sn-Glycerol-3-phosphate acyltransferases in plants

Abstract

sn-Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the acylation at sn-1 position of glycerol-3-phosphate to produce lysophosphatidic acid (LPA). LPA is an important intermediate for the formation of different types of acyl-lipids, such as extracellular lipid polyesters, storage and membrane lipids. Three types of GPAT have been found in plants, localizing to the plastid, endoplasmic reticulum, and mitochondria. These GPATs are involved in several lipid biosynthetic pathways and play important biological roles in plant development. In the present review, we will focus on the recent progress in studying the physiological functions of GPATs and their metabolic roles in glycerolipid biosynthesis.

Plant lipids are composed of a broad group of fatty acids and their derivatives, including glycerolipids, lipid polyesters and sterols. They are involved in a diverse range of metabolic reactions and play important physiological roles in plant development, as major components of cellular membranes, storage reserves, extracellular protective layers and signaling molecules. The biosynthesis of these different types of lipids is controlled by a complex network of genes and proteins. In Arabidopsis, it has been estimated that more than 600 genes encoding enzymes or regulatory factors are involved in the lipid metabolic network, which includes at least 120 enzymatic reactions.¹ The complexity of this metabolic network is caused in part by the fact that the product of one enzymatic reaction can serve as a substrate for several different subsequent reactions, resulting in the formation of a variety of final products, often with diverse physiological functions in plant development.

As an example, sn-glycerol-3-phosphate acyltransferase (GPAT) is an important enzyme in the glycerolipid synthetic pathway and is involved in different metabolic pathways and physiological functions. It catalyzes the esterification of a fatty acyl moiety from acyl-CoA or acyl-ACP (where CoA is coenzyme A and ACP is acyl carrier protein) to the sn-1 position of sn-glycerol-3-phosphate (G3P), resulting in formation of lysophosphatidate (LPA) (Fig. 1).² LPA is a substrate for the production of several important glycerolipid intermediates including extracellular lipid polyesters, storage lipids, and membrane lipids.¹ The metabolic fate of LPA is controlled in part by the subcellular localization of the GPAT-catalyzed reaction, which occurs in three distinct plant subcellular compartments, i.e., plastid, endoplasmic reticulum (ER) and mitochondria.³ These reactions are catalyzed by three different types of GPAT enzymes, a soluble form localized in plastidial stroma using acyl-ACP as its natural acyl substrate, and two membrane-bound forms localized in the ER and mitochondria with acyl-CoA and acyl-ACP as natural acyl donors, respectively.² Over the past two decades, significant progress has been made in molecular identification and biochemical and physiological characterization of plant GPAT enzymes. This review will focus on recent progress in understanding plant GPATs from biochemical and physiological perspectives and discuss the roles of GPATs in glycerolipid biosynthesis in plants.

Figure 1.

The GPAT-catalyzed reaction. GPAT catalyzes the esterification of a fatty acyl moiety from acyl-CoA or acly-ACP to the sn-1 position of G3P to form LPA. ACP, acyl carrier protein; CoA, coenzyme A; G3P, sn-glycerol-3-phosphate; GPAT, sn-glycerol-3-phosphate acyltransferase; LPA, lysophosphatidate.

Plastidial GPAT

Plastidial GPATs (also known as ATS1 in Arabidopsis) have been purified and cloned from several plant species, such as pea (Pisum sativum),^4,5 spinach (Spinacia oleracea),^4,6 squash (Cucurbita moschata),^7,8 cucumber (Cucumis sativus)⁹ and Arabidopsis.¹⁰ Most of the studies in the 1980s were focused on protein purification and investigation of the enzymatic properties of the plastidial GPAT enzymes. Results from these studies discovered that the acyl substrate preference (i.e., saturated vs. unsaturated acyl-ACPs) of the plastidial GPAT partially controlled the chilling-tolerance of plants through mediating the fatty acid composition at the sn-1 position of phosphatidylglycerol (PG), the major phospholipid of plastidial membranes, and hence affecting the membrane fluidity of the aerial tissue of plant.^11,12 Previous studies have shown that plastidial GPATs of chilling-tolerant plants such as Arabidopsis,¹⁰ spinach^4,6 or pea,^4,5 exhibited a in vitro substrate preference for unsaturated (18:1^cisΔ9) vs. saturated (16:0) acyl substrates, whereas GPAT isolated from chilling-sensitive squash¹³ or Amaranthus lividus¹⁴ displayed higher activity with 16:0 than with 18:1 acyl substrates. A study of transgenic tobacco showed that transformation with a cDNA encoding the squash (chilling-sensitive) plastidial GPAT resulted in decreased chilling-tolerance, whereas transformation with cDNA encoding Arabidopsis (chilling-tolerant) plastidial GPAT results in increased chilling-tolerance.¹⁵

