Introduction

Oil crops are a very important agricultural commodity with current value of about US $120 billion [1]. Usage of these oils has increased steadily at an annual rate of about 5 % over the last 50 years [2]. Although increased sowings and better agricultural productivity have managed to keep pace with demand until now, it is clear that, with limited fertile land, an increasing world population, and limits to productivity improvements, plant oils will soon be in short supply. In addition to the above factors, agricultural fats and oils will be needed increasingly for petrochemical replacements as biofuels or renewable chemicals [3, 4].

The major uses of biological oils are for food, as animal feeds, and as renewable chemicals or precursors thereof (79, 7, and 15 % in 2012, respectively). Four crops dominate world production of commodity oil—oil palm (Elaeis guineensis, E. oleifera, and Attalea maripa), soya bean (Glycine max), oilseed rape (Brassica napus), and sunflower (Helianthus annuus) [5, 6]. Currently these produce about 23, 22, 12, and 9 % of total commodity oils, respectively (Table 1). The rise in palm oil production over the last half century has been particularly marked, but future increases will be much smaller because of its limited growing regions and because of the praiseworthy example of Malaysia in strictly limiting future plantation expansion and the sponsoring of “sustainable palm oil” [7]. It remains to be seen if Indonesia will adopt similarly careful monitoring and regulation of oil palm plantations. The domination of world markets by four crops reduces the range of oils available. Although transgenic varieties of soybean and oilseed rape have been developed, the current public backlash against foods derived through genetic engineering means that the market is limited, especially in the European Union but also in other parts of the world including China and India. For sunflower, there are some varieties available which have altered oil composition such as high oleate (18:1Δ9cis) or high palmitate (16:0)–high oleate varieties [8], but few oil palm cultivars have been developed for oil quality, with efforts instead being focused on crop yield [7].

Table 1 World edible oil sources: trends, importance, and prices
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Many industrial applications require particular types of oil. This has led to the specialized use of many minor crops [6, 9]; for example, coconut (Cocos nucifera) oil with its high content of laurate is used for surfactant production [4]. Linseed (flax: Linum usitatissimum) enriched in α-linolenate (18:3Δ9cis,12cis,15cis) is used for paints and linoleum, and castor oil (Ricinus communis), which has 90 % ricinoleate (12-OH 18:1Δ9cis), is a useful lubricant [2, 4]. Efforts to manipulate high-yielding standard oil crops to produce specialized renewable chemicals have achieved some success. For example, some 20 years ago, Calgene produced genetically engineered oilseed rape that accumulated 60 % laurate [10], but this could not compete on the open market with coconut or, in particular, palm kernel oil. Nevertheless, as discussed below, much effort is focused on the production of specialized oils because it is certain that sooner or later there will be a use for them [4]. The use of genetically engineered oil crops for chemical purposes should attract less public opposition than their use for foods.

Eventually, the genetic manipulation of crops to produce oil more efficiently or to make novel specialized oils for particular purposes should be accepted as a good way to manage the planet’s environment. Therefore, scientists need to persevere in their efforts to understand metabolism in oil crops so that they are in a good position to inform future developments in a responsible way. There are two particular aspects that need attention: firstly, to increase total oil yields [11, 12] and, secondly, to understand how to efficiently change the fatty acid composition and channel such acyl chains into triacylglycerol (TAG) [13, 14]. Both of these aspects are discussed in the following sections.

Triacylglycerol Biosynthesis

Almost all plant oils are predominantly composed of TAG, the only significant commercial exception being the wax esters of the desert plant jojoba (Simmondsia chinensis). Basically TAG is formed in two phases. First, fatty acids are synthesized de novo in the plastid, from where they can be exported to the cytosol. Second, fatty acid modifications can take place on the endoplasmic reticulum (ER) before they are assembled into TAG end-products via either the acyl-CoA-dependent pathway [also known as the Kennedy pathway (Fig. 1)] or independent pathways (Fig. 2), or both [6, 11, 15, 16].

