A cytosolic acyltransferase contributes to triacylglycerol synthesis in sucrose-rescued Arabidopsis seed oil catabolism mutants

Abstract

Triacylglycerol (TAG) levels and oil bodies persist in sucrose (Suc)-rescued Arabidopsis (Arabidopsis thaliana) seedlings disrupted in seed oil catabolism. This study set out to establish if TAG levels persist as a metabolically inert pool when downstream catabolism is disrupted, or if other mechanisms, such as fatty acid (FA) recycling into TAG are operating. We show that TAG composition changes significantly in Suc-rescued seedlings compared with that found in dry seeds, with 18:2 and 18:3 accumulating. However, 20:1 FA is not efficiently recycled back into TAG in young seedlings, instead partitioning into the membrane lipid fraction and diacylglycerol. In the lipolysis mutant sugar dependent1and the β-oxidation double mutant acx1acx2 (for acyl-Coenzyme A oxidase), levels of TAG actually increased in seedlings growing on Suc. We performed a transcriptomic study and identified up-regulation of an acyltransferase gene, DIACYLGLYCEROL ACYLTRANSFERASE3 (DGAT3), with homology to a peanut (Arachis hypogaea) cytosolic acyltransferase. The acyl-Coenzyme A substrate for this acyltransferase accumulates in mutants that are blocked in oil breakdown postlipolysis. Transient expression in Nicotiana benthamiana confirmed involvement in TAG synthesis and specificity toward 18:3 and 18:2 FAs. Double-mutant analysis with the peroxisomal ATP-binding cassette transporter mutant peroxisomal ABC transporter1 indicated involvement of DGAT3 in the partitioning of 18:3 into TAG in mutant seedlings growing on Suc. Fusion of the DGAT3 protein with green fluorescent protein confirmed localization to the cytosol of N. benthamiana. This work has demonstrated active recycling of 18:2 and 18:3 FAs into TAG when seed oil breakdown is blocked in a process involving a soluble cytosolic acyltransferase.

Figure 1.

Total TAG content of Col-0 and mutant DS and 5-DAI seedlings. Values are means ± sd of measurements from five separate batches of 50 seeds or 30 seedlings. Significant differences (Student’s t test, P < 0.05) are indicated by an asterisk.

Figure 2.

TAG composition of Col-0 and mutant DS and 5-DAI seedlings. Values are means ± sd of measurements from five separate batches of 50 seeds or 30 seedlings. TAG molecular species are given as three concatenated FA codes, with FAs coded as follows: P = 16:0, H = 16:3, S = 18:0, O = 18:1, L = 18:2, Ln = 18:3, Ar = 20:0, G = 20:1, Gi = 20:2, Be = 22:0, and Er = 22:1. Coding order reflects molecular composition only and does not imply regiospecific arrangements. Significant differences (Student’s t test, P < 0.05) were performed for selected pxa1 TAGs and are indicated by an asterisk.

Figure 3.

Total acyl-CoA content in Col-0 and mutant DS and 5-DAI seedlings (A) and acyl-CoA composition in 5-DAI seedlings (B). Values are means ± sd of measurements from five separate batches of 30 seedlings.

Figure 4.

Glycerolipid content of Col-0 and pxa1 DS (A) and 5-DAI seedlings (B). Values are means ± sd of measurements from five separate batches of 200 seeds or 100 seedlings. Lipid classes are abbreviated as follows: PC, Phosphatidylcholine; PE, phosphatidylethanolamine; PA, phosphatidic acid; DGDG, digalactosyldiacylgalactolipid; MGDG, monogalactosyldiacylgalactolipid.

Figure 5.

Total TAG content (A) and composition (B) of N. benthamiana leaves transiently expressing DGAT1 or DGAT3 compared with the ev negative control. Values are means ± sd of measurements from five samples of approximately 40 mg leaf tissue. Total TAGs (A) were analyzed by ANOVA followed by pairwise t tests with Bonferroni correction; significantly different groupings (P < 0.05) are indicated by letters above the bars. For TAG composition (B), the same tests were performed for each individual TAG; only those species where DGAT3 was significantly different from the ev control are labeled. The triolein TAG species denoted by an asterisk is the only one that is higher in DGAT3 compared with DGAT1.

Figure 6.

FA composition calculated from TAG in Col-0, dgat3, pxa1, and pxa1dgat3 5-DAI seedlings. Values are means ± sd of measurements from five separate batches of 30 seedlings. Significant differences (P < 0.05) between pxa1 and pxa1dgat3 are indicated by an asterisk.

Figure 7.

Subcellular localization of DGAT1-GFP (A) and DGAT3-GFP (B) fusion proteins by transient expression in N. benthamiana leaves. Scale as indicated.