Arabidopsis lipins, PDAT1 acyltransferase, and SDP1 triacylglycerol lipase synergistically direct fatty acids toward β-oxidation, thereby maintaining membrane lipid homeostasis

Abstract

Triacylglycerol (TAG) metabolism is a key aspect of intracellular lipid homeostasis in yeast and mammals, but its role in vegetative tissues of plants remains poorly defined. We previously reported that PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE1 (PDAT1) is crucial for diverting fatty acids (FAs) from membrane lipid synthesis to TAG and thereby protecting against FA-induced cell death in leaves. Here, we show that overexpression of PDAT1 enhances the turnover of FAs in leaf lipids. Using the trigalactosyldiacylglycerol1-1 (tgd1-1) mutant, which displays substantially enhanced PDAT1-mediated TAG synthesis, we demonstrate that disruption of SUGAR-DEPENDENT1 (SDP1) TAG lipase or PEROXISOMAL TRANSPORTER1 (PXA1) severely decreases FA turnover, leading to increases in leaf TAG accumulation, to 9% of dry weight, and in total leaf lipid, by 3-fold. The membrane lipid composition of tgd1-1 sdp1-4 and tgd1-1 pxa1-2 double mutants is altered, and their growth and development are compromised. We also show that two Arabidopsis thaliana lipin homologs provide most of the diacylglycerol for TAG synthesis and that loss of their functions markedly reduces TAG content, but with only minor impact on eukaryotic galactolipid synthesis. Collectively, these results show that Arabidopsis lipins, along with PDAT1 and SDP1, function synergistically in directing FAs toward peroxisomal β-oxidation via TAG intermediates, thereby maintaining membrane lipid homeostasis in leaves.

Figure 1.

Overexpression of PDAT1 Enhances the Synthesis and Turnover of FAs. Initial rates of FA synthesis (A) and rates of FA turnover (B) in growing leaves of 5-week-old wild-type plants and three transgenic lines overexpressing PDAT1 in the wild-type background. Data are means of the three replicates with sd. Asterisks indicate statistically significant differences from the wild type based on Student’s t test (P < 0.05).

Figure 2.

Disruption of SDP1 or PXA1 Boosts Leaf TAG Accumulation in tgd1-1. (A) Thin layer chromatogram of neutral lipids. Lipids were visualized with 5% sulfuric acid by charring. (B) TAG content in fully mature leaves of 7-week-old wild-type, tgd1-1, tgd1-1 sdp1-4, and tgd1-1 pxa1-2 plants grown on soil. Data are means of the three replicates with sd. DW, dry weight. (C) and (D) Total FA content (C) and TAG FA composition (D) in leaves of wild-type and mutant plants. Values are means and sd of three replicates. [See online article for color version of this figure.]

Figure 3.

LD Accumulation in Leaves of tgd1-1 Lacking SDP1 or PXA1. (A) to (F) Images of LDs in wild-type (A), sdp1-4 (B), pxa1-2 (C), tgd1-1 (D) tgd1-1 sdp1-4 (E), and tgd1-1 pxa1-2 (F) leaves stained with Nile red. Bars = 10 μm. (G) to (I) Transmission electron microscopy images of leaf cells of tgd1-1 (G) tgd1-1 sdp1-4 (H), and tgd1-1 pxa1-2 (I). Bars = 2 μm. [See online article for color version of this figure.]

Figure 4.

Disruption of SDP1 or PXA1 Affects Growth and Development of tgd1-1. (A) One-week-old plants grown on agar plates in the presence of 1% Suc. (B) Four-week-old plants grown on soil. (C) Six-week-old wild-type and mutant plants grown on soil. [See online article for color version of this figure.]

Figure 5.

Disruption of PXA1 Affects Pollen Grain Germination in tgd1-1. (A) and (B) Flower morphology of tgd1-1 (A) and tgd1-1 pxa1-2 (B). (C) and (D) Pollen viability in tgd1-1 (C) and tgd1-1 pxa1-2 (D). Bars = 20 μm. (E) and (F) Germination of mature pollen grains from tgd1-1 (E) and tgd1-1 pxa1-2 (F) in liquid medium. Bars = 20 μm. (G) and (H) Pollen germination and pollen tube growth (arrows) of tgd1-1 (G) and tgd1-1 pxa1-2 (H) in the transmitting tract of the wild-type style. Bars = 20 μm. [See online article for color version of this figure.]