Fig. 1
figure 1

Triacylglycerol biosynthesis in plants via the Kennedy pathway. Fatty acid biosynthesis occurs in the plastid to generate (mainly) 16 and 18C products. These are then exported outside the plastid where, as acyl-CoAs, they participate in the Kennedy pathway to generate triacylglycerol. Alternative reactions involving phosphatidylcholine are shown in Fig. 2. For abbreviations see list at the front of the article

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Fig. 2
figure 2

Schematic of acyl-trafficking via phosphatidylcholine (PtdCho) in plant lipid biosynthesis. PtdCho is a central intermediate in the modification and flux of fatty acid into triacylglycerol (TAG) in developing seeds of oleaginous plants, and important enzymes (in bold) are involved in PtdCho-centric acyl-trafficking. For remaining abbreviations see the list at the front of the article

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De novo production of fatty acid uses photosynthate as a source of carbon either directly or via sucrose transport to the seed or fruit. Thus, one possible obvious way to increase oil crop yields is to divert carbon from carbohydrate into oil via the synthesis of acetyl-CoA [17]. The exact location of acetyl-CoA formation is controversial in some tissues [18]. Depending on the tissue, acetyl-CoA can be generated from multiple sources including amino acids, in addition to hexoses. Acetyl-CoA production, catalyzed by pyruvate dehydrogenase, also gives rise to Nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), which is used in turn during fatty acid formation. The whole topic of the interconnection between central pathways of metabolism and generation of important substrates has been the subject of considerable recent research, often using stable isotope techniques (see [19]).

In most plants, acetyl-CoA is used by a multi-subunit plastid form of acetyl-CoA carboxylase (ACCase) which is made up of four proteins–biotin carboxylase, biotin carboxylase carrier protein, and two subunits for the carboxyltransferase [20]. However, the Graminaceae have a multifunctional protein form with a molecular mass of 200–240 kDa [20]. These different forms of ACCase underlie the action of grass-specific herbicides (graminicides—aryloxyphenoxypropionates, cyclohexanediones) which specifically target the Graminaceae chloroplast ACCase [21, 22]. In many organisms, acetyl-CoA carboxylase exerts strong flux control over fatty acid (and lipid) formation. In leaves, ACCase has been shown to be extremely important [23], but the situation is less clear for oilseeds. The coincidence of high ACCase activity with the peak of oil accumulation in B. napus led Turnham and Northcote to suggest that ACCase has a major regulatory role in that species [24], but further work questioned this assumption [25]. Indeed, targeting the Arabidopsis homomeric ACCase to plastids of rapeseed only produced marginal increases in oil, even when transgenics were grown in growth chambers [26]. However, ACCase can be strongly feedback inhibited when oleate (or oleoyl-CoA/oleoyl-ACP) accumulates in embryonic cultures of B. napus, reducing oil yields by 50 % [27]. For oil palm, where most of the control over triacylglycerol synthesis is in fatty acid synthesis, ACCase was important and subject to acetyl-CoA feedback [28]. In vitro experiments in oil palm suggest that acetyl-CoA carboxylase is approximately equally important as fatty acid synthase in controlling overall carbon flux during fatty acid biosynthesis [29]. Finally, in transgenic Arabidopsis seeds accumulating hydroxyl-fatty acids, the reduction in oil content seemed to be at the level of ACCase [30]. Thus, it would be fair to conclude that, in many oil crops, ACCase is likely to exert significant flux control. A quantitative measure in vivo for normal TAG accumulation, however, has yet to be carried out.

The malonyl-CoA product of ACCase is used by a type II fatty acid synthase [20]. The sequential addition of two-carbon units to the growing fatty acid chain requires four reactions—condensation, reduction, dehydration, and a second reduction. In plants, there are three condensing enzymes, known as β-ketoacyl-acyl carrier protein (ACP) synthase I, II, and III (KASI, KASII, and KASIII). KASIII initiates the process by catalyzing the condensation of acetyl-CoA with malonyl-ACP. The majority of the other condensations are then catalyzed by KASI up to palmitoyl-ACP. The conversion of palmitoyl-ACP to stearoyl (18:0)-ACP is then catalyzed by KASII. The condensing enzymes can be inhibited (and therefore studied) by different chemicals such as arsenite and cerulenin [20, 31]. In the model plant Arabidopsis thaliana (Arabidopsis), there are genes encoding two isoforms of the first reductase, two genes for the dehydrase, but only one gene for the second (enoyl-ACP) reductase.

