Inducible expression of <scp>DEFECTIVE IN ANTHER DEHISCENCE</scp> 1 enhances triacylglycerol accumulation and lipid droplet formation in vegetative tissues

Bioengineering efforts to increase oil in non-storage vegetative tissues, which constitute the majority of plant biomass, are promising sustainable sources of renewable fuels and feedstocks. While plants typically do not accumulate significant amounts of triacylglycerol (TAG) in vegetative tissues, we report here that the expression of a plastid-localized phospholipase A1 protein, DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1), led to a substantial increase in leaf TAG in Arabidopsis. Using an inducible system to control DAD1 expression circumvented growth penalties associated with overexpressing DAD1 and resulted in a rapid burst of TAG within several hours. The increase of TAG was accompanied by the formation of oil bodies in the leaves, petioles, and stems, but not in the roots. Lipid analysis indicated that the increase in TAG was negatively correlated with plastidial galactolipid concentration. The fatty acid (FA) composition of TAG predominantly consisted of 18:3. Expression of DAD1 in the fad3fad7fad8 mutant, devoid of 18:3, resulted in comparable TAG accumulation with 18:2 as the major FA constituent, reflecting the flexible in vivo substrate use of DAD1. The transient expression of either Arabidopsis DAD1 or Nicotiana benthamiana DAD1 (NbDAD1) in N. benthamiana leaves stimulated the accumulation of TAG. Similarly, transgenic soybeans expressing Arabidopsis DAD1 exhibited an accumulation of TAG in the leaves, showcasing the biotechnological potential of this technology. In summary, inducible expression of a plastidial lipase resulted in enhanced oil production in vegetative tissues, extending our understanding of lipid remodeling mediated by DAD1 and offering a valuable tool for metabolic engineering.