Coordinate Changes in Carbon Partitioning and Plastidial Metabolism during the Development of Oilseed Rape Embryos

Eastmond, Peter J.; Rawsthorne, Stephen

doi:10.1104/pp.122.3.767

Cited by 162 publications

(166 citation statements)

References 27 publications

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“…The content of soluble protein increased continuously over more than 30 d (Fig. 2), which is also the case for B. napus embryos grown in planta (Eastmond and Rawsthorne, 2000). In summary, after growth of B. napus cv Reston embryos on S medium, gain of fresh weight and protein as well as content and composition of fatty acids were similar to embryos developing in planta.…”

Section: Formation Of Storage Products In Cultured Embryos Reflects Smentioning

confidence: 52%

“…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%

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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: Formation Of Storage Products In Cultured Embryos Reflects Smentioning

confidence: 52%

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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“…Low in vivo rates of lipid synthesis observed could be due to a limited uptake of [ 14 C]acetate into plant tissues, thus limiting the availability of radiolabeled acetate for optimum rate of incorporation into total lipids. In addition, there has been much debate over the actual metabolic source of acetyl-CoA for de novo plant fatty acid synthesis and it appears to vary considerably depending upon cell type and physiological demand (Eastmond and Rawsthorne, 2000). Others showed that exogenous pyruvate and Glc-6-P supplied to oilseed rape embryos during the maximum period of lipid synthesis had the highest rate of incorporation into fatty acid, when compared with dihydroxyacetone phosphate, malate, or acetate as substrates.…”

Section: Discussionmentioning

confidence: 99%

Biochemical and Molecular Inhibition of Plastidial Carbonic Anhydrase Reduces the Incorporation of Acetate into Lipids in Cotton Embryos and Tobacco Cell Suspensions and Leaves

Hoang

Chapman

2002

Plant Physiology

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Two cDNAs encoding functional carbonic anhydrase (CA) enzymes were recently isolated from a non-photosynthetic, cotyledon library of cotton (Gossypium hirsutum) seedlings with putative plastid-targeting sequences (GenBank accession nos. AF132854 and AF132855). Relative CA transcript abundance and enzyme activity increased 9 and 15 times, respectively, in cotton embryos during the maximum period of reserve oil accumulation. Specific sulfonamide inhibitors of CA activity significantly reduced the rate of [ 14 C]acetate incorporation into total lipids in cotton embryos in vivo, and in embryo plastids in vitro, suggesting a role for CA in plastid lipid biosynthesis. CA inhibitors did not affect acetyl-coenzyme A carboxylase activity or total storage protein synthesis. Similar results were obtained for two other plant systems: cell suspensions (and isolated plastids therefrom) of tobacco (Nicotiana tabacum), and chloroplasts isolated from leaves of transgenic CA antisensesuppressed tobacco plants (5% of wild-type CA activity). In addition, tobacco cell suspensions treated with the CA inhibitor ethoxyzolamide showed a substantial loss of CO 2 compared with controls. The rate of [ 14 C]acetate incorporation into lipid in cell suspensions was reduced by limiting external [CO 2 ] (scrubbed air), and this rate was further reduced in the presence of ethoxyzolamide. Together, these results indicate that a reduction of CA activity (biochemical or molecular inhibition) impacts the rate of plant lipid biosynthesis from acetate, perhaps by impairing the ability of CA to efficiently "trap" inorganic carbon inside plastids for utilization by acetyl-coenzyme A carboxylase and the fatty acid synthesis machinery.Carbonic anhydrase (CA, EC 4.2.1.1) is a zinccontaining metalloenzyme that catalyzes the reversible hydration of CO 2 to HCO 3 Ϫ . The widespread abundance of CA isoforms in plants, animals, and microorganisms suggest that this enzyme has many diverse roles in biological processes. CA plays a critical role in biological systems because CO 2 gas is the membrane permeable form of inorganic carbon for cells, and, in general, the uncatalyzed interconversion between HCO 3 Ϫ and CO 2 is slow when compared with the required rate in living cells (Badger and Price, 1994).In photosynthetic organisms, one generally accepted physiological role of CA is to provide sufficient levels of inorganic carbon as part of a CO 2 -concentrating mechanism for improved photosynthetic efficiency. In Chlamydomonas reinhardtii, Badger and Price (1992) suggested that chloroplastic CA plays a role in photosynthetic carbon assimilation by converting accumulated pools of HCO 3 Ϫ to CO 2 , which is the substrate for Rubisco. Moroney et al. (1985) revealed that the reduction of periplasmic CA activity by using CAspecific inhibitors significantly reduced the efficiency of external inorganic carbon utilization for photosynthesis. Like green algae, CA in cyanobacteria plays an important role in the CO 2 -concentrating mechanism and in photosynthesis (Badger an...

