Testing Models of Fatty Acid Transfer and Lipid Synthesis in Spinach Leaf Using in Vivo Oxygen-18 Labeling

Pollard, Mike; Ohlrogge, John B.

doi:10.1104/pp.121.4.1217

Cited by 74 publications

(62 citation statements)

References 29 publications

(28 reference statements)

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“…Because deuterium or tritium water labeling of fatty acids by illuminated leaf tissues shows no kinetic isotope effect and can give complete substitution of hydrogen atoms, the measured rate of FAS in wild type of 2.8 mmol H-atom h 21 mg 21 chlorophyll is a total rate and converts to 1.6 mmol C-atom h 21 mg 21 chlorophyll for the leaf strips. The rate previously measured for Arabidopsis aboveground tissues in intact seedlings was 2.3 mmol C-atom h 21 mg 21 chlorophyll (Bao et al, Pollard and Ohlrogge, 1999), fatb-ko mutant leaves incorporated 14 C into fatty acids at a rate 31% higher than wild type. Finally, intact wild-type and mutant seedlings were labeled with tracer concentrations of 14 CO 2 .…”

Section: Rate Of Fatty Acid Synthesis In Leaves Of Wild-type Arabidopmentioning

confidence: 99%

“…Production of fatty acids for export depends on the activity of acyl-ACP thioesterases (FATs) that hydrolyze acyl-acyl carrier protein (acyl-ACP) to release free fatty acids and ACP (for review, see Voelker et al, 1997). After export, the free fatty acids are re-esterified to CoA to form the cytosolic acyl-CoA pool (Pollard and Ohlrogge, 1999). In mesophyll cells, acyl-CoAs are primarily used for the biosynthesis of membrane glycerolipids in the endoplasmic reticulum (Browse and Somerville, 1991).…”

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confidence: 99%

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Metabolic Responses to the Reduction in Palmitate Caused by Disruption of the FATB Gene in Arabidopsis

et al. 2004

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Disruption of the FATB gene in Arabidopsis results in a two-thirds reduction in saturated fatty acids, largely palmitate, in the leaf extra-plastidic phospholipids and a reduction in the growth rate of the mutant compared to wild type (Bonaventure G, Salas JJ, Pollard MR, Ohlrogge JB [2003] Plant Cell 15: 1020-1033. In this study, we report that although fatb-ko seedlings grow more slowly than wild type, the rate of fatty acid synthesis in leaves of the mutant increases by 40%. This results in approximately the same amount of palmitate exported from the plastid as in wild type but an increase in oleate export of about 55%. To maintain constant amounts of fatty acids in leaves, thereby counterbalancing their higher rate of production, the mutant also increases its rate of fatty acid degradation. Although fatb-ko leaves have higher rates of fatty acid synthesis and turnover, the relative proportions of membrane lipids are similar to wild type. Thus, homeostatic mechanisms to preserve membrane compositions compensate for substantial changes in rates of fatty acid and glycerolipid metabolism in the mutant. Pulse-chase labeling studies show that in fatb-ko leaves there is a net increase in the synthesis of both prokaryotic and eukaryotic lipids and consequently of their turnover. The net loss of palmitate from phosphatidylcholine plus phosphatidylethanolamine is similar for wild type and mutant, suggesting that mechanisms are not present that can preferentially preserve the saturated fatty acids. In summary, the leaf cell responds to the loss of saturated fatty acid production in the fatb-ko mutant by increasing both fatty acid synthesis and degradation, but in doing so the mechanisms for increased fatty acid turnover contribute to the lowering of the percentage of saturated fatty acids found in eukaryotic lipids.In plants, the major site for de novo fatty acid synthesis (FAS) occurs in the plastid (Ohlrogge et al., 1979). Fatty acids are either utilized in this organelle or exported to supply diverse cytoplasmic biosynthetic pathways and cellular processes. Production of fatty acids for export depends on the activity of acyl-ACP thioesterases (FATs) that hydrolyze acyl-acyl carrier protein (acyl-ACP) to release free fatty acids and ACP (for review, see Voelker et al., 1997). After export, the free fatty acids are re-esterified to CoA to form the cytosolic acyl-CoA pool (Pollard and Ohlrogge, 1999). In mesophyll cells, acyl-CoAs are primarily used for the biosynthesis of membrane glycerolipids in the endoplasmic reticulum (Browse and Somerville, 1991). However, in other cell types exported fatty acids have different fates. For example, in embryo cells of oilseeds, the major fraction is incorporated into triacylglycerols, while in epidermal cells a large fraction is utilized for the synthesis of waxes and cutin (Post-Beittenmiller, 1996;Kolattukudy, 2003). Furthermore, all cells synthesize sphingolipids (Lynch, 1993), and we recently estimated that as much as 30% to 40% of exported palmitate is needed for sphingoid ba...

