Lipids are one of the most important groups of biological macromolecules in living cells. They have many important biological functions, including storing energy, signalling and acting as structural components of cell membranes (Fahy et al. 2009, Subramaniam et al. 2011). Thus they occur in many different forms, including fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, triglycerides, phospholipids and glycolipids. Beside surface lipids (1%) and membrane lipids (5%), most of the lipids in the seed, as the storage organ, are storage lipids (94%), which almost entirely consist of triacylglycerols
(TAGs) and are collected in oil bodies (Harwood 1996, Li-Beisson et al. 2013, Chen et al. 2015).
Triacylglycerols are composed of three fatty acids connected to a glycerol backbone. The formation of TAG can be divided into three stages. Initially fatty acids are newly built in the plastid. Subsequently these are exported in the form of acyl-CoA thioesters to the cytoplasmic endomembrane system, where the modification of fatty acids and finally the assembly of storage lipids occurs (Roscoe 2005).
2.4.1 Fatty acid synthesis
Fatty acids are built de novo in every cell of a plant. The synthesis takes place in the plastids, therefore it is referred to as the prokaryotic part of lipid synthesis (Roughan and Slack 1982).
Fatty acids are produced by the enzymes of the fatty acid synthase (FAS) complex. Using acetyl-coenzyme A (CoA) as precursor, a fatty acid carbon chain is elongated through sequential addition of two-carbon units. Each elongation process consists of four reactions: condensation, reduction, dehydration and reduction, with the acyl carrier protein (ACP) as cofactor of all reactions. Acetyl-CoA, as the major building unit of fatty acids, is mainly produced by the plastidial pyruvate dehydrogenase complex. The two carbon donor molecule necessary for fatty acid elongation is malonyl-ACP. In an ATP dependent two-step reaction acetyl-CoA and hydrogen carbonate are assembled to malonyl-CoA. This reaction is catalysed by a multisubunit heteromeric enzyme complex of prokaryotic type, the acetyl-CoA carboxylase (ACC) (Harwood 1996, Konishi et al. 1996). The first step of this reaction is catalysed by the biotin carboxylase (BC) domain of ACC, transferring CO2 from bicarbonate to a biotin prosthetic group attached to a conserved lysine residue of the biotin carboxyl carrier protein (BCCP) domain of ACC. In a second step, the carboxyl group from carboxy-biotin is transferred to acetyl-CoA to form malonyl-CoA, catalysed by the carboxyltransferase (CT) domain of ACC. Two different subunits are forming the CT domain, α-CT and β-CT. β-CT being the only component in plant lipid metabolism encoded by the plastid genome (Ohlrogge and Browse 1995). Thus, implying a coordinated production of cytosolic and plastid subunits to build a functioning ACC. To finally enter the fatty acid synthesis cycle malonyl-CoA assembled by ACC is transferred to ACP by malonyl-CoA:ACP malonyltransferase (MCMT), forming malonyl-ACP. The initial elongation cycle is assembling acetyl-CoA and malonyl-ACP, starting with a condensation reaction yielding the four-carbon product 3-ketobutyryl-ACP. The condensing enzymes of fatty acid synthesis are 3-ketoacyl-ACP synthases (KAS), while the first condensation is catalysed by KAS III, the condensation reactions of following elongation cycles are catalysed by KAS I. Elongation from palmitoyl-ACP (C16) to stearoyl-ACP (C18) is catalysed by a third isoform, namely KASII
(Pidkowich et al. 2007). After condensation 3-ketobutyryl-ACP is reduced to 3-hydroxyacyl-ACP by ketoacyl-ACP reductase (KAR). Hydroxyacyl-ACP dehydratase (HAD) then is dehydrating 3-hydroxyacyl-ACP leading to enoyl-ACP, which is finally reduced to C4 saturated fatty acid-ACP, the precursor for the next elongation cycle, by enoyl-ACP reductase (ENR) (Mou et al. 2000).
Assembling of the C4 acyl-ACP with another malonyl-ACP initiates the next elongation cycle. The serial addition of two-carbon units to the growing fatty acid chain catalysed by the enzymes of the FAS complex is finally terminated by hydrolysation of 16:0-ACP and 18:0-ACP. Hydrolysation is catalysed by two different acyl-ACP thioesterases and results in free fatty acids. Fatty acid thioesterase B (FATB) produces palmitic acid by the hydrolysation of 16:0-ACP, while fatty acid thioesterase A (FATA) hydrolyses 18:0-ACP, which was produced by an additional cycle in the FAS machinery utilising KASII for condensation, releasing the 18:0 free fatty acid, stearic acid.
