The synergistic roles of MsRCI2B and MsRCI2E in the regulation of ion balance and ROS homeostasis in alfalfa under salt stress.

Under salt stress, plasma membrane proteins regulate ion homeostasis and the balance between reactive oxygen species (ROS). In this study, we investigated the functions of two small membrane proteins-MsRCI2B (tailless) and MsRCI2E (tailed)-encoded by the RCI2 (Rare Cold Inducible 2) gene family in Medicago sativa (alfalfa). We identified the distinct subcellular localization and expression patterns of these proteins under salt stress. Using yeast two-hybrid (Y2H), GST pull-down, and bimolecular fluorescence complementation (BiFC) assays, we confirmed the physical interactions between MsRCI2B and MsRCI2E. Transgenic alfalfa lines overexpressing MsRCI2(OE#RCI2) and co-expressing both MsRCI2B and MsRCI2E (OE#RCI2E-2B) were developed to explore their roles in salt tolerance. Interestingly, the C-terminal tail of MsRCI2E negatively affects salt tolerance; however, its interaction with MsRCI2B mitigates this adverse effect. To further understand the regulatory mechanisms, we screened for plasma membrane proteins (PMPs) that interact with MsRCI2B or MsRCI2E using a DUALmembrane yeast two-hybrid system. MsCaM1 interacts with MsRCI2B, whereas MsPIP1;4 and MsHVP1 specifically interact with MsRCI2E. Notably, the MsRCI2E-PIP1;4 interaction influenced the intracellular trafficking of PIP1;4, reducing its presence on the plasma membrane and thereby limiting the export of HO, which helps maintain ROS homeostasis. Additionally, the interaction between MsRCI2E and HVP1 stabilized ion homeostasis by decreasing Na concentration in the cytoplasm under salt stress. Overall, our study provides new insights into the molecular mechanisms through which MsRCI2B and MsRCI2E coordinate the ion and ROS balance under salt stress and offering promising strategies for enhancing crop tolerance to salinity.