Identification of Arabidopsis GPAT9 (At5g60620) as an Essential Gene Involved in Triacylglycerol Biosynthesis

Abstract

The first step in the biosynthesis of nearly all plant membrane phospholipids and storage triacylglycerols is catalyzed by a glycerol-3-phosphate acyltransferase (GPAT). The requirement for an endoplasmic reticulum (ER)-localized GPAT for both of these critical metabolic pathways was recognized more than 60 years ago. However, identification of the gene(s) encoding this GPAT activity has remained elusive. Here, we present the results of a series of in vivo, in vitro, and in silico experiments in Arabidopsis (Arabidopsis thaliana) designed to assign this essential function to AtGPAT9. This gene has been highly conserved throughout evolution and is largely present as a single copy in most plants, features consistent with essential housekeeping functions. A knockout mutant of AtGPAT9 demonstrates both male and female gametophytic lethality phenotypes, consistent with the role in essential membrane lipid synthesis. Significant expression of developing seed AtGPAT9 is required for wild-type levels of triacylglycerol accumulation, and the transcript level is directly correlated to the level of microsomal GPAT enzymatic activity in seeds. Finally, the AtGPAT9 protein interacts with other enzymes involved in ER glycerolipid biosynthesis, suggesting the possibility of ER-localized lipid biosynthetic complexes. Together, these results suggest that GPAT9 is the ER-localized GPAT enzyme responsible for plant membrane lipid and oil biosynthesis.

Figure 1.

Phylogenetic comparison of GPAT9, LPEAT1, and LPEAT2 from various monocot and dicot plant species. Protein sequences were aligned using ClustalX version 1.8.1 (Thompson et al., 1997). An unrooted phylogenetic tree was created from the alignment, using TreeView version 1.6.6 (http://taxonomy.zoology.gla.ac.uk/rod/rod.html; Page, 1996). The branch lengths are proportional to the degree of divergence, with the scale of 0.1 representing 10% change.

Figure 2.

Aborted ovules in GPAT9-2/gpat9-2 siliques. The white arrows indicate aborted ovules in GPAT9-2/gpat9-2 siliques. The Col-0 silique did not contain aborted ovules.

Figure 3.

Reduced pollen size and pollen tube growth of GPAT9-2/gpat9-2 pollen in the qrt1 background. A, Tetrad pollen in the qrt1 mutant can germinate to produce up to four pollen tubes, one from each attached pollen grain. B, Tetrad pollen in the qrt1 GPAT9-2/gpat9-2 line does not produce more than two pollen tubes per tetrad. C, Size of individual tetrad pollen grains from qrt1 and qrt1 GPAT9-2/gpat9-2 plants. Pollen grain length and width were measured to calculate the apparent surface area of each pollen grain.

Figure 4.

Segregating red and brown T2 GPAT9 amiRNA seed oil content and size. A, Oil content of segregating brown (not transformed; black bars) and red (transformed; red bars) T2 seeds from 20 individual T1 plant lines. Twenty red or brown seeds were used per analysis. FAME, FA methyl ester. B, Line 7 seeds under white light. C, The same seeds as in B under green light with a red filter. In B and C, the red GPAT9 amiRNA seeds are smaller than the brown untransformed seeds.

Figure 5.

Oil quantity and composition of homozygous T3 GPAT9 amiRNA lines. A, Distribution of whole-seed FA methyl ester (FAME) content among the wild type (Col-0) and different knockdown lines. Each diamond represents a 50-seed sample from an individual plant. Red bars indicate averages. B, FA composition of seed lipid analysis from A. Wild-type Col-0 (n = 36) and amiRNA knockdown lines 12 (n = 3) and 2 (n = 2) were chosen for further analysis. Values are averages ± sd.

Figure 6.

Developing silique microsome GPAT activity. GPAT assays were done with microsomes isolated from whole developing siliques 5 to 7 and 9 to 11 d after flowering (DAF) from Col-0 and GPAT9 amiRNA knockdown lines 2 and 12. The assay utilized 3.55 nmol of [¹⁴C]G3P and 25 nmol of palmitoyl-CoA for 15 min at 24°C. A, Phosphor image of a thin-layer chromatography (TLC) plate indicating that [¹⁴C]LPA and [¹⁴C]PA are the major products of whole-silique microsome GPAT assays utilizing palmitoyl-CoA as an acyl donor. B and C, Quantification of total products from GPAT activity in whole-silique microsomes. Two representative GPAT assays from a total of six GPAT assays were performed with different batches of plants grown at different times. Total GPAT activity was dependent on growth conditions and stage of development. However, with each batch of plants or stage of development, the GPAT activity of amiRNA lines was always less than that of the wild-type control. An additional two representative GPAT assays are shown in Supplemental Figure S11. MAG, Monoacylglycerol.