Alternatively, 18:0-ACP can first be desaturated by Δ9 stearoyl-ACP desaturase (SAD) producing 18:1-ACP, which is then hydrolysed by FATA to oleic acid. All free long-chain fatty acids produced are esterified with CoA by a long chain acyl-CoA synthetase (LACS) and exported from the plastid to the endoplasmic reticulum, where they are used to build storage lipids.
DNA microarray data indicated co-regulation of core enzymes of fatty acid synthesis at the transcriptional level (Mentzen et al. 2008) and further investigations identified the transcription factor WRINKLED1 (WRI1) directly activating the fatty acid biosynthesis pathway (Cernac and Benning 2004, Baud et al. 2007; Maeo et al. 2009). However, not only transcriptional control but also optimization of enzyme activity is regulating fatty acid biosynthesis (Buckhout and Thimm 2003).
2.4.2 Modifications of fatty acids
In B. napus two types of modification of fatty acids can be distinguished, the desaturation and the sequential elongation of oleic acid (18:1). Desaturation of 18:1 is conducted by two specialized microsomal membrane-associated desaturases, FAD2 (Δ12) and FAD3 (Δ15), which form 18:2 and 18:3, respectively. However, the elongation of 18:1 leads to the production of the long chain unsaturated fatty acids (LUFAs), eicosenoic (20:1) and erucic acid (22:1). The elongation from 18:1 to 22:1 takes place in the cytoplasm and is catalysed by the membrane-bound oleoyl-CoA elongation complex. Four successive reactions are included in the elongation process. In a first step β-ketoacyl-CoA synthase (KCS) is catalysing the condensation of malonyl-CoA with the long chain (18:1 or 20:1) acyl-CoA, resulting in the formation of β-ketoacyl-CoA. The second step comprises the reduction of β-ketoacyl-CoA by β-ketoacyl-CoA reductase using NAD(P)H as
reductant. The reduction leads to β-hydroxyacyl-CoA which in a third step is dehydrated to an enoyl-CoA by β-OH-acyl-CoA dehydratase. Enoyl-CoA finally undergoes a second reduction, which is mediated by trans-2,3-enoyl-CoA reductase also using NAD(P)H as reductant. The second reduction forms the long chain (20:1 or 22:1) acyl-CoA (Fehling and Mukherjee 1991, Harwood 1996). This elongation process is adding a two-carbon fragment to the carboxyl end of oleic acid forming eicosenoic acid in a first cycle and erucic acid after a second two-carbon addition (Downey and Craig 1964, Jönsson 1977, Sasongko et al. 2003). The erucic acid biosynthesis has been well characterized in A. thaliana identifying fatty acid elongase 1 (FAE1), encoding the condensing enzyme KCS, as key-regulator (Lemieux et al. 1990, Kunst et al. 1992, Rahman et al. 2008).
2.4.3 Triacylglycerol synthesis
After fatty acids have been synthesised they are exported from the plastid to the ER were they enter the so called Kennedy pathway or glycerol phosphate pathway to form triacylglycerols (TAGs). Beside fatty acid chains the main component of these storage lipids is glycerol-3-phosphate (G3P). The first reaction of the glycerol glycerol-3-phosphate pathway is the acylation of G3P at its sn-1 position, which is catalysed by glycerol-3-phosphate acyltransferase (GPAT). In a second acylation step 2-lysophosphatidic acid acyltransferase (LPAAT) is transferring a second fatty acid from the acyl-CoA pool to the sn-2 position. The resulting phosphatidic acid afterwards is dephosphorylated by phosphatidate phosphatase (PP) forming diacylglycerol (DAG). A particular characteristic of the Brassica LPAAT is its specificity, making the utilization of erucoyl-CoA as an acyl donor incapable (Bernerth and Frentzen 1990, Taylor et al. 1992). Thus, erucoyl moieties are typically excluded from the central sn-2 position of the triacylglycerol molecule in B. napus (Nath et al. 2008). The resulting DAGs are representing important intermediates not only for storage but also for membrane lipid synthesis. Therefore, the final acylation of the sn-3 position of the glycerol backbone is the unique and specific reaction in TAG biosynthesis.