Though plastidial GPATs from different plant species have been biochemically characterized in vitro, the physiological functions of these enzymes were not fully understood until recently. The Arabidopsis plastidial GPAT (ATS1) deficient mutant lines (chemically mutagenized) were first isolated and characterized by Kunst et al.¹⁶ In this study, the mutant Arabidopsis plants exhibited a slight reduction (10–25%) of PG content and modified fatty acid composition in typical plastidial lipids such as the galactolipids of the leaves. Xu et al.¹⁷ further characterized the molecular and physiological defects of several Arabidopsis ats1 T-DNA/RNAi lines. Similar to the result from the earlier study, a reduction (~20%) of PG content was observed in the ats1 lines which was accompanied by delayed plant growth and aborted seed development. Furthermore, when lysophosphatidate acyltransferase (LPAAT, encoded by ATS2) catalyzing the subsequent acylation of LPA at the sn-2 position was downregulated in the ats1 mutant, the Arabidopsis double mutant line (ats1 ats2) exhibited strongly reduced PG content and severe reduction of growth. ATS1 and ATS2 were thus suggested to be involved in a coordinated regulation of plastidial PG synthesis, which is essential for plant development.¹⁷ Interestingly, an early study of the Arabidopsis ats2 T-DNA lines demonstrated that the loss of the plastidial LPAAT (ATS2) also caused embryo-lethality.¹⁸ Nevertheless, it is not clear that at what level a deficiency of only ATS2 would affect the PG biosynthesis in plant.

ER membrane-bound GPAT

Although the presence of GPAT activities in the ER of plant cells has been known for decades,^19-21 it was not easy to purify these enzymes due to their membrane-bound nature.¹² Thus, this limited studies of GPAT at biochemical or molecular level. In light of the Arabidopsis whole genome sequence being available, plant ER-bound GPATs were first identified in Arabidopsis (AtGPAT4–7) based on the amino acid sequence similarities to the conserved regions of known GPATs from bacteria, yeast and mammals.² To date, six Arabidopsis GPAT genes (AtGPAT4–9) have been identified with four of them (AtGPAT4,5,6,8) being functionally characterized in planta.^2,3,21 Recently, orthologous genes of AtGPAT4 have also been cloned from Brassica napus²³ and Echium pitardii²⁴ with confirmed GPAT enzyme activities. These studies revealed an important physiological role of the ER-bound GPAT enzymes in formation of extracellular lipid polyesters (i.e., cutin and suberin).^22-26 Additionally, because the biosynthesis of triacylglycerol (TAG) and membrane lipids mainly occurs in the ER, the ER-bound GPAT is also believed to be involved in the biosynthesis of these lipids.³ In the following sections, the metabolic roles and physiological functions of several ER membrane-bound GPATs will be discussed.

