BnLEA4 and BnMYB96 target BnLTP2 to enhance drought tolerance and oil accumulation in Brassica napus L.

Brassica napus is one of the most important oilseed crops worldwide and improving its oil content is a key research focus. The transcription factor (TF) BnMYB96 was found to be upregulated during oil accumulation under drought conditions, but the molecular regulation pathway remains unclear. Here, a cDNA library was constructed in a B. napus line with high oil content. BnLEA4 was first screened using BnMYB96 as bait, which was confirmed by yeast two-hybrid (Y2H), co-immunoprecipitation (Co-IP), bimolecular fluorescence complementation (BiFC), and pull-down assays. BnMYB96 can interact with BnLEA4 and regulate downstream BnLTP2, as confirmed by yeast one-hybrid (Y1H), dual-luciferase reporter (LUC) assay, electrophoretic mobility shift assay (EMSA), and β-glucuronidase (GUS) assay. Overexpression of BnMYB96 and BnLTP2 increased oil content and drought resistance through photosynthetic physiological processes and reactive oxygen species (ROS) metabolism. In contrast, opposite trends were observed in the CRISPR/Cas9 knockout lines. Hybrids (six-line crosses) between the two genes (BnMYB96 and BnLTP2) with increased or reduced expression showed stronger trends in drought tolerance and lipid accumulation. Single-cell and bulk transcriptome sequencing analyses showed that genes involved in carbon fixation and fatty acid (FA) synthesis in photosynthetic organisms were upregulated by BnMYB96 and BnLTP2, enhancing photosynthesis and FA synthesis. This study elucidates the BnLEA4-BnMYB96-BnLTP2 regulatory pathway that coordinates oil accumulation and drought resistance in B. napus. These findings provide a theoretical basis for improving the drought resistance and oil content of plants.