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Lipidomic analysis of Arabidopsis seed genetically engineered to contain DHA.

Zhou XR, Callahan DL, Shrestha P, Liu Q, Petrie JR, Singh SP - Front Plant Sci (2014)

Bottom Line: Metabolic engineering of omega-3 long-chain (≥C20) polyunsaturated fatty acids (ω3 LC-PUFA) in oilseeds has been one of the key targets in recent years.Liquid chromatography-mass spectrometry (LC-MS) was used to characterize the triacylglycerol (TAG), diacylglycerol (DAG) and phospholipid (PL) lipid classes in the transgenic and wild type Arabidopsis seeds at both developing and mature stages.Trace amounts of di-DHA PC and tri-DHA TAG were identified and confirmed by high resolution MS/MS.

View Article: PubMed Central - PubMed

Affiliation: Food Futures National Research Flagship, Commonwealth Scientific and Industrial Research Organisation Canberra, ACT, Australia ; Plant Industry, Commonwealth Scientific and Industrial Research Organisation Canberra, ACT, Australia.

ABSTRACT
Metabolic engineering of omega-3 long-chain (≥C20) polyunsaturated fatty acids (ω3 LC-PUFA) in oilseeds has been one of the key targets in recent years. By expressing a transgenic pathway for enhancing the synthesis of the ω3 LC-PUFA docosahexaenoic acid (DHA) from endogenous α-linolenic acid (ALA), we obtained the production of fish oil-like proportions of DHA in Arabidopsis seed oil. Liquid chromatography-mass spectrometry (LC-MS) was used to characterize the triacylglycerol (TAG), diacylglycerol (DAG) and phospholipid (PL) lipid classes in the transgenic and wild type Arabidopsis seeds at both developing and mature stages. The analysis identified the appearance of several abundant DHA-containing phosphatidylcholine (PC), DAG and TAG molecular species in mature seeds. The relative abundances of PL, DAG, and TAG species showed a preferred combination of LC-PUFA with ALA in the transgenic seeds, where LC-PUFA were esterified in positions usually occupied by 20:1ω9. Trace amounts of di-DHA PC and tri-DHA TAG were identified and confirmed by high resolution MS/MS. Studying the lipidome in transgenic seeds provided insights into where DHA accumulated and combined with other fatty acids of neutral and phospholipids from the developing and mature seeds.

No MeSH data available.


Simplified representation of lipid metabolism in plant cells. Lipid pools are acyl-CoA, acyl co-enzyme A; LPC, lysophosphatidylcholine; PC, phosphatidylcholine; G-3-P, glycerol-3-phosphate; LPA, lysophosphatidic acid; PA, phosphatidic acid; DAG, diacylglycerol; TAG, triacylglycerol. The enzymes are LPCAT, lysophosphatidylcholine acyltransferase; PLD, phospholipase D; CPT, CDP-choline:diacylglycerol cholinephosphotransferase; PDCT, phosphatidylcholine:diacylglycerol cholinephosphotransferase; PLC, phospholipase C; PDAT, phospholipid:diacylglycerol acyltransferase; GPAT, glycerol-3-phosphate acyltransferase; LPAAT, lysophosphatidic acid acyltransferase; PAP, phosphatidic acid phosphatase; DGAT, diacylglycerol acyltransferase.
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Figure 1: Simplified representation of lipid metabolism in plant cells. Lipid pools are acyl-CoA, acyl co-enzyme A; LPC, lysophosphatidylcholine; PC, phosphatidylcholine; G-3-P, glycerol-3-phosphate; LPA, lysophosphatidic acid; PA, phosphatidic acid; DAG, diacylglycerol; TAG, triacylglycerol. The enzymes are LPCAT, lysophosphatidylcholine acyltransferase; PLD, phospholipase D; CPT, CDP-choline:diacylglycerol cholinephosphotransferase; PDCT, phosphatidylcholine:diacylglycerol cholinephosphotransferase; PLC, phospholipase C; PDAT, phospholipid:diacylglycerol acyltransferase; GPAT, glycerol-3-phosphate acyltransferase; LPAAT, lysophosphatidic acid acyltransferase; PAP, phosphatidic acid phosphatase; DGAT, diacylglycerol acyltransferase.