GPAT4 and GPAT8

In Arabidopsis, GPAT4 and GPAT8 exhibit high sequence identity (over 80% between the cDNAs), and are suggested to be duplicated genes²⁷ with functional redundancy in cutin (a type of fatty acyl and glycerol-based polyester) synthesis in leaves and stems.²² Neither of the single T-DNA mutant lines of gpat4 or gpat8 exhibit any obvious cuticle defect; however, the gpat4 gpat8 double T-DNA line of Arabidopsis exhibited a strong decrease in cutin content of the leaves and stems. Among all the cutin monomers, α, ω-18:2 dicarboxylic acid exhibited the most significant decrease in these gpat4 gpat8 lines. When GPAT4 or GPAT8 was overexpressed in Arabidopsis, however, the cutin monomers exhibiting the most significant increases were, α, ω-16:0 and 18:0 dicarboxylic acids. These results suggested that the GPAT-catalyzed cutin synthesis process is coupled with other enzymes that provide further modifications such as desaturation and carboxylation to the fatty acyl chains.^22,28 In addition to acylating at the sn-1 position of G3P, GPAT4 was also shown to catalyze the esterification of α,ω-dicarboxylic acid-CoA (DCA-CoA) to the sn-2 postion of G3P forming sn-2 DCA LPA.²⁹ In the same study, the researchers also found that AtGPAT4 has an additional phosphatase activity, resulting in a portion of the LPA being further converted to monoacylglycerol (sn-1 or 2 MAG), which was suggested to be an important intermediate for polyester assembly.²⁹ Furthermore, the GPAT4 orthologs in Brassica napus were found to be highly expressed in seed coat and the periderm and endodermis of root, and thus were suggested to be likely involved in the synthesis of another type of lipid polyester, suberin, in seed coat and root.²³

GPAT5

GPAT5 was demonstrated to be essential for suberin synthesis in root and seed coat.²⁵ Suberin is another type of fatty acyl and glycerol-based polyester that can be distinguished from cutin by the monomer composition and the deposit location in plants.³⁰ By analyzing the polyester monomer profiles in seed coat and root of the gpat5 T-DNA Arabidopsis mutant lines, the authors observed strong reductions in 22:0 and 24:0 fatty acids and their derivatives, and therefore suggested that the physiological role of GPAT5 is to provide acyl-glycerols containing 22–24 carbon groups to the suberin synthetic pathway. This substrate preference for C22 or C24 acyl chains of GPAT5 was further supported by the fact that Arabidopsis overexpressing GPAT5 had increased accumulation of very long chain saturated fatty acids in suberin.²⁵ In addition, AtGPAT5 can also acylate G3P at the sn-2 position with DCA-CoA; however, unlike GPAT4, it does not have a phosphatase activity.³⁰

GPAT6

GPAT6 is involved in cutin synthesis of the flower petals.²⁶ In the gpat6 T-DNA mutant Arabidopsis line, a strong reduction in cutin content was detected in the flowers, resulting in a lack of nanoridges on the petal surfaces.²⁷ By analyzing the cutin monomer profiles of the gpat6 T-DNA and GPAT6 overexpression lines, the authors proposed that GPAT6 is involved in using 16:0 and its derivatives for petal cutin synthesis.²⁶ Similar to AtGPAT4, AtGPAT6 was also confirmed to be able to esterify DCA-CoA to the sn-2 position of G3P and further convert LPA to MAG by its phosphatase activity.²⁹

The putative GPAT9 and other proposed metabolic functions of ER-bound GPAT

In addition to being essential for plant extracellular lipid polyester synthesis, the ER-bound GPAT is also believed to be involved in the formation of membrane and storage lipids.³ In mammalian cells, four membrane-bound GPATs, including two localized in mitochondria (GPAT1–2) and two localized in the ER (GPAT3–4), have been confirmed to play important roles in storage lipid biosynthesis.³¹ The phylogenetic analysis of the polypeptide sequences of the mammalian GPATs and Arabidopsis GPATs revealed that the membrane-bound AtGPAT1–8 have evolved quite distantly from mammalian GPAT1–4 (Fig. 2). Recently, a putative GPAT9 was identified in Arabidopsis by a bioinformatics approach, and exhibited a much closer evolutionary relationship with the mammalian GPATs (Fig. 2).³ Although the enzyme activity of this GPAT9 has not been directly confirmed and its physiological function is unknown, polypeptide sequence alignment, phylogenetic analysis, conserved domain analysis and gene expression data suggested this AtGPAT9 may be a functional enzyme, playing an essential role in plant membrane and storage lipid biosynthesis.³

Figure 2.