Most plants produce stearate and palmitate (in about a 3:1 ratio) as end-products. However, there are important exceptions; For example, oil palm mesocarp produces a ratio near 1:1, which accounts for the ~44 % palmitate in palm oil. On the other hand, the accumulation of medium-chain fatty acids (e.g., 6:0–12:0 in Cuphea species or 10:0 and 12:0 in coconut) is probably due to a distinct thioesterase which was first demonstrated in California bay (Umbellularia californica) [10, 32].

The plastid contains a soluble Δ9-acyl-ACP desaturase which usually catalyzes the conversion of stearate to oleate. In most plants, this desaturase also has activity with palmitate to form palmitoleate (16:1Δ9cis), a reaction which can be significant in some cases such as in cat’s claw (Doxantha unguis-cati) and macadamia nut (Macadamia integrifolia) [33, 34]. The Δ9-desaturase has been studied by John Shanklin and his group (e.g., [35]), who were particularly interested in substrate selectivity [36]. The Δ9-desaturase has high activity, therefore significant accumulation of stearate occurs in a limited number of plant species, such as cocoa (Theobroma cacao), where levels of approximately 35 % have been reported [2, 37].

Generally, there are two plastid thioesterases in plants, FAT A and FAT B, which catalyze the hydrolysis of newly formed acyl-ACPs, releasing nonesterified (free) fatty acid and ACP. These two thioesterases differ from each other in their acyl-ACP selectivity. FAT A catalyzes the hydrolysis of oleoyl-ACP preferentially, while FAT B also catalyzes the hydrolysis of saturated acyl-ACPs such as palmitoyl-ACP [38]. The plastid free fatty acids are then transported across both inner and outer plastid envelope membranes. This may be catalyzed by fatty acid export 1 (FAX1) [39], although the whole subject has been extensively discussed by Allen et al. [19]. It is generally assumed that the free fatty acids released are then used by a plastid-envelope long-chain acyl-CoA synthetase (LACS) [40–43]. There may be other acyl-CoA synthases found elsewhere in the cell (see [8]). The acyl-CoAs then accumulate in the cytosol, probably bound to acyl-CoA binding protein(s) [44], where they can be used by the acyltransferases of the Kennedy pathway (Fig. 1) [11, 15] or by the fatty acid elongation system [45], or may be partially used by acyl lipid synthesis in plastid [46].

The basic pathway of TAG biosynthesis is widely known as the Kennedy pathway [11, 47]. However, for historical accuracy, it should be noted that the initial two acylations to yield phosphatidate (Fig. 1) were reported first by Kornberg and Pricer [48]. Nevertheless, Eugene Kennedy’s seminal contributions included the continuation reactions to yield TAG as well as the various reactions used in the biosynthesis of phosphoglycerides [47, 49]. The Kennedy pathway (Fig. 1) has three acyltransferase reactions in addition to a phosphatidate phosphatase. Each acyltransferase has its own substrate selectivity. For plants producing unusual oils, substrate selectivity of the acyltransferase is pivotal in utilizing the unusual fatty acids that will be incorporated into their TAGs. In cases where genetic manipulation of “standard” agricultural oil crops is attempted, the substrate selectivity of acyltransferases is often an inherent problem which has to be considered seriously during development of the final transgenic crop. Diacylglycerol acyltransferase (DGAT) catalyzes the acyl-CoA-dependent acylation of DAG to produce TAG, and its activity may have a substantial effect on the flow of carbon into seed oil in some species [11]. In addition to the basic Kennedy pathway, there are a number of important additional enzymes with using phosphatidylcholine (PtdCho) as a central intermediate that may play a significant role in the generation of TAG—depending on the oil crop concerned. These are discussed in the following sections.

