Metabolism of glucose-6-phosphate and utilization of multiple metabolites for fatty acid synthesis by plastids from developing oilseed rape embryos

Fan, Kangqi; Rawsthorne, Stephen

doi:10.1007/bf00196575

Cited by 77 publications

(73 citation statements)

References 18 publications

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“…The oxidative pentose phosphate pathway (OPPP) provides an alternative pathway to glycolysis for metabolism of hexose to acetyl-CoA and has been proposed as a source of reductant for fatty acid synthesis in oilseeds (Kang and Rawsthorne, 1996;Eastmond and Rawsthorne, 2000). Earlier studies attempted to assess OPPP activity by measurement of the reduction of label from [1-13 C]Glc in metabolic products by Glc 6-phosphate dehydrogenase reaction relative to the label from [6-13 C]Glc.…”

Section: Glycolytic Breakdown Of Glc Dominates In Plastidic Acetyl-comentioning

confidence: 99%

Probing in Vivo Metabolism by Stable Isotope Labeling of Storage Lipids and Proteins in Developing Brassica napusEmbryos

2002

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Developing embryos of Brassica napus accumulate both triacylglycerols and proteins as major storage reserves. To evaluate metabolic fluxes during embryo development, we have established conditions for stable isotope labeling of cultured embryos under steady-state conditions. Sucrose supplied via the endosperm is considered to be the main carbon and energy source for seed metabolism. However, in addition to 220 to 270 mm carbohydrates (sucrose, glucose, and fructose), analysis of endosperm liquid revealed up to 70 mm amino acids as well as 6 to 15 mm malic acid. Therefore, a labeling approach with multiple carbon sources is a precondition to quantitatively reflect fluxes of central carbon metabolism in developing embryos. Mid-cotyledon stage B. napus embryos were dissected from plants and cultured for 15 d on a complex liquid medium containing 13 C-labeled carbohydrates. The 13 C enrichment of fatty acids and amino acids (after hydrolysis of the seed proteins) was determined by gas chromatography/mass spectrometry. Analysis of 13 C isotope isomers of labeled fatty acids and plastid-derived amino acids indicated that direct glycolysis provides at least 90% of precursors of plastid acetyl-coenzyme A (CoA). Unlabeled amino acids, when added to the growth medium, did not reduce incorporation of 13 C label into plastid-formed fatty acids, but substantially diluted 13 C label in seed protein. Approximately 30% of carbon in seed protein was derived from exogenous amino acids and as a consequence, the use of amino acids as a carbon source may have significant influence on the total carbon and energy balance in seed metabolism. 13 C label in the terminal acetate units of C 20 and C 22 fatty acids that derive from cytosolic acetyl-CoA was also significantly diluted by unlabeled amino acids. We conclude that cytosolic acetyl-CoA has a more complex biogenetic origin than plastidic acetyl-CoA. Malic acid in the growth medium did not dilute 13 C label incorporation into fatty acids or proteins and can be ruled out as a source of carbon for the major storage components of B. napus embryos.Plant oils represent the largest renewable resource of highly reduced carbon chains and there is interest in increasing their production by oilseed crops. Brassica napus (canola, oilseed rape) is a major oil crop and a multitude of literature focuses on the biochemistry and physiology of oil accumulation in developing seeds of B. napus (see Singal et al., 1987;Murphy and Cummis, 1989;Kang and Rawsthorne, 1994;Eastmond and Rawsthorne, 1998;King et al., 1998). Although the biochemical pathways leading from Suc to oil storage are largely understood, a number of questions remain regarding, for example, the subcellular organization of reactions, and the origin of acetyl-CoA, reducing power, and ATP for fatty acid synthesis. These questions are particularly difficult to address using standard in vitro or organellar biochemical analyses that often result in loss of key activities. In addition, due to several reasons including dilution of isotopes by ...

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Section: Glycolytic Breakdown Of Glc Dominates In Plastidic Acetyl-comentioning

confidence: 99%

Probing in Vivo Metabolism by Stable Isotope Labeling of Storage Lipids and Proteins in Developing Brassica napusEmbryos

2002

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“…Pyruvate and acetyl-CoA, the precursors for FA biosynthesis, are synthesized via multiple metabolic routes. For instance, glycolysis and pentose phosphate pathways are the major contributors for pyruvate production in vascular plants, and a minor amount of pyruvate can also be synthesized from malate by NADP-dependent malic enzyme (Kang and Rawsthorne, 1996;Alonso et al, 2007). Pyruvate is then converted to acetyl-CoA by the pyruvate dehydrogenase complex (PDHC) for de novo FA biosynthesis in the plastid (Lutziger and Oliver, 2000;Lin et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Choreography of Transcriptomes and Lipidomes ofNannochloropsisReveals the Mechanisms of Oil Synthesis in Microalgae

