Tracking synthesis and turnover of triacylglycerol in leaves

Abstract

Triacylglycerol (TAG), typically represents <1% of leaf glycerolipids but can accumulate under stress and other conditions or if leaves are supplied with fatty acids, or in plants transformed with regulators or enzymes of lipid metabolism. To better understand the metabolism of TAG in leaves, pulse-chase radiolabelling experiments were designed to probe its synthesis and turnover. When Arabidopsis leaves were incubated with [(14)C]lauric acid (12:0), a major initial product was [(14)C]TAG. Thus, despite low steady-state levels, leaves possess substantial TAG biosynthetic capacity. The contributions of diacylglycerol acyltransferase1 and phospholipid:diacylglycerol acyltransferase1 to leaf TAG synthesis were examined by labelling of dgat1 and pdat1 mutants. The dgat1 mutant displayed a major (76%) reduction in [(14)C]TAG accumulation whereas pdat1 TAG labelling was only slightly reduced. Thus, DGAT1 has a principal role in TAG biosynthesis in young leaves. During a 4h chase period, radioactivity in TAG declined 70%, whereas the turnover of [(14)C]acyl chains of phosphatidylcholine (PC) and other polar lipids was much lower. Sixty percent of [(14)C]12:0 was directly incorporated into glycerolipids without modification, whereas 40% was elongated and desaturated to 16:0 and 18:1 by plastids. The unmodified [(14)C]12:0 and the plastid products of [(14)C]12:0 metabolism entered different pathways. Although plastid-modified (14)C-labelled products accumulated in monogalactosyldiacylglycerol, PC, phosphatidylethanolamine, and diacylglcerol (DAG), there was almost no accumulation of [(14)C]16:0 and [(14)C]18:1 in TAG. Because DAG and acyl-CoA are direct precursors of TAG, the differential labelling of polar glycerolipids and TAG by [(14)C]12:0 and its plastid-modified products provides evidence for multiple subcellular pools of both acyl-CoA and DAG.

Keywords: Acyl-CoA; DGAT; diacylglycerol acyltransferase; leaf TAG; lipids; triacylglycerol..

Fig. 1.

Synthesis and turnover of triacylglycerol in leaves of the wild type (WT) and dgat1 and pdat1 mutants. Incorporation of [¹⁴C]fatty acids into triacylglycerols (A), diacylglycerol (B), and phosphatidylcholine (C) in Arabidopsis WT and pdat1 and dgat1 mutants. The dotted line represents the end of the pulse period and the beginning of the chase period. The inset graph presents the percentage of WT incorporation after 60min of labelling. The data represent the average of three biological replicates. Additional data including all identified glycerolipids and free fatty acids with error bars are presented in Supplementar Fig. S2 at JXB online. (Figure 1 is available in colour at JXB online.)

Fig. 2.

Unsaturated [¹⁴C]fatty acids are not incorporated into triacylglycerol after a pulse–chase labelling experiment. Fatty acid methyl esters after 60min (end of pulse) and 300min (end of chase) of PC, DAG, and TAG separated by argentation TLC. Each lane was loaded with radioactivity corresponding to 10 kdpm. PC, phosphatidylcholine; DAG, diacylglycerol; TAG, triacylglycerol; S, saturated FA; M, mono-unsaturated FA; D, di-unsaturated FA; T, tri-unsaturated FA. Representative autoradiograms of TLC plates are shown.

Fig. 3.

Distinct differences in [¹⁴C]fatty acid composition of triacylglycerol (TAG) compared with diacylglycerol (DAG) and phosphatidylcholine (PC). Arabidopsis wild-type (WT) leaves were labelled with [¹⁴C]12:0 for 60min. [¹⁴C]FA composition determined by argentation and reverse-phase TLC is shown for: (A) TAG, (B) DAG, and (C) PC. Data represent the average of three biological replicates. Tabulated averages ±SD are presented in Supplementary Table S1 at JXB online. (Figure 3 is available in colour at JXB online.)

Fig. 4.

[ ¹²C]Diacylglycerel (DAG) molecular species precursor for [¹⁴C]triacylglycerol (TAG) synthesis. Molecular species of [¹⁴C]TAG were separated by argentation TLC. The major [¹⁴C]TAG species contain two double bonds (MMS or SSD). Because all radiolabelled [¹⁴C]FAs in TAG are saturated (Fig. 3A), the molecular species of the unlabelled [¹²C]DAG precursor of [¹⁴C]TAG must also possess two double bonds (MM or SD). The data represent the average of three biological replicates. S, saturated FA; M, mono-unsaturated FA; D, di-unsaturated FA; T, tri-unsaturated FA. No regiochemistry is specified. A representative TLC plate is shown.