Due to an increasing interest in engineering crops to accumulate vegetative oils (particularly in leaves) [50] as a way of boosting their energy content for animal feed and other purposes, the so-called 16:3 and 18:3 plants need some comment. 16:3 crops accumulate hexadecatrienoate in the monogalactosyldiacylglycerol (MGDG) in their plastids, while the MGDG of 18:3 crops is instead dominated by α-linolenate. This difference is due to there being two alternative routes for the assembly of fatty acids onto the glycerol backbone. The first route, known as the eukaryotic pathway, uses acyl-CoA substrates for lipid assembly via the Kennedy pathway in ER, as described above. The second pathway is located entirely in the plastid and is known as the prokaryotic pathway. In this case, acyl-ACPs formed by fatty acid synthase are used directly for assembly onto a glycerol backbone without first undergoing hydrolysis (Fig. 1). Diacylglycerol (DAG) produced by each of these two routes differs in its fatty acid composition. DAG produced by the prokaryotic pathway has a characteristic distribution of fatty acid on the glycerol backbone of 18-carbon and 16-carbon at positions sn-1 and sn-2, respectively, while DAG originating from the eukaryotic pathway is enriched in 18-carbon fatty acid at both the sn-1 and sn-2 positions. The difference in plastid membrane lipid composition of 16:3 and 18:3 plants results from preferential use of DAG from the prokaryotic and eukaryotic pathways, respectively, to synthesize the plastid glycosylglycerides and phosphatidylglycerol [51, 52]. These processes underpin leaf TAG accumulation and are therefore important to consider for genetic engineering purposes.

Early Studies on the Role that Phosphatidylcholine Can Play in Fatty Acid Metabolism

Experiments in Tony James’ lab at Unilever around 50 years ago implicated PtdCho (and other glycerolipids) in the production of polyunsaturated fatty acids (PUFA) in the green microalga Chlorella vulgaris [53]. Further work confirmed that PtdCho was an important intermediate in the conversion of oleate to linoleate (18:2Δ9cis,12cis) in Chlorella vulgaris [54], and a similar lipid involvement was postulated for the conversion of linoleate to α-linolenate in pumpkin (Cucurbita pepo) [55]. The possible use of complex lipids as substrates for fatty acid modifications in algae and plants as proposed originally by Nichols, James, and coworkers had a precedent. Law and colleagues had previously examined cyclopropane synthase from Clostridium butyricum in 1964 and showed that the substrate for methyl group addition was attached to phosphatidylethanolanolamine, the major lipid of the organism [56].

After the initial observations at Unilever, the Δ12-desaturation of oleate was studied in a number of systems, and there was general agreement that PtdCho is the main substrate in most tissues [57–61]. However, there was more controversy for the Δ15-desaturation of linoleate to α-linoleate. Although PtdCho was suggested to be an important substrate for Δ15-desaturation [55], the most definite evidence was initially obtained with photosynthetic tissues where MGDG was the preferred substrate [62–64]. A more detailed discussion is available in Harwood [31].

Experiments with other tissues showed clearly that PtdCho was an effective substrate for fatty acid desaturation. Initially, the best evidence was reported for Candida lipolytica [65], but later research included animal systems [66]. The protozoon Acanthamoeba castellanii has been studied in considerable detail [67, 68] as well as the desaturase gene involved, which encoded a bifunctional desaturase with both Δ12- and Δ15-desaturase activities [69]. The initial controversy about the relative importance of PtdCho versus MGDG as a substrate for Δ15-desaturation in plants was finally resolved by the identification of the genes encoding the desaturases. Thus, the FAD3 gene on the ER uses PtdCho, while the FAD7/8 genes on the plastid thylakoids use MGDG [52]. Clearly, in most seeds accumulating TAG, the enzymes on the ER will be more important than their plastid counterparts and, therefore, expression of FAD2 (coding the Δ12-desaturase) and FAD3 will be critical.