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Section: Rate Of Fatty Acid Synthesis In Leaves Of Wild-type Arabidopmentioning

confidence: 99%

mentioning

confidence: 99%

Metabolic Responses to the Reduction in Palmitate Caused by Disruption of the FATB Gene in Arabidopsis

et al. 2004

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“…This reaction competes with the direct transacylation of ACP by glycerol-3-phosphate acyltransferases for the formation of phosphatidate. An increase in FAT1 activity, therefore, could be indicative of increased fatty acid export from the chloroplast to the ER, where TAG assembly occurs, as acyl-ACPs have to be hydrolyzed prior to export (Pollard and Ohlrogge, 1999).…”

Section: Specific Changes In Gene Expression Related To General Lipidmentioning

confidence: 99%

Changes in Transcript Abundance in Chlamydomonas reinhardtii following Nitrogen Deprivation Predict Diversion of Metabolism

et al. 2010

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Like many microalgae, Chlamydomonas reinhardtii forms lipid droplets rich in triacylglycerols when nutrient deprived. To begin studying the mechanisms underlying this process, nitrogen (N) deprivation was used to induce triacylglycerol accumulation and changes in developmental programs such as gametogenesis. Comparative global analysis of transcripts under induced and noninduced conditions was applied as a first approach to studying molecular changes that promote or accompany triacylglycerol accumulation in cells encountering a new nutrient environment. Towards this goal, high-throughput sequencing technology was employed to generate large numbers of expressed sequence tags of eight biologically independent libraries, four for each condition, N replete and N deprived, allowing a statistically sound comparison of expression levels under the two tested conditions. As expected, N deprivation activated a subset of control genes involved in gametogenesis while down-regulating protein biosynthesis. Genes for components of photosynthesis were also down-regulated, with the exception of the PSBS gene. N deprivation led to a marked redirection of metabolism: the primary carbon source, acetate, was no longer converted to cell building blocks by the glyoxylate cycle and gluconeogenesis but funneled directly into fatty acid biosynthesis. Additional fatty acids may be produced by membrane remodeling, a process that is suggested by the changes observed in transcript abundance of putative lipase genes. Inferences on metabolism based on transcriptional analysis are indirect, but biochemical experiments supported some of these deductions. The data provided here represent a rich source for the exploration of the mechanism of oil accumulation in microalgae.

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“…Mass 75 has a much higher abundance than expected from natural isotopes of C 3 H 6 O 2 (m/z 74), due to a second proton transfer to the same fragment resulting in C 3 H 7 O 2 m/z 75 (Murphy, 1993). For saturated fatty acid methyl esters of different chain lengths, this fragment was used to measure incorporation of stable isotopes into the terminal acetate unit (Schmid et al, 1988;Pollard and Ohlrogge, 1999).…”

Section: Mclafferty Ion (M/z 74) In Fatty Acidsmentioning

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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Testing Models of Fatty Acid Transfer and Lipid Synthesis in Spinach Leaf Using in Vivo Oxygen-18 Labeling

Abstract: Oxygen-18 labeling has been applied to the study of plant lipid biosynthesis for the first time.

Cited by 74 publications

References 29 publications

Metabolic Responses to the Reduction in Palmitate Caused by Disruption of the FATB Gene in Arabidopsis

Metabolic Responses to the Reduction in Palmitate Caused by Disruption of the FATB Gene in Arabidopsis

Changes in Transcript Abundance in Chlamydomonas reinhardtii following Nitrogen Deprivation Predict Diversion of Metabolism

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

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