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In vivo isotopic tracing indicates Physaria fendleri incorporates industrially valuable unusual fatty acids into seed storage oil through a dynamic triacylglycerol remodeling process., Oilseed plants accumulate triacylglycerol (TAG) up to 80% of seed weight with the TAG fatty acid composition determining its nutritional value or use in the biofuel or chemical industries. Two major pathways for production of diacylglycerol (DAG), the immediate precursor to TAG, have been identified in plants: de novo DAG synthesis and conversion of the membrane lipid phosphatidylcholine (PC) to DAG, with each pathway producing distinct TAG compositions. However, neither pathway fits with previous biochemical and transcriptomic results from developing Physaria fendleri seeds for accumulation of TAG containing >60% lesquerolic acid (an unusual 20 carbon hydroxylated fatty acid), which accumulates at only the sn-1 and sn-3 positions of TAG. Isotopic tracing of developing P. fendleri seed lipid metabolism identified that PC-derived DAG is utilized to initially produce TAG with only one lesquerolic acid. Subsequently a nonhydroxylated fatty acid is removed from TAG (transiently reproducing DAG) and a second lesquerolic acid is incorporated. Thus, a dynamic TAG remodeling process involving anabolic and catabolic reactions controls the final TAG fatty acid composition. Reinterpretation of P. fendleri transcriptomic data identified potential genes involved in TAG remodeling that could provide a new approach for oilseed engineering by altering oil fatty acid composition after initial TAG synthesis; and the comparison of current results to that of related Brassicaceae species in the literature suggests the possibility of TAG remodeling involved in incorporation of very long-chain fatty acids into the TAG sn-1 position in various plants.

The triacylglycerols (TAGs; i.e. oils) that accumulate in plants represent the most energy-dense form of biological carbon storage, and are used for food, fuels, and chemicals. The increasing human population and decreasing amount of arable land have amplified the need to produce plant oil more efficiently. Engineering plants to accumulate oils in vegetative tissues is a novel strategy, because most plants only accumulate large amounts of lipids in the seeds. Recently, tobacco (Nicotiana tabacum) leaves were engineered to accumulate oil at 15% of dry weight due to a push (increased fatty acid synthesis)-and-pull (increased final step of TAG biosynthesis) engineering strategy. However, to accumulate both TAG and essential membrane lipids, fatty acid flux through nonengineered reactions of the endogenous metabolic network must also adapt, which is not evident from total oil analysis. To increase our understanding of endogenous leaf lipid metabolism and its ability to adapt to metabolic engineering, we utilized a series of in vitro and in vivo experiments to characterize the path of acyl flux in wild-type and transgenic oil-accumulating tobacco leaves. Acyl flux around the phosphatidylcholine acyl editing cycle was the largest acyl flux reaction in wild-type and engineered tobacco leaves. In oil-accumulating leaves, acyl flux into the eukaryotic pathway of glycerolipid assembly was enhanced at the expense of the prokaryotic pathway. However, a direct Kennedy pathway of TAG biosynthesis was not detected, as acyl flux through phosphatidylcholine preceded the incorporation into TAG. These results provide insight into the plasticity and control of acyl lipid metabolism in leaves.