The Contribution of Acyl-Trafficking via Phosphatidylcholine to Plant Lipid Biosynthesis

Since the first report of the function of PtdCho in the interrelationship between fatty acid biosynthesis and acyl-lipid synthesis [53], many biochemical and molecular studies have demonstrated that PtdCho is a central intermediate in the modification and flux of fatty acid into TAG in developing seeds of oleaginous plants [11, 15]. In this PtdCho-centric acyl-trafficking, acyl groups can be channeled into PtdCho from the acyl-CoA pool via the activity of lysophosphatidylcholine acyltransferase (LPCAT) or from DAG (Fig. 2) [15, 16]. Acyl groups are then modified on PtdCho in desaturation, epoxidation, conjugation or hydroxylation reactions catalyzed by FAD2/FAD3-like enzymes [3]. The modified acyl groups are subsequently transferred: (1) to the acyl-CoA pool by the combined forward/reverse reactions catalyzed by LPCAT and phospholipase As (PLAs) and LACS, which can be used for lipid biosynthesis on both ER and chloroplast envelope [46, 70–74]; (2) the DAG pool by the catalytic action of phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT), phospholipase C (PLC), or the combined activities of phospholipase D (PLD) and phosphatidate phosphohydrolase (PAPase) [15, 75, 76]; or (3) to TAG by the catalytic action of phospholipid:diacylglycerol acyltransferase (PDAT), which uses acyl groups from sn-2 position of PtdCho [77]. In addition to the above mechanisms, acyl chains can also be channeled directly from glycerophosphocholine (GPC) to LPC by acyl-CoA:glycerophosphocholine acyltransferase (GPCAT), and LPCAT can be utilized in LPC:LPC transacylation reactions by lysophosphatidylcholine transacylase (LPCT) activities creating PtdCho and GPC [70]. The physiological functions of the GPC-LPC-PtdCho reactions and the enzymes involved are not yet certain. Key enzymes involved in PtdCho-centric acyl-trafficking are discussed in the next section.

Lysophosphatidylcholine Acyltransferase and Phospholipase A

The rapid exchange of fatty acid between PtdCho and the acyl-CoA pool via PtdCho-deacylation and a lysophosphatidylcholine (LPC)-reacylation cycle without net PtdCho synthesis or degradation is a major component of the acyl editing process. The forward reaction catalyzed by LPCAT is involved in the LPC-reacylation. In contrast, PtdCho-deacylation may be catalyzed by both the reverse activity of LPCAT and PLA2 + LACS action within the Lands cycle [46, 73, 78–81].

In oleaginous plants producing seed oils enriched in PUFA or unusual fatty acids, the majority of fatty acids newly exported from the plastid in the form of acyl-CoA enter PtdCho rather than the Kennedy pathway [19, 82–84]. The forward reaction catalyzed by LPCAT is responsible for this reaction [81, 84]. Microsomes prepared from developing seeds/embryos of different plant species have been used to study the substrate selectivity of LPCATs (see [85]). Recently, recombinant plant LPCATs were produced in yeast, and their biochemical properties were rigorously tested by radioactive labeling experiments [79]. In vitro assay results indicated that LPCAT-mediated acyl editing of PtdCho is a result of the combined forward and reverse reactions catalyzed by the enzyme. In the forward reaction (LPC-reacylation), LPCATs can incorporate acyl groups at both sn-1 and sn-2 positions with a preference for C18-unsaturated acyl-CoAs but not C16-CoAs or ricinoleoyl-CoA. In the reverse reaction (PtdCho-deacylation), however, all LPCATs prefer ricinoleoyl-PtdCho when an equimolar mixture of ricinoleoyl-PtdCho and oleoyl-PtdCho is used as substrate [79]. In vivo studies with Arabidopsis lpcat1 lpcat2 double knockout mutants are also strongly supportive of the role of LPCAT in acyl exchange between the acyl-CoA pool and PtdCho [81, 84].

Plants have up to 29 PLAs with diverse structure and substrate preferences [71]. The involvement of PLA2 in PtdCho deacylation was first proposed in the Lands cycle [86]. Recent in vitro and in vivo analyses suggest that two PLAs, patatin-like PLA-IIIδ (pPLA-IIIδ) and the small molecular PLA2α (sPLA2α), may be involved in acyl editing [72, 73, 78]. pPLAIIIδ is a membrane-binding protein with a typical Asp-Gly-Gly catalytic dyad motif. It can catalyze hydrolysis of PtdCho at both the sn-1 and sn-2 positions with a preference for sn-2 position [73]. This enzyme also has thiolesterase activity, as it can catalyze the hydrolysis of acyl-CoA [73]. In Arabidopsis, ppla-IIIδ T-DNA insertional knockout mutant seeds have significantly lower oil contents, whereas Arabidopsis mutants with pPLA-IIIδ overexpressed have increased TAG content without any detrimental effect on overall seed yield per plant [73]. In addition, overexpression of pPLAIIIδ under the control of a seed-specific promoter boosted seed oil content without compromising plant growth in Camelina sativa [72].

