BnPUB12 Enhances Drought Tolerance by Improving Water Retention and ROS Scavenging in Brassica napus

Drought stress severely limits the growth, yield, and seed quality of rapeseed. Plant U-box (PUB) proteins are a class of E3 ubiquitin ligases involved in abiotic stress responses, but the function of BnPUBs in B. napus drought tolerance remains largely unknown. We obtained bnpub12 knockout mutants by CRISPR/Cas9 technology, and constructed BnPUB12 overexpression lines. This study investigated the role of BnPUB12 in drought resistance through phenotypic, physiological, and biochemical analyses. Under drought stress, bnpub12 mutants exhibited a significantly lower survival rate during germination, whereas overexpression lines showed a higher survival rate. During the seedling stage, bnpub12 mutants displayed lower relative water content, a lower water holding capacity, and a faster water loss rate; conversely, overexpression lines showed higher relative water content, an enhanced water holding capacity, and a slower water loss rate. DAB and NBT staining revealed less reactive oxygen species accumulation in overexpression lines but stronger staining in mutants. Physiological measurements further indicated that BnPUB12 overexpression increased SOD and POD activities and proline content, while decreasing MDA content; the opposite trends were observed in mutants. Overall, these results demonstrated that BnPUB12 positively regulates drought tolerance in B. napus during both the germination and seedling stages.