A Novel MsEOBI-MsPAL1 Module Enhances Salinity Stress Tolerance, Floral Scent Emission and Seed Yield in Alfalfa

Alfalfa (Medicago sativa L.) is an important and widely cultivated forage legume, yet its yield is constrained by salinity stress. In this study, we characterized an R2R3-MYB transcription factor MsEOBI in alfalfa. Its salt tolerance function and regulatory pathways were investigated. The nuclear-localized MsEOBI functions as a transcriptional activator, enhancing salinity tolerance by promoting the biosynthesis of flavonoids and lignin, as well as facilitating the scavenging of reactive oxygen species (ROS). Additionally, MsEOBI promotes pollinator attraction and increases seed yield by activating the biosynthesis of volatile phenylpropanoids. Yeast one-hybrid (Y1H), dual-luciferase reporter and chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) assays demonstrated that MsEOBI directly binds to the promoter regions of MsPAL1, a key gene in the phenylpropanoid pathway, thereby activating its expression. Overexpression of MsPAL1 enhances salinity tolerance in alfalfa. These findings elucidate the role of the MsEOBI-MsPAL1 regulatory module and provide valuable genetic resources for the future breeding of salt-tolerant alfalfa varieties.