U-box-type E3 ubiquitin ligases (PUBs) are critical for signaling homeostasis and abiotic stress responses via substrate ubiquitination, yet their upstream transcriptional regulation remains largely unexplored, especially in alfalfa drought adaptation. Through GWAS of 171 alfalfa accessions, we identified a novel U-box gene, MsPUB6, on chromosome 3, which encodes a protein with E3 ubiquitin ligase activity and is induced by drought, cold, salt, and ABA. Overexpression and virus-induced gene silencing (VIGS) revealed that MsPUB6 positively regulates drought tolerance by enhancing antioxidant enzymes (SOD and CAT), reducing malondialdehyde (MDA) and reactive oxygen species (ROS) levels, and maintaining chlorophyll content and leaf water content. Further mechanistic investigation showed that the transcription factor MsDREB1C specifically binds to the CRT/DRE element in the MsPUB6 promoter and activates its transcription. CRISPR-Cas9-mediated MsDREB1C mutants exhibited a drought-sensitive phenotype similar to that of MsPUB6-silenced lines, accompanied by significantly reduced MsPUB6 expression. In contrast, MsDREB1C-overexpressing lines showed enhanced drought tolerance and upregulated MsPUB6 expression, and similarly promoted drought tolerance by alleviating oxidative damage and membrane damage, and by maintaining chlorophyll content and relative leaf water content. Collectively, this study reveals a novel regulatory module, MsDREB1C-MsPUB6, in which a DREB1 transcription factor positively regulates a U-box-type E3 ligase gene to contribute to drought tolerance in alfalfa. These findings not only provide important genetic resources for molecular breeding of drought-tolerant alfalfa but also expand our understanding of the functional diversity of plant U-box proteins.