The function of castor sPLA2α in acyl editing has been identified recently [78]. During seed development, castor can accumulate a high level of the hydroxy fatty acid ricinoleate in TAG. RcsPLA 2 α has much higher expression in castor endosperm transcriptome compared with that of Arabidopsis, and it is the highest among all castor PLAs. In vitro assays showed a high selectivity of RcsPLA2α for ricinoleic acid, superior to that of AtsPLA2α. Coexpression of RcsPLA 2 α in Arabidopsis with castor hydroxylase RcFAH12 resulted in a dramatic decrease in hydroxy fatty acid content in both PtdCho and the nonpolar lipid fraction of seeds when compared with RcFAH12 expression alone, whereas the coexpression of AtsPLA 2 α with RcFAH12 did not. Therefore, RcsPLA2α functions as a PLA2 with enhanced selectivity for catalyzing the removal of hydroxy fatty acid from PtdCho. As sPLA2α generally exists in plants, further studies of this enzyme in transgenic plants accumulating other unusual fatty acids (such as conjugated fatty acids) would improve our understanding of its function in acyl editing.

In summary, the LPC-reacylation cycle is catalyzed by the forward reaction of LPCAT. In contrast, the PtdCho-deacylation cycle may be catalyzed by LPCAT and/or PLA [72, 73, 78, 79, 81]. This conclusion is further supported by a recent in vitro assay of Arabidopsis mutants: in the PAPase lpcat double knockout mutant, PLA2s were massively induced as a response to a need for increased PtdCho deacylation in the absence of the LPCAT reverse reaction, whereas none of the PLA transcripts showed a significant change in the PAPase single mutant where the reverse reaction of LPCAT was enough for PtdCho deacylation [80]. A conclusive in vivo determination of the relative contribution of the reverse reaction of LPCAT versus PLA in PtdCho-deacylation would substantially expand our understanding of PtdCho-centric acyl-trafficking.

Cholinephosphotransferase and Phosphatidylcholine:Diacylglycerol Cholinephosphotransferase

It has been firmly established that PtdCho and DAG can be readily interconverted. Two enzymes, cholinephosphotransferase (CPT) and PDCT, have been proposed to catalyze this interconversion [75, 76]. PLC and/or PLD+PAPase may also be involved in the conversion of PtdCho to DAG [15, 16].

CPT catalyzes the final condensation reaction of DAG with Cytidine diphosphate-choline (CDP-choline) in the CDP-choline pathway for PtdCho synthesis [87, 88]. In 1955, Kennedy and Weiss first reported this enzyme in rat liver, and somewhat later it was shown that CPT is an ER membrane-bound protein and that the CPT reaction is reversible in mammalian cells [89, 90]. In 1971, CPT was characterized from plant tissues (spinach leaf microsomes) for the first time [91]. The results showed that spinach CPT has many similar biochemical properties to animal microsomal CPT, including an optimum pH at about 8.0 and a K m of 10μM for CDP-choline. Spinach CPT also requires either Mn2+ or Mg2+ as cofactor, and the saturation concentration for enzymatic activity was 0.3 mM for Mn2+ and 13 mM for Mg2+. Further studies of this enzyme were made shortly afterwards by Kates’ group [92, 93]. In 1982, Jolliot et al. reported that CPT of potato microsomes was a phospholipid-dependent enzyme, the activity of which could be regulated by the surrounding lipid microenvironment [94]. Based on metabolic labeling experiments, in 1983, Slack et al. demonstrated the reversibility of the CPT reaction in developing linseed cotyledons and proposed that this reaction provides a mechanism for the production of a highly unsaturation DAG pool for synthesis of TAG with enriched 18:2 and 18:3 [95]. The possible synthesis of DAG catalyzed by CPT, however, represents a thermodynamically unfavorable process when compared with the formation of PtdCho. Because CPT is a key enzyme involved in PtdCho synthesis, one attractive hypothesis for this enzyme is that it might have the ability to exclude DAG molecules with unusual fatty acids from being used for membrane lipid biosynthesis [96]. However, CPT from different oilseeds, including safflower [76], Cuphea, castor bean, and rapeseed [97], showed little or no preference with a broad range of DAG substrates.

