Synergistic interaction between MsRCI2D/E and HVP1 confers enhanced salt tolerance in alfalfa by regulating ion...

Salt stress is a major constraint of alfalfa (Medicago sativa) growth, disrupting ion homeostasis and inducing oxidative damage. Here, we investigated the roles of the small hydrophobic membrane proteins MsRCI2D and MsRCI2E in enhancing salt tolerance through interaction with the vacuolar H⁺-pyrophosphatase (HVP1). Yeast two-hybrid, luciferase complementation imaging (LCI), and BiFC assays confirmed their physical interaction and membrane co-localization. Transgenic alfalfa lines overexpressing MsRCI2D/E alone or co-expressing with HVP1 were evaluated under 200 mmol L^-1 NaCl. Co-expression lines exhibited improved growth performance, with reduced ROS accumulation, lower lipid peroxidation and higher activities of antioxidant enzymes (SOD, CAT, POD). They also maintained superior ionic balance, characterized by lower Na⁺/K⁺ ratios, enhanced Na⁺ efflux and greater K⁺ influx. Early in salt stress, qPCR analysis revealed the upregulation of ion transporters (NHX1, SOS1, AKT1, H⁺ -ATPase) and salt-responsive signaling genes (GIPC, PYR). Together, these results support a model in which MsRCI2D/E act as scaffold-like adaptors that promote HVP1 activity and contribute to both antioxidant defense and ion transport under salt stress. This study provides new insights into the cooperative functions of membrane protein complexes and highlights promising genetic targets for engineering salt-tolerant alfalfa cultivars.