Converging GWAS and Genome Editing Decrypts MsCBF8 ‐ MsGolS2 Regulatory Module for Drought Tolerance Without Yield Penalty in Medicago sativa

ABSTRACT Drought constitutes a major abiotic stress limiting alfalfa productivity. To elucidate the genetic basis of drought tolerance, a genome‐wide association study (GWAS) was conducted in alfalfa, which identified a CBF/DREB1 family transcription factor designated MsCBF8. Utilizing an optimized CRISPR‐Cas9 system, mutants were successfully generated in the complex autotetraploid background. Combined with overexpression and Arabidopsis complementation assays, this confirmed that MsCBF8 functions as a positive regulator of drought tolerance, with its overexpression enhancing drought tolerance without compromising plant growth. Physiological analyses revealed that MsCBF8 improves drought tolerance by coordinating antioxidant defence (elevated SOD and CAT activities, decreased H 2 O 2 and MDA contents), dynamically regulating stomatal movement (increased water‐use efficiency), and maintaining photosynthetic performance. At the molecular level, MsCBF8 directly and specifically binds to the GCCGAC cis ‐element in the MsGolS2 promoter and activates its expression. Silencing MsGolS2 via virus‐induced gene silencing (VIGS) reduced drought tolerance and aggravated oxidative damage, providing the first functional evidence for the essential role of this gene in the drought response of alfalfa. This study systematically elucidates a novel MsCBF8‐ MsGolS2 regulatory module, offering crucial insights into the drought adaptation mechanisms in alfalfa and providing valuable genetic resources and breeding targets for developing high‐yield, water‐efficient and drought‐tolerant alfalfa cultivars.