Figure 7.

Silique microsome LPAAT assays. LPAAT assays were done with microsomes isolated from whole developing siliques of Col-0 and GPAT9 amiRNA knockdown lines 2 and 12. Two assays were done with siliques aged 5 to 7 (left) and 9 to 11 (right) DAF. The assays utilized the same conditions as the GPAT assays in the text except that 3.33 nmol of [¹⁴C]oleoyl-CoA was the acyl donor and 25 nmol of 18:1-LPA was used as the acyl acceptor for 15 min at 24°C. The [¹⁴C]oleoyl-CoA can also be used by other acyltransferases and endogenous lipid acceptors found within the microsomes. However, PA was the major product of the assay in all samples. A and B, Phosphor images of TLC separation of products. C and D, Quantitation of PA within each lane of the TLC image. NL, Neutral lipids; FFA, free fatty acids.

Figure 8.

Testing of protein-protein interactions between AtGPAT9 and other lipid acyltransferases. Coding sequences for each of two different membrane proteins of interest were ligated in frame to either the C-terminal half of ubiquitin (Cub)-LexA transcription factor protein fusion or the N-terminal half of ubiquitin (Nub). Nub may be represented as either native polypeptide sequence (NubI) or a mutant form of Nub containing an Ile/Gly conversion point mutation (NubG). NubI strongly interacts with Cub. NubG has very weak affinity for Cub and must be brought into close proximity to Cub to allow for interaction of the two halves of ubiquitin, release of the LexA transcription factor, and, finally, activation of the reporter genes (His and adenine prototrophic markers and β-galactosidase, for quantitative analysis). A and B, Prototrophic growth assay of yeast strains containing various combinations of AtGPAT9 bait plasmid coexpressed with potential prey NubG-acyltransferase plasmids. Serial dilutions of cells expressing different bait-prey combinations were plated on nonselective (A) or selective (B) medium conditions. C, Quantitative measurement of β-galactosidase activity from cell lysates of the strains used in the serial dilution assays. LPCAT, Acyl-CoA:Lysophosphatidylcholine acyltransferase; DGAT, acyl-CoA:Diacylglycerol acyltransferase.

Figure 9.

Models of GPAT9-dependent glycerol flux and the spatial organization of TAG biosynthesis. The model is based on our results and previous in vivo labeling experiments (Allen et al., 2015). Wild-type (WT) TAG biosynthesis is dependent on the flux of G3P through GPAT9 for the initial incorporation of the glycerol backbone into glycerolipids (wide blue arrows). GPAT9, LPAAT2, and LPCAT2 are localized together for the efficient flux of acyl groups out of PC into de novo glycerolipid synthesis (ER complex 1). De novo DAG [DAG(1)] is utilized to produce PC. After traversing through the ER membrane, where desaturation can take place (large dashed arrow), PC is converted to PC-derived DAG [DAG(2)] and is incorporated into TAG through DGAT1. DGAT1 is spatially separated from GPAT9 but also associates with LPCAT2 in ER complex 2. Here, de novo DAG and PC-derived DAG are spatially separated and localized within ER complexes 1 and 2, respectively. The amount of 18:2/18:3 in TAG is dependent on the residence time of acyl groups in PC for desaturation versus the flux out of PC for incorporation into TAG. In GPAT9 amiRNA knockdown (KD) lines, the flux of G3P into glycerolipid synthesis is reduced (thin orange arrows embedded in large blue arrows), lowering total TAG accumulation. However, the rate of acyl exchange through acyl editing, and DAG exchange into/out of PC by phosphatidylcholine:diacyglycerol cholinephosphotransferase (PDCT), is not changed (green arrows). Therefore, the residence time of acyl groups in PC for desaturation increases as the overall flux of DAG to TAG slows down, leading to both higher overall levels of polyunsaturated FAs and a significantly higher ratio of 18:3 to 18:2 in the knockdown lines.