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“…Possible sources of acetyl-CoA for plastid and cytosolic ACCase and related problems of carbon partitioning in plastid metabolism have been investigated Eastmond and Rawsthorne, 2000;Ke et al, 2000;Ohlrogge et al, 2000;Rawsthorne, 2002). Most of these studies focused on the expression of the de novo fatty acid biosynthetic pathway in seeds accumulating oil.…”

mentioning

confidence: 99%

Expression of Cytosolic and Plastid Acetyl-Coenzyme A Carboxylase Genes in Young Wheat Plants,

et al. 2003

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Expression of cytosolic and plastid acetyl-coenzyme A carboxylase (ACCase) gene families at the mRNA level was analyzed in developing wheat (Triticum aestivum) plants. The major plastid ACCase mRNA level is high in the middle part of the plant and low in roots and leaf blades. An alternative plastid ACCase transcript initiated at a different promoter and using an alternative 5Ј splice site for the first intron accumulates to its highest level in roots. Cytosolic ACCase mRNA also consists of two species, one of which is present at approximately a constant level, whereas the other accumulates to a high level in the lower sheath section. It is likely that different promoters are also responsible for the two forms of cytosolic ACCase mRNA. The abundances of cytosolic and plastid ACCase mRNAs in the sheath section of the plant are similar. ACCase protein level is significantly lower in the leaf blades, in parallel with changes in the total ACCase mRNA level. Homoeologous ACCase genes show the same expression patterns and similar mRNA levels, suggesting that none of the genes was silenced or acquired new tissue specificity after polyploidization.Acetyl-CoA carboxylase (ACCase) directs the flow of carbon from photosynthesis to primary and secondary metabolites in plants. One ACCase isozyme supplies malonyl-CoA for de novo fatty acid biosynthesis in plastids. A second ACCase isozyme supplies malonyl-CoA for fatty acid elongation and flavonoid biosynthesis in the cytosol. Their activity is expected to be fine tuned, responding to the demand for specific metabolites determined by plant development and by the environment. Our study focused on correlations between major developmental processes in the young wheat (Triticum aestivum) plant and expression of the ACCase gene families. The wheat leaf provides a succession of cells of different age and characteristics, from dividing cells of the leaf meristem located at the leaf base, to cells undergoing rapid enlargement accompanied by chloroplast maturation, to fully photosynthetically active cells in the leaf blade. These cells can be contrasted with photosynthetically inactive cells in the roots.ACCase and its product, malonyl-CoA, play a key role in regulating metabolite flux through the fatty acid biosynthetic and degradative pathways, and are important regulators of carbon allocation and energy homeostasis. In mammals, ACCase is regulated by multiple mechanisms including tissue-specific promoters, reversible phosphorylation, and feedback by a number of metabolites (Kim, 1997;Munday and Hemingway, 2001). In Escherichia coli, transcription of ACCase genes is correlated with the rate of cellular growth, overexpression of all four subunits of ACCase increasing the rate of fatty acid biosynthesis (Davis et al., 2000). The expression of the ACCase gene in yeast (Saccharomyces cerevisiae) is coordinated with phospholipid metabolism (Haslacher et al., 1993). For plants, it has been suggested that plastid ACCase activity controls flux through the de novo fatty acid biosynthetic pat...

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Coordinate Changes in Carbon Partitioning and Plastidial Metabolism during the Development of Oilseed Rape Embryos

Cited by 162 publications

References 27 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

Biochemical and Molecular Inhibition of Plastidial Carbonic Anhydrase Reduces the Incorporation of Acetate into Lipids in Cotton Embryos and Tobacco Cell Suspensions and Leaves

Expression of Cytosolic and Plastid Acetyl-Coenzyme A Carboxylase Genes in Young Wheat Plants,

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