Mentions: The DHA synthesis pathway consists of a series of fatty acid desaturation and elongation steps that are thought to occur in discrete seed lipid pools. Therefore, effects of transgenic expression of this pathway are expected to be expressed throughout the lipidome of the developing seed. Oil accumulation in the developing seed requires cross talk between the ER membrane lipids, predominantly represented by PC and other phospholipids, such as phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI) and phosphatidylserine (PS), soluble acyl CoA fatty acids and DAG lipid pools. The fatty acids in the acyl-CoA pool are then utilized by the acyl-CoA-dependent Kennedy pathway to be assembled into TAG. This involves the sequential acylation of glycerol-3-phosphate to produce phosphatidic acid (PA) by glycerol-3-phosphate acyltransferase (GPAT) and 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), dephosphorylation of PA to diacylglycerol (DAG) by phosphatidic acid phosphatase (PAP), and the final acylation of DAG to TAG by diacylglycerol acyltransferase (DGAT) (Figure 1). In addition, de novo synthesized DAG can be used for synthesis of PC by a reversible cytidine-5′-diphosphocholine:diacylglycerol cholinephosphotransferase (CPT) (Slack et al., 1983). However, recent studies (Williams et al., 2000; Bates et al., 2012; Wang et al., 2012) have demonstrated that the acyl-editing cycle is the major pathway for PC synthesis. This cycle proceeds through the acylation of lysophosphatidylcholine (LPC) and deacylation of PC by acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) (Stymne and Stobart, 1984; Bates et al., 2012; Wang et al., 2012). On the other hand, there is accumulating evidence of the use of PC-derived DAG for synthesis of PUFA-containing TAG in plants (Bates and Browse, 2012). The PC to DAG conversion is believed to be mainly carried out by phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT) through phosphocholine head group exchange (Bates and Browse, 2012; Hu et al., 2012), although PC-derived DAG can also be synthesized by CPT, or phospholipase C.


Lipidomic analysis of Arabidopsis seed genetically engineered to contain DHA.

Zhou XR, Callahan DL, Shrestha P, Liu Q, Petrie JR, Singh SP - Front Plant Sci (2014)

Simplified representation of lipid metabolism in plant cells. Lipid pools are acyl-CoA, acyl co-enzyme A; LPC, lysophosphatidylcholine; PC, phosphatidylcholine; G-3-P, glycerol-3-phosphate; LPA, lysophosphatidic acid; PA, phosphatidic acid; DAG, diacylglycerol; TAG, triacylglycerol. The enzymes are LPCAT, lysophosphatidylcholine acyltransferase; PLD, phospholipase D; CPT, CDP-choline:diacylglycerol cholinephosphotransferase; PDCT, phosphatidylcholine:diacylglycerol cholinephosphotransferase; PLC, phospholipase C; PDAT, phospholipid:diacylglycerol acyltransferase; GPAT, glycerol-3-phosphate acyltransferase; LPAAT, lysophosphatidic acid acyltransferase; PAP, phosphatidic acid phosphatase; DGAT, diacylglycerol acyltransferase.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150447&req=5

