesting that <i>MsRCI2B</i> contributes to ion homeostasis through interactions with CaM and V-H⁺-ATPase, thereby promoting Na⁺/K⁺ balance and enhancing salt tolerance. These findings provide new insights into the role of <i>MsRCI2B</i> in salt stress responses and underscore its potential as a genetic target for enhancing salinity tolerance in forage crops.