Ectopic Expression of WRI1 Affects Fatty Acid Homeostasis in Brachypodium distachyon Vegetative Tissues

Triacylglycerol (TAG) is the major storage lipid used as a biofuel feedstock for renewable energy. WRINKLED1 (WRI1) is a transcription factor, which is important for TAG accumulation not only in seeds, but also in vegetative tissues. In this study, we identified the WRI1 ortholog in Brachypodium distachyon and ectopically expressed BdWRI1 in Brachypodium vegetative tissues under the control of the maize ubiquitin (ZmUBIL) promoter. The transcription levels of key genes involved in the de novo fatty acid biosynthesis pathway, such as ACP1, KASІ, and PI-PKβ1, were highly induced in 8-week-old leaf blades of T₃-generation homozygous plants, and TAG content was also drastically increased (up to 37.5 fold compared to wild type, 1.51±0.35 μg mg^-1 Dry Weight) compared to wild type. We also found that ectopic expression of BdWRI1 caused premature cell death in vegetative tissues. Necrosis became first visible in 4-week-old leaf blades of transgenic lines. Lipid analysis indicated that the free fatty acid (FFA) content was increased 2.5 fold in Ub::BdWRI1 expressing leaf blades. The transcription levels of key genes involved in β-oxidation pathways were also induced. In addition, 18:2 fatty acid treatment also caused cell death in Brachypodium wild-type leaf blades, a phenotype that could be inhibited by the fatty acid biosynthesis inhibitor cerulenin. Taken together, our data suggest that BdWRI plays an important role in fatty acid biosynthesis and oil accumulation. The induction of fatty acid biosynthesis increases the FFA content in vegetative tissues and leads to cytotoxic effects in Brachypodium.