Photosynthetic Carbon Fixation Characteristics of Fruiting Structures of <i>Brassica campestris</i> L.

Singal, H. R.; Sheoran, I. S.; Singh, Randhir

doi:10.1104/pp.83.4.1043

Cited by 32 publications

(24 citation statements)

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“…Measured rates of oxygen evolution of developing B. napus embryos (Eastmond et al, 1996;King et al, 1998) as well as Rubisco activity (King et al, 1998) are at least theoretically sufficient to refix the CO 2 that is produced by maximal oil synthesis. In addition to Rubisco, high activities of PEP carboxylase and malic enzyme are found in Brassica campestris in the developing seeds (Singal et al, 1987(Singal et al, , 1995King et al, 1998). The latter enzyme activities are essential to the CO 2 concentration mechanism in C 4 photosynthesis.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

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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: Discussionmentioning

confidence: 99%

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confidence: 99%

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

2002

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“…Plastidic malate dehydrogenase and plastidic malic enzyme (EC 1.1.1.39) were proposed to supply NADPH and pyruvate to the plastidic biosynthesis of fatty acids (20). However, in isolated plastids of B. napus embryos, incorporation of label into fatty acids from malate is less than from Glc 6-phosphate, DHAP, or pyruvate (9).…”

Section: Defining the Metabolic Networkmentioning

confidence: 99%

“…The Absence of Fatty Acid Synthesis from Malate-It has been suggested that in B. napus embryos, malate produced by the sequential actions of cytosolic PEP carboxylase (EC 4.1.1.31) and malate dehydrogenase (EC 1.1.1.37) enters the plastids to supply fatty acid synthesis (20). Plastidic malate dehydrogenase and plastidic malic enzyme (EC 1.1.1.39) were proposed to supply NADPH and pyruvate to the plastidic biosynthesis of fatty acids (20).…”

Section: Defining the Metabolic Networkmentioning

confidence: 99%

A Flux Model of Glycolysis and the Oxidative Pentosephosphate Pathway in Developing Brassica napus Embryos

Schwender

Ohlrogge

Shachar‐Hill

2003

Journal of Biological Chemistry

242

252

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Computer simulation of the steady state distribution of isotopomers in intermediates of the glycolysis/OPPP network was used to fit metabolic flux parameters to the experimental data. The observed distribution of label in cytosolic and plastidic metabolites indicated that key intermediates of glycolysis and OPPP have similar labeling in these two compartments, suggesting rapid exchange of metabolites between these compartments compared with net fluxes into end products. Cycling between hexose phosphate and triose phosphate and reversible transketolase velocity were similar to net glycolytic flux, whereas reversible transaldolase velocity was minimal. Flux parameters were overdetermined by analyzing labeling in different metabolites and by using data from different labeling experiments, which increased the reliability of the findings. Net flux of glucose through the OPPP accounts for close to 10% of the total hexose influx into the embryo. Therefore, the reductant produced by the OPPP accounts for at most 44% of the NADPH and 22% of total reductant needed for fatty acid synthesis.Brassica napus (rapeseed, canola) is one of the world's major oilseed crops and is also a well studied model for oilseed metabolism (1-21). The main storage compounds in seeds of B. napus are oil (triacylglycerols) and storage proteins, which are derived from sugars and amino acids taken up from the surrounding endosperm liquid (11,12,22,23). Because of its high oil content and ease of genetic transformation, B. napus has also been a target for metabolic engineering of oil metabolism. However, some attempts to engineer plant oils have had limited success (for a review, see Ref. 24). In order to make advances in improving oil yield and quality, a more detailed understanding of metabolism during seed development is needed. In particular, a number of fundamental metabolic issues remain unresolved. These include the source(s) of reductant and ATP for fatty acid synthesis; the degree to which cytosolic, plastidial, and mitochondrial metabolic fluxes are integrated; and the chief metabolic and transport route(s) by which carbon flows from maternal sources to seed storage products.

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“…Developing seeds of B. napus have also been the subject of numerous biochemical studies and are a model for oil accumulating seeds (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). In previous studies we have used intact developing embryos to make a quantitative analysis of steadystate metabolic fluxes during the conversion of carbohydrates to fatty acids in vivo (1, 14 -17).…”

mentioning

confidence: 99%

Mitochondrial Metabolism in Developing Embryos of Brassica napus

Schwender

Shachar‐Hill

Ohlrogge

2006

Journal of Biological Chemistry

216

283

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The metabolism of developing plant seeds is directed toward transforming primary assimilatory products (sugars and amino acids) into seed storage compounds. To understand the role of mitochondria in this metabolism, metabolic fluxes were determined in developing embryos of Brassica napus. After labeling with [1,2-13 C 2 ]glucose ؉ [U- 13 N]glutamine, the resulting labeling patterns in protein amino acids and in fatty acids were analyzed by gas chromatography-mass spectrometry. Fluxes through mitochondrial metabolism were quantified using a steady state flux model. Labeling information from experiments using different labeled substrates was essential for model validation and reliable flux estimation. The resulting flux map shows that mitochondrial metabolism in these developing seeds is very different from that in either heterotrophic or autotrophic plant tissues or in most other organisms: (i) flux around the tricarboxylic acid cycle is absent and the small fluxes through oxidative reactions in the mitochondrion can generate (via oxidative phosphorylation) at most 22% of the ATP needed for biosynthesis; (ii) isocitrate dehydrogenase is reversible in vivo; (iii) about 40% of mitochondrial pyruvate is produced by malic enzyme rather than being imported from the cytosol; (iv) mitochondrial flux is largely devoted to providing precursors for cytosolic fatty acid elongation; and (v) the uptake of amino acids rather than anaplerosis via PEP carboxylase determines carbon flow into storage proteins.Directly or indirectly plant seeds provide most of the food consumed by humans. Although the metabolism of developing seeds has been extensively studied, quantitative understanding of fluxes through central metabolism is still quite limited. Brassica napus (canola, oilseed rape) is a major oil crop and is amenable to detailed quantitative flux analysis under conditions that closely mimic in planta seed development (1). The main storage compounds in seeds of B. napus are oil (triacylglycerols) and proteins, which are synthesized by the developing embryo from sugars and amino acids taken up from the surrounding endosperm liquid.Developing seeds of B. napus have also been the subject of numerous biochemical studies and are a model for oil accumulating seeds (2-13). In previous studies we have used intact developing embryos to make a quantitative analysis of steadystate metabolic fluxes during the conversion of carbohydrates to fatty acids in vivo (1, 14 -17). These studies introduced a labeling approach using multiple carbon sources (1), quantified the contribution of the oxidative pentose phosphate pathway to biosynthetic NADPH demands (14), and revealed that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) 2 operates in a novel context of carbohydrate conversion to fatty acids, bypassing glycolytic reactions and increasing the efficiency of seed carbon metabolism (16). However, a detailed description of fluxes through mitochondrial metabolism in B. napus or other developing seeds is lacking.Several functions of mitoc...

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Photosynthetic Carbon Fixation Characteristics of Fruiting Structures of Brassica campestris L.

Cited by 32 publications

References 13 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

A Flux Model of Glycolysis and the Oxidative Pentosephosphate Pathway in Developing Brassica napus Embryos

Mitochondrial Metabolism in Developing Embryos of Brassica napus

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