Figure 1: Simplified representation of lipid metabolism in plant cells. Lipid pools are acyl-CoA, acyl co-enzyme A; LPC, lysophosphatidylcholine; PC, phosphatidylcholine; G-3-P, glycerol-3-phosphate; LPA, lysophosphatidic acid; PA, phosphatidic acid; DAG, diacylglycerol; TAG, triacylglycerol. The enzymes are LPCAT, lysophosphatidylcholine acyltransferase; PLD, phospholipase D; CPT, CDP-choline:diacylglycerol cholinephosphotransferase; PDCT, phosphatidylcholine:diacylglycerol cholinephosphotransferase; PLC, phospholipase C; PDAT, phospholipid:diacylglycerol acyltransferase; GPAT, glycerol-3-phosphate acyltransferase; LPAAT, lysophosphatidic acid acyltransferase; PAP, phosphatidic acid phosphatase; DGAT, diacylglycerol acyltransferase.
Mentions: The DHA synthesis pathway consists of a series of fatty acid desaturation and elongation steps that are thought to occur in discrete seed lipid pools. Therefore, effects of transgenic expression of this pathway are expected to be expressed throughout the lipidome of the developing seed. Oil accumulation in the developing seed requires cross talk between the ER membrane lipids, predominantly represented by PC and other phospholipids, such as phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI) and phosphatidylserine (PS), soluble acyl CoA fatty acids and DAG lipid pools. The fatty acids in the acyl-CoA pool are then utilized by the acyl-CoA-dependent Kennedy pathway to be assembled into TAG. This involves the sequential acylation of glycerol-3-phosphate to produce phosphatidic acid (PA) by glycerol-3-phosphate acyltransferase (GPAT) and 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), dephosphorylation of PA to diacylglycerol (DAG) by phosphatidic acid phosphatase (PAP), and the final acylation of DAG to TAG by diacylglycerol acyltransferase (DGAT) (Figure 1). In addition, de novo synthesized DAG can be used for synthesis of PC by a reversible cytidine-5′-diphosphocholine:diacylglycerol cholinephosphotransferase (CPT) (Slack et al., 1983). However, recent studies (Williams et al., 2000; Bates et al., 2012; Wang et al., 2012) have demonstrated that the acyl-editing cycle is the major pathway for PC synthesis. This cycle proceeds through the acylation of lysophosphatidylcholine (LPC) and deacylation of PC by acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) (Stymne and Stobart, 1984; Bates et al., 2012; Wang et al., 2012). On the other hand, there is accumulating evidence of the use of PC-derived DAG for synthesis of PUFA-containing TAG in plants (Bates and Browse, 2012). The PC to DAG conversion is believed to be mainly carried out by phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT) through phosphocholine head group exchange (Bates and Browse, 2012; Hu et al., 2012), although PC-derived DAG can also be synthesized by CPT, or phospholipase C.

Bottom Line: Metabolic engineering of omega-3 long-chain (≥C20) polyunsaturated fatty acids (ω3 LC-PUFA) in oilseeds has been one of the key targets in recent years.Liquid chromatography-mass spectrometry (LC-MS) was used to characterize the triacylglycerol (TAG), diacylglycerol (DAG) and phospholipid (PL) lipid classes in the transgenic and wild type Arabidopsis seeds at both developing and mature stages.Trace amounts of di-DHA PC and tri-DHA TAG were identified and confirmed by high resolution MS/MS.

View Article: PubMed Central - PubMed

Affiliation: Food Futures National Research Flagship, Commonwealth Scientific and Industrial Research Organisation Canberra, ACT, Australia ; Plant Industry, Commonwealth Scientific and Industrial Research Organisation Canberra, ACT, Australia.

ABSTRACT
Metabolic engineering of omega-3 long-chain (≥C20) polyunsaturated fatty acids (ω3 LC-PUFA) in oilseeds has been one of the key targets in recent years. By expressing a transgenic pathway for enhancing the synthesis of the ω3 LC-PUFA docosahexaenoic acid (DHA) from endogenous α-linolenic acid (ALA), we obtained the production of fish oil-like proportions of DHA in Arabidopsis seed oil. Liquid chromatography-mass spectrometry (LC-MS) was used to characterize the triacylglycerol (TAG), diacylglycerol (DAG) and phospholipid (PL) lipid classes in the transgenic and wild type Arabidopsis seeds at both developing and mature stages. The analysis identified the appearance of several abundant DHA-containing phosphatidylcholine (PC), DAG and TAG molecular species in mature seeds. The relative abundances of PL, DAG, and TAG species showed a preferred combination of LC-PUFA with ALA in the transgenic seeds, where LC-PUFA were esterified in positions usually occupied by 20:1ω9. Trace amounts of di-DHA PC and tri-DHA TAG were identified and confirmed by high resolution MS/MS. Studying the lipidome in transgenic seeds provided insights into where DHA accumulated and combined with other fatty acids of neutral and phospholipids from the developing and mature seeds.

No MeSH data available.