Isolation and characterization of an Arabidopsis thaliana knockout line for phospholipid: diacylglycerol transacylase gene (At5g13640)

Mhaske, V. B.; Beldjilali, Khedidja; Ohlrogge, John B.; Pollard, Mike

doi:10.1016/j.plaphy.2005.01.013

Cited by 111 publications

(105 citation statements)

References 22 publications

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“…White triangles, Wild type; black squares, 5-2; black circles, 9-5. cell function and therefore are expressed in multiple tissues and during different developmental stages, mutations such as T-DNA knockouts could have a profound impact on the plant, including cell death, making it difficult to distinguish between cause and consequence of the lesion (Mou et al, 2000). Conversely, other knockout mutations do not show any phenotype due to gene redundancy (Mhaske et al, 2005).…”

Section: Discussion Targeted Genetic Perturbation To Study Metabolic mentioning

confidence: 99%

System Analysis of an Arabidopsis Mutant Altered in de Novo Fatty Acid Synthesis Reveals Diverse Changes in Seed Composition and Metabolism

et al. 2009

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Embryo-specific overexpression of biotin carboxyl carrier protein 2 (BCCP2) inhibited plastid acetyl-coenzyme A carboxylase (ACCase), resulting in altered oil, protein, and carbohydrate composition in mature Arabidopsis (Arabidopsis thaliana) seed. To characterize gene and protein regulatory consequences of this mutation, global microarray, two-dimensional difference gel electrophoresis, iTRAQ, and quantitative immunoblotting were performed in parallel. These analyses revealed that (1) transgenic overexpression of BCCP2 did not affect the expression of three other ACCase subunits; (2) four subunits to plastid pyruvate dehydrogenase complex were 25% to 70% down-regulated at protein but not transcript levels; (3) key glycolysis and de novo fatty acid/lipid synthesis enzymes were induced; (4) multiple storage proteins, but not cognate transcripts, were upregulated; and (5) the biotin synthesis pathway was up-regulated at both transcript and protein levels. Biotin production appears closely matched to endogenous BCCP levels, since overexpression of BCCP2 produced mostly apo-BCCP2 and the resulting ACCase-compromised, low-oil phenotype. Differential expression of glycolysis, plastid pyruvate dehydrogenase complex, fatty acid, and lipid synthesis activities indicate multiple, complex regulatory responses including feedback as well as futile "feed-forward" elicitation in the case of fatty acid and lipid biosynthetic enzymes. Induction of storage proteins reveals that oil and protein synthesis share carbon intermediate(s) and that reducing malonyl-coenzyme A flow into fatty acids diverts carbon into amino acid and protein synthesis.

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Section: Discussion Targeted Genetic Perturbation To Study Metabolic mentioning

confidence: 99%

System Analysis of an Arabidopsis Mutant Altered in de Novo Fatty Acid Synthesis Reveals Diverse Changes in Seed Composition and Metabolism

et al. 2009

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“…For Arabidopsis, neither PDAT overexpression nor knockout leads to any changes in total lipid content or fatty acid composition of the seed oil (Ståhl et al, 2004;Mhaske et al, 2005;Banas et al, 2014). Overexpression of flax PDAT in Arabidopsis, on the one hand, gives an increase in 18:3n-3 as well as, to a lesser degree, 18:2n-6 (Pan et al, 2013).…”

Section: Pdat Overexpressionmentioning

confidence: 99%

Two Acyltransferases Contribute Differently to Linolenic Acid Levels in Seed Oil

et al. 2017

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ORCID IDs: 0000-0001-6929-7859 (S.M.); 0000-0003-3152-0394 (D.S.); 0000-0001-8255-3255 (C.H.); 0000-0003-0489-3072 (K.C.); 0000-0002-9888-7003 (I.F.).Acyltransferases are key contributors to triacylglycerol (TAG) synthesis and, thus, are of great importance for seed oil quality. The effects of increased or decreased expression of ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1) or PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE (PDAT) on seed lipid composition were assessed in several Camelina sativa lines. Furthermore, in vitro assays of acyltransferases in microsomal fractions prepared from developing seeds of some of these lines were performed. Decreased expression of DGAT1 led to an increased percentage of 18:3n-3 without any change in total lipid content of the seed. The tri-18:3 TAG increase occurred predominantly in the cotyledon, as determined with matrix-assisted laser desorption/ionization-mass spectrometry, whereas species with two 18:3n-3 acyl groups were elevated in both cotyledon and embryonal axis. PDAT overexpression led to a relative increase of 18:2n-6 at the expense of 18:3n-3, also without affecting the total lipid content. Differential distributions of TAG species also were observed in different parts of the seed. The microsomal assays revealed that C. sativa seeds have very high activity of diacylglycerol-phosphatidylcholine interconversion. The combination of analytical and biochemical data suggests that the higher 18:2n-6 content in the seed oil of the PDAT overexpressors is due to the channeling of fatty acids from phosphatidylcholine into TAG before being desaturated to 18:3n-3, caused by the high activity of PDAT in general and by PDAT specificity for 18:2n-6. The higher levels of 18:3n-3 in DGAT1-silencing lines are likely due to the compensatory activity of a TAG-synthesizing enzyme with specificity for this acyl group and more desaturation of acyl groups occurring on phosphatidylcholine.

