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Cytosol

Formation of PUFA occurs FA on PC

Acyl-exchange with FA-CoA pool may occur at sn-2 position of PC via LPCAT action

FIGURE 1.1 Generalized scheme for fatty acid (FA) and triacylglycerol (TAG) biosynthesis in developing seeds of oil crops. Reactions having a substantial effect on the flow of carbon into seed oil are indicated with an asterisk. The formation of TAG can occur through acyl-coenzyme A (acyl-CoA)-dependent processes (enzyme 8) and acyl-CoA-independent processes (enzymes 9 and 10). Enzymes 1-12 are 1, acetyl-CoA carboxylase; 2, fatty acid synthase complex; 3, acyl-CoA synthetase; 4, sn-glycerol-3-phosphate dehydrogenase; 5, sn-glycerol-3-phosphate acyltransferase; 6, lysophosphatidic acid acyltransferase; 7, phospha-tidic acid phosphatase; 8, diacylglycerol acyltransferase; 9, diacylglycerol transacylase; 10, phospholipid:diacylglycerol acyltransferase; 11, choline phosphotransferase; 12, phospholi-pase A2. Additional abbreviations: ATP, adenosine triphosphate; CoA, coenzyme A; DAG, sn-1, 2-diacylglycerol; DHAP, dihydroxyacetone phosphate; ER, endoplasmic reticulum; FA, fatty acid; FA-ACP; fatty acyl-acyl carrier protein; FA-CoA, fatty acyl-coenzyme A; G3P, sn-glycerol-3-phosphate; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; LPCAT, lysophosphatidylcholine acyltransferase; MAG, monoacylglycerol; PA, phosphatidic acid; PC, phosphatidylcholine; PUFA, polyunsaturated fatty acid. The depicted scheme is based on information from Stymne and Stobart (1987), Ohlrogge and Browse (1995), Harwood (2005), and Weselake (2005).

The cytoplasmic pool of plastidially derived acyl-CoAs can in turn serve as substrates for the reactions of TAG assembly, which occur through the catalytic action of membrane-bound enzymes in the ER in a process that involves membrane metabolism (Weselake 2005). The glycerol backbone used for TAG bioassembly is derived from sn-glycerol-3-phosphate (G3P), which is produced from dihydroxyacetone phosphate (DHAP) and reduced nicotinamide adenine dinucleotide (NADH)

through the catalytic action of sn-glycerol-3-phosphate dehydrogenase. sn-Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the acyl-CoA-dependent acylation of the sn-1 position of G3P to produce lysophosphatidic acid (LPA), which is further acylated at the sn-2 position by the catalytic action of acyl-CoA-dependent lyso-phosphatidic acid acyltransferase (LPAAT) to produce phosphatidic acid (PA). The removal of the phosphate group from PA is catalyzed by PA phosphatase. The resulting sn-1,2-diacylglycerol (DAG) can be converted to TAG by acyl-CoA-dependent and acyl-CoA-independent processes. Diacyglycerol acyltransferase (DGAT) catalyzes the acyl-CoA-dependent acylation of DAG. Two genes encoding two isoforms of membrane-bound DGAT (type 1 and type 2) have been identified in a number of organisms, including plants. DGAT1 does not share homology with DGAT2 (Lardizabal et al. 2001). In addition, Saha et al. (2006) described the molecular cloning and expression of a soluble DGAT from developing peanut (Arachis hypogaea) cotyledons. This third isoform of DGAT shared less than 10% identity with previously identified forms of DGAT1 or DGAT2 from other organisms.

Acyl-CoA-independent processes have been identified that depend on either phosphatidylcholine (PC) or DAG as donors of fatty acyl moieties to DAG. These reactions are catalyzed by phospholipid:diacylglycerol acyltransferase (PDAT) or diacylglycerol transacylase (DGTA), respectively. Complementary DNAs (cDNAs) encoding PDAT have been isolated (Dahlqvist et al. 2000, Stáhl et al.

2004), whereas a cDNA encoding DGTA has not been identified. A study with a knockout for PDAT in Arabidopsis, however, has suggested that the enzyme does not play a major role in TAG biosynthesis during seed development (Mhaske et al.

TAG produced through acyltransferase action accumulates as droplets between the leaflets of the ER. Oil bodies surrounded by a half-unit membrane and ranging in size from 0.2 to 2 micrometers eventually bud off the surface of the ER.

DAG produced via the action of PA phosphatase can also be converted to PC by the catalytic action of choline phosphotransferase (CPT). Production of polyun-saturated FAs via the catalytic action of membrane-bound desaturases occurs while monounsaturated FAs are esterified to the glycerol backbone of PC. The combined forward and reverse reactions catalyzed by lysophosphatidylcholine acyltransferase (LPCAT) are believed to facilitate acyl exchange at the sn-2 position of PC, with the acyl-CoA pool thereby allowing incorporation of polyunsaturated FA into TAG via the acyl-CoA-dependent acyltransferase reactions in the mainstream of TAG biosynthesis. The reverse reaction of CPT to produce DAG provides a second opportunity to introduce polyunsaturated FA into TAG. In addition, phospholipase A2 may catalyze the release of free FA from PC. The liberated FA could be reesterified as acyl-CoA for use in the mainstream of TAG biosynthesis.

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