Triacylglycerol remodeling in Physaria fendleri indicates oil accumulation is dynamic and not a metabolic endpoint

Abstract

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.

Figure 1

Plant lipid metabolic network of TAG assembly. TAG is synthesized from multiple substrate pools including at least three DAG pools and at least two acyl donors (acyl-CoA and PC). The path of acyl flux through the network into TAG ultimately determines the final oil fatty acid composition. Dotted lines indicate acyl group transfer, solid lines indicate flux of the glycerol backbone. A, Fatty acid modification on PC (e.g. desaturation, hydroxylation). B, Acyl editing cycle exchanging fatty acids between PC and acyl-CoA pools. C, Fatty acid elongation. D, De novo DAG(1) can be used directly for TAG synthesis as in the Kennedy pathway (gray shade), or can be used to produce PC and subsequently PC-derived DAG(2) prior to TAG synthesis (blue shade). A third pool of DAG(3) may be associated with the oil body and made up of both sources and can be used for TAG biosynthesis. G3P, glycerol-3-phosphate; LPA, lyso-phosphatidic acid; PA, phosphatidic acid.

Figure 2

Fatty acid composition and TAG accumulation across P. fendleri embryo development. A, Fatty acid composition as µg per embryo. B, Fatty acid composition as mole percentage. C, TAG accumulation per embryo. Data are mean ± sem of five replicates for each stage except for 24 DAP, three replicates.

Figure 3

¹⁴C-lipid accumulation from continuous [¹⁴C]acetate labeling of developing P. fendleri embryos. A, Total lipid radiolabeled fatty acid composition. B, Stereochemistry of labeled fatty acid accumulation in PC. C, Accumulation of radiolabeled fatty acids in PLs. D, Accumulation of radiolabeled fatty acids in neutral lipids and total PLs. Each data point is mean ± sem of three individual labeling replicates. Each time point replicate contained 10 embryos at 30 DAP, 120 total embryos for the time course.

Figure 4

[¹⁴C]acetate pulse-chase labeling of developing P. fendleri embryos. One hour pulse with 30 DAP embryos followed by chase time points of 2, 24, 72, 120, and 168 h. A, Total lipid accumulation (dotted line) and radioactivity incorporated in total lipids (solid line). B, Proportion of total ¹⁴C-HFAs and ¹⁴C-non-HFAs. ¹⁴C-fatty acid accumulation into neutral lipids and total PLs measured as (C) DPM per embryo and (D) ¹⁴C Percentage. ¹⁴C-fatty acid accumulation into individual PLs measured as (E) DPM per embryo (F) ¹⁴C Percentage. Data are mean ± sem of three individual labeling replicates of 10 embryos for each time point replicate, 180 total embryos for the time course.

Figure 5

Relative ¹⁴C-fatty acid accumulation in different lipid species during [¹⁴C]acetate pulse-chase labeling of developing P. fendleri embryos. A, PC. B, 0HFA-TAG. C, 1HFA-TAG. D, 2HFA-TAG. Data are mean ± sem of three individual labeling replicates of 10 embryos for each time point replicate, 180 total embryos for the time course.

Figure 6

[¹⁴C]glycerol pulse-chase labeling of developing P. fendleri embryos. One hour pulse followed by chase time points of 0, 2, 24, 74, and 120 h. A, Total lipid accumulation (dotted line) and radioactivity incorporated in total lipid (solid line). B, ¹⁴C incorporation into the acyl and backbone fraction of total lipids. [¹⁴C]glycerol incorporation into backbones of neutral lipids and PC measured as (C) DPM per embryo (D) ¹⁴C Percentage. [¹⁴C]glycerol incorporation into backbones of individual PLs measured as (E) DPM per embryo (F) ¹⁴C percentage. Data are mean ± sem of three individual labeling replicates of 10 embryos for each time point replicate, 150 total embryos for the time course.

Figure 7

Metabolic pathway for incorporation of HFA into P. fendleri TAG. Solid lines indicate glycerol backbone transfer where thicker lines represent larger flux. Dotted lines indicate acyl group transfer. A, Hydroxylation of oleic acid (18:1) to ricinoleic acid (18:1OH) in PC. B, Acyl editing cycle to transfer 18:1OH to the acyl CoA pool. C, Elongation of 18:1OH to lesquorelic acid (20:1OH). D, De novo DAG is predominantly utilized for PC synthesis. E, Initial TAG is predominantly produced from PC-derived DAG (sn-1/2) not containing an HFA. F, TAG remodeling initially removes non-HFAs from TAG generating sn-1/2 or sn-2/3 DAG from 0HFA-TAG, and sn-2/3 DAG from 1HFA-TAG remodeling. G, Acyl groups removed by TAG remodeling are reincorporated into the acyl-CoA pool and may be further modified on PC and elongated. Enzyme abbreviations: DGAT, diacylglycerol acyltansferase; GPAT, glycerol-3-phosphate acyltransferase; LPAT, lyso-phosphatidic acid acyltransferase; PAP, phosphatidic acid phosphatase; TAGL1, TAGLipase like-1. Substrate abbreviations: G3P, glycerol-3-phosphate; PA, phosphatidic acid.