The contribution of PDCT to the dynamic interactions between the PtdCho and DAG pools was demonstrated recently [75, 98]. PDCT is encoded by the REDUCED OLEATE DESATURASE1 (ROD1) gene and was first isolated from Arabidopsis [75]. PDCT is closely related to the mammalian sphingomyelin synthease within the large family of lipid phosphatase/phosphotransferase proteins. It transfers the phosphocholine head group from PtdCho to DAG, thereby catalyzing a symmetrical interconversion between PtdCho and DAG. In contrast to the CPT-catalyzed reaction that leads to a net production of PtdCho from DAG, PDCT action generates new molecular species of PtdCho and DAG, and as a result, there is no net accumulation of PtdCho or DAG [99]. The substrate specificity of PDCT allows oleate flux through PtdCho for further desaturation and movement of linoleate and linolenate back into the DAG pool, thus providing the DAG pool with PtdCho-modified fatty acids as substrates for PUFA-enriched TAG synthesis. A loss-of-function mutation of this gene resulted in significantly reduced linoleate and linolenate and a concomitant increase in oleate in seed TAG. Further analysis of the rod1/lpcat1/lpcat2 Arabidopsis triple mutant indicated that PDCT-mediated PtdCho-DAG interconversion together with LPCAT-mediated acyl editing control the major fluxes of PUFA from PtdCho to TAG (approximately 66 %) in Arabidopsis seeds [84]. Recently, overexpression of flax PDCT in wild-type Arabidopsis was shown to enhance the PUFA content of the seed oil [100]. In addition to the contribution of PDCT in channeling PUFA from PtdCho to DAG, PDCT is also required for efficiently shuffling unusual fatty acids (such as hydroxy fatty acids) from PtdCho to DAG and eventually increasing the amount of hydroxy fatty acids in TAG [98]. Given the potential difficulty associated with CPT-catalyzed formation of DAG, the channeling of PUFA-enriched acyl groups from PtdCho to DAG is probably more associated with PDCT action.

Phospholipid:Diacylglycerol Acyltransferase

In addition to the Kennedy pathway, TAG can also be formed via an acyl-CoA-independent pathway [77, 101]. This pathway is mediated by PDAT, which catalyzes transfer of an acyl group from a phospholipid to DAG, yielding TAG and a lysophospholipid. The PDAT gene family is present widely in yeast, microalgae, and plants [102, 103]. It is noteworthy that PDAT activity has also been detected in the bacterium Streptomyces coelicolor [104], but this reaction has no counterpart in mammalian cells.

