MsSGR interacts with MsMSRB5 to antagonistically regulate salt tolerance in Medicago sativa

Photosynthesis is the basis of plant growth and development. It directly influences forage yield and quality; however, salt stress severely inhibits photosynthetic efficiency, accelerates leaf senescence, and reduces alfalfa yield and quality. Stay-green proteins (SGR) are important regulators of chlorophyll degradation and leaf senescence. RT-qPCR analysis of alfalfa seedlings revealed that MsSGR expression is significantly induced by salt stress in leaves and roots, suggesting its potential involvement in salt tolerance. Nevertheless, the specific role of MsSGR in the salt stress response of alfalfa remains unclear. We found that MsSGR is a negative regulator of salt tolerance in alfalfa. Compared with wild-type lines, MsSGR-overexpression (MsSGR-OE) lines exhibited more severe damage to photosynthesis, increased ROS accumulation, reduced antioxidant enzyme activity, and a higher mortality rate. Conversely, the MsSGR knockout (MsSGR-KO) line showed enhanced salt tolerance and survival rates. RNA-seq analysis revealed that the salt-sensitive phenotype of the MsSGR-OE lines was primarily affected by photosynthetic and antioxidant pathways. In addition, yeast two-hybrid, split luciferase complementation, and pull-down assays confirmed an interaction between MsSGR and MsMSRB5. Moreover, chlorophyll degradation assays indicated that MsMSRB5 can inhibit MsSGR-mediated chlorophyll degradation. MsMSRB5 overexpression significantly improved alfalfa salt tolerance. Based on these findings, we propose a mechanism for alfalfa salt tolerance mediated by the MsSGR-MsMSRB5 molecular module. This study enhances our understanding of how MsSGR regulates alfalfa salt tolerance and provides breeding strategies for developing salt-tolerant alfalfa varieties.