Functional Characterization of the 14-3-3 Gene Family in Alfalfa and the Role of MsGRF2 in Drought Response Mechanisms

Drought stress affects crop growth and development, significantly reducing crop yield and quality. Alfalfa ( L.), the most widely cultivated forage crop, is particularly susceptible to drought. The general regulatory factor (GRF) protein 14-3-3, a highly conserved family in plants, specifically recognizes and binds to phosphoserine residues in target proteins, regulating both plant development and responses to environmental stressors. In this study, 66 alfalfa 14-3-3 proteins were identified, and the full-length gene was cloned and functionally analyzed. The expression of was highest in alfalfa inflorescences and lowest in roots. Transgenic tobacco overexpressing exhibited increased tolerance to low temperature and drought stress, evidenced by physiological indicators including low levels of active oxygen species and increased activity of antioxidant enzymes and osmoregulatory substances. Under drought stress conditions, compared to wild-type plants, -overexpressing tobacco plants exhibited significantly increased expression of drought stress-related genes and , while the expression of , / , , and was significantly reduced. Together, these results provide new insights into the roles of the 14-3-3 protein MsGRF2 in plant drought response mechanisms.