Phylogenetic analysis of the GPATs from Arabidopsis (At), Homo sapiens (Hs) and Saccharomyces cerevisiae (Sc). Arabidopsis GPAT1–8 (AtGPAT1–8) evolved quite distantly from the human GPATs (HsGPAT1–4) and yeast GPATs (ScGPAT1–2). Unlike other AtGPATs, the putative AtGPAT9 exhibits a closer evolutionary relationship with the human ER membrane-bound GPAT (HsGPAT3–4). AtGPAT1–3 and HsGPAT1–2 are mitochondrial membrane-bound proteins. AtGPAT4–9, HsGPAT3–4 and ScGPAT1–2 are ER membrane-bound proteins. ATS1 is a soluble GPAT protein located in the plastid stroma of Arabidopsis. The GPAT amino acid sequences were subjected to phylogenetic analysis using the PHYML program³³ of the Geneious software.

Mitochondrial GPAT

Among all three types of GPAT in the plant, the mitochondrial isoforms have been the least studied at molecular and biochemical levels. Three mitochondrial GPATs (AtGPAT1–3) were first identified in Arabidopsis together with the ER membrane-bound AtGPAT4–7.² To date, only AtGPAT1 has been characterized in detail. By analyzing the gpat1 T-DNA Arabidopsis lines, the AtGPAT1 was found to be important for tapetal differentiation and nutrient secretion, which are crucial for the development of pollen grains.² Additionally, the deficiency of GPAT1 activity in Arabidopsis also caused reduced seed set and altered fatty acid compositions of the storage or membrane lipids in a number of plant organs, i.e., pollen grain, flower bud and seed. These results suggested that in addition to ER-bound GPATs, the mitochondrial GPAT1 is also involved in the biosynthesis of eukaryotic glycerolipids,² although the mechanism behind this pathway is still unknown. Notably, in mammalian liver cells, LPA produced by the mitochondrial GPAT1 is transported from mitochondria to the ER for TAG assembly by a fatty acid binding protein.³² To date, there is no report of acyltransferase activity existing in plant mitochondria for subsequent acylations of LPA, thus, it is possible that the mitochondria-produced LPA is transported to the ER for eukaryotic glycerolipid biosynthesis with the help of certain unknown glycerolipid transporters.

Future prospects

Recent progress in reverse-genetics studies of Arabidopsis T-DNA lines has revealed a diverse range of biological processes that plant GPATs are involved in. These new insights and discoveries also lead to new questions and challenges for researchers. At the biochemical level, several ER-bound GPATs exhibited sn-2 acylation activity for G3P when assayed with DCA-CoA in vitro, thus, it will be interesting to know whether this sn-2 acylation activity is also present with regular fatty acyl-CoA. Two related questions are: 1) does the GPAT have an acyl substrate preference (i.e., DCA-CoA vs. regular acyl-CoA) for stereospecific (i.e., sn-1 vs. sn-2) acylation of G3P and 2) will the plastidial and mitochondrial GPATs also possess this sn-2 acylation activity? At the physiological level, although most of the GPAT family members have been characterized in Arabidopsis, more studies are needed for answering the unresolved questions. Given that the deficiency of plastidial ATS resulted in ~20% reduction in PG content, what are the alternative sources or biosynthetic routes for PG in Arabidopsis? There are also questions related to the membrane-bound GPATs. Since most of the enzymes involved in eukaryotic glycerolipid assembly are ER membrane-bound, the logical GPAT candidate for eukayotic glycerolipid biosynthesis should also be located in the ER. Only the mitochondrial GPAT1, however, has been shown to be involved in membrane and storage lipid synthesis; while a few ER GPATs have only been shown to play important roles in extracellular lipid polyester biosynthesis. Thus, it will be of interest to investigate the metabolic functions of ER GPATs in membrane and storage glycerolipid biosynthesis pathways. Additionally, insights into lipid transport between mitochondria and ER, which allows the mitochondrial AtGPAT1 to be involved in the eukaryotic glycerolipid synthesis pathway, may also contribute our knowledge for further understanding the glycerolipid biosynthesis in plants.