Acyl editing and headgroup exchange are the major mechanisms that direct polyunsaturated fatty acid flux into triacylglycerols

Abstract

Triacylglycerols (TAG) in seeds of Arabidopsis (Arabidopsis thaliana) and many plant species contain large amounts of polyunsaturated fatty acids (PUFA). These PUFA are synthesized on the membrane lipid phosphatidylcholine (PC). However, the exact mechanisms of how fatty acids enter PC and how they are removed from PC after being modified to participate in the TAG assembly are unclear, nor are the identities of the key enzymes/genes that control these fluxes known. By reverse genetics and metabolic labeling experiments, we demonstrate that two genes encoding the lysophosphatidylcholine acyltransferases LPCAT1 and LPCAT2 in Arabidopsis control the previously identified "acyl-editing" process, the main entry of fatty acids into PC. The lpcat1/lpcat2 mutant showed increased contents of very-long-chain fatty acids and decreased PUFA in TAG and the accumulation of small amounts of lysophosphatidylcholine in developing seeds revealed by [¹⁴C]acetate-labeling experiments. We also showed that mutations in LPCATs and the PC diacylglycerol cholinephosphotransferase in the reduced oleate desaturation1 (rod1)/lpcat1/lpcat2 mutant resulted in a drastic reduction of PUFA content in seed TAG, accumulating only one-third of the wild-type level. These results indicate that PC acyl editing and phosphocholine headgroup exchange between PC and diacylglycerols control the majority of acyl fluxes through PC to provide PUFA for TAG synthesis.

Figure 1.

Reactions involved in the flux of fatty acids into TAG. De novo glycerolipid synthesis is shown in white arrows, acyl transfer reactions are indicated by dashed lines, and the movement of the lipid glycerol backbone through the pathway is shown in solid lines. Major reactions (in thick lines) controlling the flux of fatty acid from PC into TAG are as follows: LPC acylation reaction of acyl editing by LPCAT (A); PC deacylation reaction of acyl editing by the reverse action of LPCAT or phospholipase A (B); and the interconversion of DAG and PC by PDCT (C). Substrates are in boldface, enzymatic reactions are in italics. FAD, Fatty acid desaturase; FAS, fatty acid synthase; GPAT, acyl-CoA:G3P acyltransferase; LPA, lysophosphatidic acid; LPAT, acyl-CoA:LPA acyltransferase; PA, phosphatidic acid; PLC, phospholipase C; PLD, phospholipase D.

Figure 2.

[¹⁴C]Acetate incorporation into fatty acids of Col-0 and lpcat1/lpcat2 developing seeds. A and C, Total radioactivity incorporation into seed fatty acids at 3, 6, 10, 30, and 60 min of incubation. Triplicate labelings for each time point and best fit line are shown. A, Col-0. C, lpcat1/lpcat2. B and D, Composition of radiolabeled fatty acids. Average values plus se of triplicate labeled samples are shown. Sat., Total saturated fatty acids. B, Col-0. D, lpcat1/lpcat2.

Figure 3.

Accumulation of [¹⁴C]fatty acids into developing seed glycerolipids of Col-0 and lpcat1/lpcat2. A and C, Full 60-min time course. B and D, Initial 15-min of time course. A and B, Col-0. C and D, lpcat1/lpcat2. Average values and se of triplicate labeled samples are shown. Radiolabeled glycerolipids are indicated as follows: TAG (closed circles and solid line); PC (open squares and solid line); DAG (closed diamonds and double line); phosphatidic acid (PA)/PE (closed triangles and solid line); LPC (open circles and dashed line).

Figure 4.

Stereochemical incorporation of [¹⁴C]fatty acids into DAG and PC of Col-0 and lpcat1/lpcat2 developing seeds. Percentage of total lipid radiolabeled fatty acids at the sn-1 position of the glycerol backbone, diamonds and dashed line; percentage of total lipid radiolabeled fatty acids at the sn-2 position of the glycerol backbone, squares and solid line. A, Col-0 DAG. B, Col-0 PC. C, lpcat1/lpcat2 DAG. D, lpcat1/lpcat2 PC. Average values and se of triplicate labeled samples are shown.

Figure 5.

Fatty acid composition of seed PC. The mol % fatty acid composition of seed PC from Col-0, lpcat1/lpcat2, rod1, and rod1/lpcat1/lpcat2 is shown. Average values and se of triplicate samples are shown.