A Novel Alfalfa MsWRKY75 Transcription Factor Negatively Regulates Seed Germination via an ABA-Dependent Pathway Under Salinity Stress

Abstract Salinity stress severely impairs seed germination in plants. In this study, we identified MsWRKY75, a salt-responsive WRKY transcription factor from alfalfa (Medicago sativa L.), and characterized its role in modulating abscisic acid (ABA) signaling to regulate seed germination under salinity stress. Phylogenetic and structural analyses revealed that MsWRKY75, homologous to Medicago truncatula MtWRKY75, contains a conserved WRKYGQK motif and a C2H2-type zinc finger domain. Subcellular localization confirmed its nuclear localization, while yeast assays demonstrated transcriptional activation activity, supporting its function as a transcription factor. Overexpression of MsWRKY75 in Arabidopsis and M. truncatula led to enhanced suppression of seed germination under both salinity and ABA treatments, in a dose-dependent manner. Interestingly, MsWRKY75 did not affect ABA biosynthesis or catabolism, but specifically upregulated core components of the ABA signaling pathway. qRT-PCR analysis revealed strong induction of MtABI5 a key repressor of seed germination and its upstream receptor MtPYL4 in MsWRKY75-overexpressing lines. Yeast one-hybrid, dual-luciferase, and chromatin immunoprecipitation (ChIP) assays demonstrated that MsWRKY75 directly binds to the W-box cis-element within the MtABI5 promoter, activating its transcription. Mutation of the W-box motif abolished this interaction, confirming its essential role in transcriptional activation. Together, these results establish MsWRKY75 as a negative regulator of seed germination under salinity stress, acting through direct enhancement of ABA signaling. This study provides new mechanistic insights into ABA-mediated stress responses and identifies MsWRKY75 as a promising candidate for improving stress resilience through genetic engineering in legume crops.