PDAT activity was originally detected in microsomal preparations from three different oilseeds: castor bean, Crepis palaestina, and sunflower [77]. In the same study, the first PDAT gene was cloned from yeast (Saccharomyces cerevisiae) as a homolog of lecithin:cholesterol acyltransferase, which is a soluble enzyme responsible for catalyzing the synthesis of cholesteryl-ester in blood plasma. Knowledge of the yeast PDAT sequences enabled the subsequent identification of two PDAT orthologs, AtPDAT1 (At5g13640) and AtPDAT2 (At3g44830), in Arabidopsis. The expression level of AtPDAT1 was higher in leaves than in seeds [103]. Overexpression or knockout of AtPDAT1 in Arabidopsis resulted in significant changes in oil content and fatty acid composition in leaves but not in seeds [105]. A further study indicated that the AtPDAT1-mediated TAG synthesis is involved in the process of diverting fatty acids from membrane lipids towards peroxisomal β-oxidation and, thereby, is important for maintaining membrane lipid homeostasis in Arabidopsis leaves [106]. The role of AtPDAT1 in TAG synthesis has also been studied in the absence of DGAT1 activity. When PDAT1 was suppressed using RNAi-mediated gene silencing under a dgat1 knockout background, the oil content was further reduced by 63 % compared with the dgat1 control [107]. This result suggested that AtPDAT1 is the major contributor to seed oil synthesis when AtDGAT1 activity is compromised. The embryonic lethality in dgat1/pdat1 double mutant suggested that PDAT1 and DGAT1 have overlapping functions for TAG synthesis in pollen and seed and their expression is essential for pollen and seed viability. Studies comparing the relative contribution of AtPDAT1 versus AtDGAT1 to TAG synthesis in leaves have resulted in different conclusions. Fan et al. compared leaf TAG levels of the wild type with that of dgat1 and pdat1 mutants as well as with that of 35S-PDAT1 and 35S-DGAT1 overexpression lines [102]. The results suggested that AtPDAT1 plays a more important role than AtDGAT1 in TAG synthesis in young Arabidopsis leaves. In contrast, pulse-chase labeling experiments showed that [14C]12:0 was incorporated into TAG by leaves of the pdat1 mutant at a much higher rate than that of the dgat1 mutant, indicating that DGAT1 is the predominate enzyme involved in TAG synthesis in young Arabidopsis leaves [108]. The relative contribution of AtPDAT1 and AtDGAT1 to lipid metabolism needs to be further explored. The other ortholog, AtPDAT2, has no substantial role in TAG biosynthesis even though the encoding transcript is highly expressed in developing seed [107].

In addition to Arabidopsis PDAT, three castor [109, 110], six flax [111], and one green alga (Chlamydomonas reinhardtii) [112] PDAT have also been functionally characterized. Overall, these results support the idea that multiple PDAT paralogs arising from the core eudicot-shared ancient genome duplication may have evolved with different TAG-synthesizing abilities and developed divergent expression patterns due to varied selection pressures [103]. It is also important to note that both in vivo and in vitro approaches revealed that PDAT appears to have enhanced preferences for modified fatty acids, including PUFA and unusual fatty acids. Dahlqvist et al. demonstrated that the microsomal PDAT from yeast has a higher preference for ricinoleoyl- or vernoloyl (cis-12-epoxy-octadeca-cis-9-enoyl)-DAG over dioleoyl-DAG [77]. The PDAT activity in microsomal preparations from leaves from the AtPDAT1 overexpresser showed that AtPDAT1 has a strong preference for PtdCho containing acyl groups with several double bonds, epoxy, or hydroxyl groups [113]. Recently, a pair of flax PDAT genes were identified, which are preferentially expressed in seeds and encode enzymes with the unique ability to efficiently channel α-linolenic acid into TAG [111]. Similarly, it appears that castor also contains a specialized PDAT for the selective transfer of hydroxy fatty acids into TAG [109, 110]. Together, these results suggest that the contribution of PDAT to TAG synthesis in seeds might be significant in some oilseeds which contain TAG enriched in PUFA or unusual fatty acids.

Closing Comments

Although our understanding of plant oil accumulation has increased substantially over the last two decades, many important questions remain unanswered. There appear to be differences in the relative contributions of fatty acid synthesis versus TAG assembly in different species in the control of the flow of carbon into oil. More detail is required regarding the properties of the enzymes catalyzing the four steps of the Kennedy pathway. The transport of acyl chain and lipid fractions between plastids and the ER needs to be extensively explored. In addition, to inform future genetic manipulation, more in-depth information is required on both the genetic and biochemical regulation of the lipid accumulation process in plants.

PtdCho, as the main membrane lipid in most seeds, has an important role mediating fatty acid desaturation and receiving and donating acyl chains for TAG production. Moreover, for some 30 years this phosphoglyceride has been known to be a key metabolic substrate for desaturation and other fatty acid modifications. Because of this, the passage of fatty acid substrates onto PtdCho has been recognized to be dynamic. In recent years this acyl-editing process has been further detailed with a multitude of ancillary enzymes that can participate. However, delineation of the overall process, its refinement in different plants, and the properties of the individual enzymes involved are in need of further investigation.

Knowledge of these metabolic details will be crucial to understanding plant oil accumulation and in the design of new strategies for biotechnological modification of plant lipids.