The tree peony DREB transcription factor PrDREB2D regulates seed α-linolenic acid accumulation

α-Linolenic acid (ALA), an essential fatty acid (FA) for human health, serves as the precursor of 2 nutritional benefits, docosahexaenoic acid and eicosapentaenoic acid, and can only be obtained from plant foods. We previously found that phospholipid:diacylglycerol acyltransferase 2 (PrPDAT2) derived from ALA-rich tree peony (Paeonia rockii) can promote seed ALA accumulation. However, the regulatory mechanism underlying its promoting effect on ALA accumulation remains unknown. Here, we revealed a tree peony dehydration-responsive element binding transcription factor, PrDREB2D, as an upstream regulator of PrPDAT2, which is involved in regulating seed ALA accumulation. Our findings demonstrated that PrDREB2D serves as a nucleus-localized transcriptional activator that directly activates PrPDAT2 expression. PrDREB2D altered the FA composition in transient overexpression Nicotiana benthamiana leaves and stable transgenic Arabidopsis (Arabidopsis thaliana) seeds. Repressing PrDREB2D expression in P. rockii resulted in decreased PrPDAT2 expression and ALA accumulation. In addition, PrDREB2D strengthened its regulation of ALA accumulation by recruiting the cofactor ABA-response element binding factor PrABF2b. Collectively, the study findings provide insights into the mechanism of seed ALA accumulation and avenues for enhancing ALA yield via biotechnological manipulation.