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“…Six PDAT-like genes were identified in Arabidopsis, and one, designated PDAT1, accounted for most PDAT activity in plant tissues (61). However, there was no significant impact on TAG accumulation in pdat1 loss-of-function mutants or in plants overexpressing A. thaliana PDAT, so the role in overall TAG accumulation in Arabidopsis was uncertain (61,62). A clear demonstration of a role for PDAT1 in TAG biosynthesis was provided more recently when its expression was silenced by RNA interference in a dgat1-1 mutant background (63).…”

Section: Acyl-coa-independent Mechanisms Involved In Tag Synthesismentioning

confidence: 99%

Compartmentation of Triacylglycerol Accumulation in Plants

Chapman

Ohlrogge

2012

Journal of Biological Chemistry

406

352

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Triacylglycerols from plants, familiar to most people as vegetable oils, supply 25% of dietary calories to the developed world and are increasingly a source for renewable biomaterials and fuels. Demand for vegetable oils will double by 2030, which can be met only by increased oil production. Triacylglycerol synthesis is accomplished through the coordinate action of multiple pathways in multiple subcellular compartments. Recent information has revealed an underappreciated complexity in pathways for synthesis and accumulation of this important energyrich class of molecules. Regulation of Fatty Acid Supply by PlastidsPlant fatty acid (FA) 2 synthesis differs from almost all other eukaryotes in two fundamental features. First, unlike the cytosolic location in other kingdoms, FAs for triacylglycerol (TAG) and membrane synthesis are produced in the plastid compartment of plant cells: chloroplasts in green tissues and proplastids (or leucoplasts) in non-green tissues. Second, the plant FA synthase (FAS) is a dissociable complex with separate proteins for the acyl carrier protein (ACP) and each enzyme (1). After assembly of C 16 and C 18 acyl chains by FAS and desaturation of C 18:0 to C 18:1 , FAs destined for TAG assembly are released from ACP in the plastid stroma by chain-terminating acyl-ACP thioesterases. Two classes of thioesterases designated FATA and FATB are responsible for hydrolysis of unsaturated and saturated acyl-ACPs, respectively, and thus determine in large part the chain length and saturated FA content of plant oils (2). The FA products of FATA and FATB are activated to CoA before export to the endoplasmic reticulum (ER). The subsequent reactions of TAG synthesis belong to the so-called "eukaryotic" pathway of glycerolipid synthesis, which occurs outside the plastid (3, 4). An overview of the compartmentalization of FA supply for TAG is shown in Fig. 1. FA production by plastids can limit TAG accumulation in seeds (5, 6), so increasing flux through FA biosynthesis may perhaps have the single greatest influence on the amount of TAG produced in plant tissues. As in bacteria, fungi, and animals, both in vitro and in vivo evidence indicates that acetylCoA carboxylase (ACCase) is a key rate-determining step that controls FA biosynthesis. ACCase activity is under complex regulation by light, phosphorylation, thioredoxin, PII protein, and product feedback control (7,8). Of course, the flux of carbon to FA synthesis can have multiple regulatory steps, which may explain why efforts to increase seed oil by up-regulating ACCase were only modestly successful (9).Transcriptional regulation of the production of FA for TAG biosynthesis is most directly controlled by the WRINKLED1 transcription factor (7, 10, 11). Arabidopsis wri1 mutants are reduced by 80% in seed oil, and overexpression of WRI1 can increase oil content in seeds of several plants. Targets of WRI1 include ACCase, many enzymes of FAS, and key enzymes and transporters that provide pyruvate and acetyl-CoA in the plastid. Thus, WRI1 can be considered as ...

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Isolation and characterization of an Arabidopsis thaliana knockout line for phospholipid: diacylglycerol transacylase gene (At5g13640)

Cited by 111 publications

References 22 publications

System Analysis of an Arabidopsis Mutant Altered in de Novo Fatty Acid Synthesis Reveals Diverse Changes in Seed Composition and Metabolism

System Analysis of an Arabidopsis Mutant Altered in de Novo Fatty Acid Synthesis Reveals Diverse Changes in Seed Composition and Metabolism

Two Acyltransferases Contribute Differently to Linolenic Acid Levels in Seed Oil

Compartmentation of Triacylglycerol Accumulation in Plants

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