Abstract
In expanding pea leaves, over 95% of fatty acids (FA) synthesized in the plastid are exported for assembly of eukaryotic glycerolipids. It is often assumed that the major products of plastid FA synthesis (18:1 and 16:0) are first incorporated into 16:0/18:1 and 18:1/18:1 molecular species of phosphatidic acid (PA), which are then converted to phosphatidylcholine (PC), the major eukaryotic phospholipid and site of acyl desaturation. However, by labeling lipids of pea leaves with [(14)C]acetate, [(14)C]glycerol, and [(14)C]carbon dioxide, we demonstrate that acyl editing is an integral component of eukaryotic glycerolipid synthesis. First, no precursor-product relationship between PA and PC [(14)C]acyl chains was observed at very early time points. Second, analysis of PC molecular species at these early time points showed that >90% of newly synthesized [(14)C]18:1 and [(14)C]16:0 acyl groups were incorporated into PC alongside a previously synthesized unlabeled acyl group (18:2, 18:3, or 16:0). And third, [(14)C]glycerol labeling produced PC molecular species highly enriched with 18:2, 18:3, and 16:0 FA, and not 18:1, the major product of plastid fatty acid synthesis. In conclusion, we propose that most newly synthesized 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. Additionally, the acyl groups removed by acyl editing are largely used for the net synthesis of PC through glycerol 3-phosphate acylation.
Highlights
Directly by acyltransferases of the “prokaryotic” pathway within the plastid, or (ii) the acyl-acyl carrier protein (ACP) thioester bond is hydrolyzed during acyl export from the plastid prior to fatty acids (FA) reactivation and incorporation into glycerolipids by acyltransferases of the “eukaryotic” pathway outside the plastid
We address whether the initial incorporation of nascent fatty acids occurs via acyl editing, or if there is a rapid incorporation by de novo PC synthesis via glycerol-3-P (G3P) and DAG, which is followed by rapid acyl editing of PC. 2) We perform both total molecular species and stereochemical analyses on acyl-labeled PC using in vivo experiments with expanding pea leaves and seedlings
Through analyses of the kinetics, molecular species and stereochemistry of the immediately labeled products of eukaryotic glycerolipid synthesis we demonstrate that, in rapidly expanding pea leaves, 1) there was no detectable precursorproduct relationship for phosphatidic acid (PA) and DAG in the incorporation of nascent FA into PC, 2) nascent FA were incorporated into PC
Summary
Directly by acyltransferases of the “prokaryotic” pathway within the plastid, or (ii) the acyl-ACP thioester bond is hydrolyzed during acyl export from the plastid prior to FA reactivation and incorporation into glycerolipids by acyltransferases of the “eukaryotic” pathway outside the plastid. The eukaryotic pathway for de novo PC synthesis utilizes this pool of newly synthesized acyl-CoAs for sequential sn-1 and sn-2 acylations of glycerol 3-phosphate to produce 18:1/18:1 and 16:0/ 18:1 molecular species of phosphatidic acid (PA). LPCAT allows for a mechanism for acyl editing, the in vitro results do not indicate how prevalent the reaction might be in vivo In this context, isolated pea chloroplasts incubated with [14C]acetate immediately label PC with newly synthesized FA through a channeled pool of acyl-CoA [21]. The most direct line of evidence of acyl editing in plants comes from a careful analysis of the molecular species of monogalactosyldiacylglycerol and PC after labeling leaf disks of the 16:3 plant Brassica napus with carbon dioxide [23]. The results parallel those obtained with excised tissue assays, indicating that there are no wound responses that compromise the metabolic conclusions obtained from excised tissue experiments
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