Figure 6.

Disruption of SDP1 or PXA1 Enhances FA Flux toward TAG Storage in tgd1-1. (A) Decreases in total labeled FAs during the chase period in tgd1-1 and double mutants. Detached leaves were labeled with [¹⁴C]acetate for 1 h, and the label was chased for 3 d. Values are means and sd of three replicates. Asterisks indicate statistically significant differences from tgd1-1 based on Student’s t test (P < 0.05). (B) and (C) Autoradiographs of radiolabeled lipids separated by thin layer chromatography using a double development. PI/SL, phosphatidylinositol/sulfoquinovosyldiacylglycerol.

Figure 7.

Disruption of SDP1 or PXA1 Alters Membrane Lipid Content and Galactolipid FA Positional Distribution in tgd1-1. (A) Polar lipid content in tgd1-1 and double mutants. DW, dry weight; PI/SL, phosphatidylinositol/sulfoquinovosyldiacylglycerol. (B) FA composition exclusively at the sn-2 position of the glycerol backbone of galactolipids from leaves of wild-type and mutant plants. Data are means of the three replicates with sd. Asterisks indicate statistically significant differences from tgd1-1 based on Student’s t test (P < 0.05).

Figure 8.

Disruption of PAH1 and PAH2 Compromises TAG but Not the Eukaryotic Galactolipid Synthesis. (A) Leaf TAG accumulation in the wild type, tgd1-1, and PDAT1 overexpressing line 3 (PDAT1 #3) disrupted in PAH1/2. Data are means of the three replicates with sd. DW, dry weight. (B) Leaf galactolipid content in the wild type and mutants. Data are means of the three replicates with sd. Asterisks indicate statistically significant differences from act1 based on Student’s t test (P < 0.05). FW, fresh weight.

Figure 9.

Disruption of SFR2 Affects Membrane Lipid Content and FA Turnover in tgd1-1. Detached leaves were labeled with [¹⁴C]acetate for 1 h, and the label was chased for 3 d. Values are means and sd of three replicates. Asterisks indicate statistically significant differences from tgd1-1 based on Student’s t test (P < 0.05). (A) Membrane lipid content in leaves of wild-type and mutant plants. DW, dry weight. (B) Changes in radiolabel in membrane lipids and TAG during the chase period. SL/PI, sulfoquinovosyldiacylglycerol/phosphatidylinositol. (C) Changes in total radiolabeled FAs during the chase period.

Figure 10.

Model for the Proposed Pathway of FA Oxidation in Leaves of the Wild Type and the tgd1-1 Mutant. The model proposes that FAs released by SDP1 from TAG stored in cytosolic LDs are the direct substrates for peroxisomal β-oxidation and that PDAT1 plays a critical role in diverting FAs from membrane lipid synthesis to TAG storage. FAs exported from the plastid are first incorporated into PC via an acyl editing cycle. Acyl groups released from PC acyl editing are used predominantly for sequential acylation of glycerol-3-phosphate to produce PA. Dephosphorylation of PA by PAH1/2 generates DAG, which in the wild-type leaves is mostly used for the de novo synthesis of extraplastidic phospholipids, including PC. A major portion of PC derivatives are returned to the plastid to support the eukaryotic pathway of thylakoid glycolipid (GL) synthesis. In the tgd1-1 mutant, a compromised (X) eukaryotic GL pathway enhances (green arrows) not only FA synthesis but also FA peroxisomal β-oxidation through PC and TAG. A substantial fraction of acyl chains in tgd1-1 are channeled into peroxisomal β-oxidation through SFR2-mediated DAG production. LPC, lysophosphatidylcholine; LPL, lysophospholipid; OG, oligogalactolipid; PL, phospholipid.