et al. 2014

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To reveal the molecular mechanisms of oleaginousness in microalgae, transcriptomic and lipidomic dynamics of the oleaginous microalga Nannochloropsis oceanica IMET1 under nitrogen-replete (N+) and N-depleted (N-) conditions were simultaneously tracked. At the transcript level, enhanced triacylglycerol (TAG) synthesis under N-conditions primarily involved upregulation of seven putative diacylglycerol acyltransferase (DGAT) genes and downregulation of six other DGAT genes, with a simultaneous elevation of the other Kennedy pathway genes. Under N-conditions, despite downregulation of most de novo fatty acid synthesis genes, the pathways that shunt carbon precursors from protein and carbohydrate metabolic pathways into glycerolipid synthesis were stimulated at the transcript level. In particular, the genes involved in supplying carbon precursors and energy for de novo fatty acid synthesis, including those encoding components of the pyruvate dehydrogenase complex (PDHC), glycolysis, and PDHC bypass, and suites of specific transporters, were substantially upregulated under N-conditions, resulting in increased overall TAG production. Moreover, genes involved in the citric acid cycle and b-oxidation in mitochondria were greatly enhanced to utilize the carbon skeletons derived from membrane lipids and proteins to produce additional TAG or its precursors. This temporal and spatial regulation model of oil accumulation in microalgae provides a basis for improving our understanding of TAG synthesis in microalgae and will also enable more rational genetic engineering of TAG production.

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“…The embryos (200-300) were harvested from seeds, approximately 22-33 days after anthesis (mid-cotyledonary stage embryos, 1.5-2.5 mg), incubated in 2-3 ml of plastid isolation medium (PIM ; 0.5 M sorbitol, 20 mM Hepes\NaOH, pH 7.4, 10 mM KCl, 1 mM MgCl # , 1 mM sodium EDTA) [5], without BSA and finely chopped. The solution was passed through two layers of Miracloth (Calbiochem-Novabiochem Ltd, Beeston, Nottinghamshire, U.K.) and the chopping\filtering process repeated five times.…”

Section: Preparation Of Plastidsmentioning

confidence: 99%

“…In itro, oilseed rape plastids can utilize a variety of carbon precursors to synthesize long-chain fatty acids [5], the latter being predominantly oleic acid (C ") : " ) with a little palmitic (C "' : ! ) and stearic acid (C ") : ! )…”

Section: Introductionmentioning

confidence: 99%

“…[6]. These carbon precursors include -glucose 6-phosphate (Glc6P), malate, pyruvate, dihydroxyacetone phosphate and acetate [5,7]. The majority of the fatty acids synthesized in the plastids are exported [8] and may be modified by enzymes such as the ∆12 and ∆15 desaturases on the endoplasmic reticulum [9] before incorporation into storage oil via the enzymes of the Kennedy pathway [10,11].…”

Section: Introductionmentioning

confidence: 99%

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Inhibition of the glucose-6-phosphate transporter in oilseed rape (Brassica napus L.) plastids by acyl-CoA thioesters reduces fatty acid synthesis

Fox

Hill

Rawsthorne

et al. 2000

Biochemical Journal

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Addition of oleoyl-CoA (1µM), or other acyl-CoA thioesters with a chain length of C16 or greater, to oilseed rape plastids (Brassica napus L.) inhibited the rate of D-glucose 6-phosphate (Glc6P) uptake by 70% after 2min. The IC50 value for oleoyl-CoA inhibition of the transporter was approx. 0.2–0.3µM. Inhibition was alleviated by the addition of acyl-CoA binding protein (ACBP) or BSA at slightly higher concentrations. Oleic acid (5–25µM), Tween 40 (10µM), Triton-X 100 (10µM) and palmitoylcarnitine (5µM) had no effect on Glc6P uptake. The uptake of [1-14C]Glc6P occurred in exchange for Pi, 3-phosphoglycerate or Glc6P at a typical rate of 30nmol Glc6P/min per unit of glyceraldehyde-3-phosphate dehydrogenase (NADP+). The Km(app) of the Glc6P transporter for Glc6P was 100µM. Neither CoA (0.3mM) nor ATP (3mM) inhibited Glc6P uptake, but the transporter was inhibited by 72% when ATP and CoA were added together. This inhibition was attributable to the synthesis of acyl-CoA thioesters, predominantly oleoyl-CoA and palmitoyl-CoA, by long-chain fatty acid-CoA ligase (EC 6.2.1.3) from endogenous fatty acids in the plastid preparations. Acyl-CoA thioesters did not inhibit the uptake of [2-14C]pyruvate or D-[1-14C]glucose into plastids. In vivo quantities of oleoyl-CoA and other long-chain acyl-CoA thioesters were lower than those for ACBP in early cotyledonary embryos, 0.7±0.2pmol/embryo and 2.2±0.2pmol/embryo respectively, but in late cotyledonary embryos quantities of long-chain acyl-CoA thioesters were greater than ACBP, 3±0.4pmol/embryo and 1.9±0.2pmol/embryo respectively.

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Metabolism of glucose-6-phosphate and utilization of multiple metabolites for fatty acid synthesis by plastids from developing oilseed rape embryos

Cited by 77 publications

References 18 publications

Probing in Vivo Metabolism by Stable Isotope Labeling of Storage Lipids and Proteins in Developing Brassica napusEmbryos

Probing in Vivo Metabolism by Stable Isotope Labeling of Storage Lipids and Proteins in Developing Brassica napusEmbryos

Choreography of Transcriptomes and Lipidomes ofNannochloropsisReveals the Mechanisms of Oil Synthesis in Microalgae

Inhibition of the glucose-6-phosphate transporter in oilseed rape (Brassica napus L.) plastids by acyl-CoA thioesters reduces fatty acid synthesis

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