Re-examining the initial steps of membrane and storage lipid assembly in pea leaves and soybean embryos: The dominant flux of newly synthesized fatty acid incorporation into extra-plastidic glycerolipids is through phosphatidylcholine acyl editing

In expanding pea leaves and developing soybean embryos over 95% of fatty acids (FA) synthesized in the plastid are exported for assembly of eukaryotic membrane and storage glycerolipids. It is often assumed that the major products of plastid FA synthesis (18:1 and 16:0) are first acylated to glycerol-3-phosphate (G3P) producing 16:0/18:1 and 18:1/18:1 initial molecular species of phosphatidic acid (PA) which are then converted to phosphatidylcholine (PC), the major eukaryotic phospholipid and site of acyl desaturation. However, by rapid labeling of pea leaf and soybean embryo lipids with [14C]acetate and [14C]glycerol we demonstrate that acyl editing is an integral component of eukaryotic glycerolipid synthesis. First, no precursor-product relationship between diacylglycerol (DAG) and PC [14C]acyl chains was observed at very early time points. Second, analysis of PC molecular species and stereochemistry at these early time points showed that the majority of newly synthesized acyl groups (>90% of the [14C]18:1 and [14C]saturated acyl groups in pea, and >60% in soybeans) were incorporated into PC alongside a previously synthesized unlabeled acyl group (18:2, 18:3 or 16:0). The nascent FA occupied 62% of the sn-2 position in pea leaves and 86% in soybean embryos. Third, [14C]glycerol labeling produced PC molecular species highly enriched with 18:2, 18:3 and saturated acyl chains and not 18:1, the major product of plastid fatty acid synthesis. And forth, the kinetics of [14C]glycerol and the stereochemistry of newly synthesized FA incorporation into triacylglycerol (TAG) of soybean embryos indicated that TAG is synthesized from a DAG molecule generated by removal of the PC phosphocholine head group and not de novo synthesized DAG. In conclusion, we propose that most newly synthesized and plastid exported acyl groups are not immediately utilized for PA synthesis, but instead are incorporated directly into PC through an acyl editing mechanism that operates at both sn-1 and sn-2 positions. The acyl groups removed by acyl-editing are largely used for the net synthesis of PC and other glycerolipids through (G3P) acylation. Very little if any TAG is produced by three consecutive acylations as in the traditional Kennedy pathway. These results provide a more quantitative basis to demonstrate in vivo that FA flux in/out of PC by acyl editing, and the inter-converstion of DAG with PC are dominate fluxes in the assembly of eukaryotic membrane and storage glycerolipids in plants.