Depending on the acyl donor source three different mechanisms have been identified contributing to this step. Using a fatty acyl-CoA molecule acetylation of the sn-3 position of DAG is catalysed by diacylglycerol acyltransferase (DGAT). There have been two classes of DGAT identified, DGAT1 and DGAT2 respectively, which are differing in their sequence and membrane topology as well as their substrate discrimination. Up to now only DGAT1 has been shown to play a role in seed oil accumulation, while the role of DGAT2 remains to be confirmed. A second way of DAG acylation is catalysed by a phospholipid:diacylglycerol acyltransferase (PDAT) that is utilizing phosphatidylcholine (PC) as acyl source. PC is generated from lyso-PC by
lysophosphatidylcholine acyltransferase (LPCAT). Hence, TAG formation by PDAT depends on LPCAT activity. Dahlqvist et al. (2000) detected PDAT activity in plants and the gene encoding PDAT was identified in A. thaliana by Ståhl et al. (2004). Examination of mutants by Zhang et al.
(2009) indicated that PDAT1 is capable of compensating absence of DGAT1, because double mutants of PDAT1 and DGAT1 were lethal, and RNAi suppression of either gene in a mutant background lacking the other gene resulted in severe defects in pollen and seed development, including greatly reduced oil bodies and oil content, but dgat1 mutants only showed a minor reduction of oil content. Diacylglycerol:diacylglycerol transacylase (DAGTA) is catalysing the third reaction mechanism synthesising TAG by transferring a acyl group from one DAG to another (Roscoe 2005 ). Synthesised TAGs converge and are released from the ER enclosed by a phospholipid monolayer as the so called oil bodies or lipid droplets. The phospholipid monolayer is also containing different types of proteins, including oleosins, caleosins and steroleosins (Jolivet et al. 2004). Oleosins, which build the most abundant group, are regulating the size of oil bodies and thus enabling the mobilization of the TAG storage during seed germination by maximizing the surface-to-volume ratio of the oil bodies (Siloto et al. 2006, Shimada et al. 2008). While Caleosins also seem to play a role in TAG mobilisation during germination through mediating interactions with vacuoles (Poxleitner et al. 2006), steroleosins appear to play a role in signal transduction (Lin et al. 2002).
2.4.4 Candidate genes of oil biosynthesis
To reveal the mechanisms and characterize the genes involved in plant lipid biosynthesis a number of different genetic, molecular and biochemical studies were performed on the model plant and close relative of B. napus, A. thaliana (Ohlrogge et al. 2000). Information on the genes found to be involved in the lipid biosynthesis was thereupon collected in the Arabidopsis Lipid Gene Database (Beisson et al. 2003, http://aralip.plantbiology.msu.edu/pathways/pathways).
The comparison of the A. thaliana wild type to collections of A. thaliana mutants identified the tag1-mutant (Zou et al. 1999) and the wrinkled1-mutant (Focks and Benning 1998) with reduced seed oil content. The tag1-mutant which additionally showed an altered fatty acid composition (Katavic et al. 1995) was traced back to the DGAT-gene. Seed-specific overexpression of the DGAT-gene increased the oil content (Jako et al. 2001). However, the decreased oil content in the wrinkled1-mutant of about 80% was caused by the mutation of the wri1-locus, presumably encoding a transcription factor (Cernac and Benning 2004b, Bach 2007).
Microarray experiments of Ruuska et al. (2002) allowed the simultaneous investigation of >100 genes involved in lipid metabolism, enabling a broad overview of the transcriptional regulation
of the pathway. The researchers identified an expression cascade for specific groups of genes involved in oil biosynthesis during seed maturation. The comparison of wild type expression to the expression of the wri1-mutant identified 45 genes showing clear differences in their expression patterns. Most of these genes appeared to encode key regulators of fatty acid synthesis or carbon metabolism like BCCP2, KASI, enoyl-ACP reductase (ENR), two ACP isoforms, FAD2 (Ruuska et al. 2002). Thelen and Ohlrogge (2002) also realized the important role of the BCCP2-gene, since overexpression as well as antisense mediated reduction inactivated the plastid acetyl-CoA-carboxylase, which led to decreased oil content and changed fatty acid composition. Voelker and Kinney (2001) reviewing the “Variation in the Biosynthesis of Seed-storage Lipids” demonstrated that nearly every modification of the enzymes involved in oil biosynthesis, including fatty acid synthesis, modification and elongation as well as TAG formation, changes seed oil production.