A Flux Model of Glycolysis and the Oxidative Pentosephosphate Pathway in Developing Brassica napus Embryos
2003; Elsevier BV; Volume: 278; Issue: 32 Linguagem: Inglês
10.1074/jbc.m303432200
ISSN1083-351X
AutoresJörg Schwender, John B. Ohlrogge, Yair Shachar‐Hill,
Tópico(s)Enzyme Catalysis and Immobilization
ResumoDeveloping oilseeds synthesize large quantities of triacylglycerol from sucrose and hexose. To understand the fluxes involved in this conversion, a quantitative metabolic flux model was developed and tested for the reaction network of glycolysis and the oxidative pentose phosphate pathway (OPPP). Developing Brassica napus embryos were cultured with [U-13C6]glucose, [1-13C]glucose, [6-13C]glucose, [U-13C12]sucrose, and/or [1,2-13C2]glucose and the labeling patterns in amino acids, lipids, sucrose, and starch were measured by gas chromatography/mass spectrometry and NMR. Data were used to verify a reaction network of central carbon metabolism distributed between the cytosol and plastid. 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. Developing oilseeds synthesize large quantities of triacylglycerol from sucrose and hexose. To understand the fluxes involved in this conversion, a quantitative metabolic flux model was developed and tested for the reaction network of glycolysis and the oxidative pentose phosphate pathway (OPPP). Developing Brassica napus embryos were cultured with [U-13C6]glucose, [1-13C]glucose, [6-13C]glucose, [U-13C12]sucrose, and/or [1,2-13C2]glucose and the labeling patterns in amino acids, lipids, sucrose, and starch were measured by gas chromatography/mass spectrometry and NMR. Data were used to verify a reaction network of central carbon metabolism distributed between the cytosol and plastid. 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 (1Hill L.M. Rawsthorne S. Biochem. Soc. Trans. 2000; 28: 667-669Crossref PubMed Scopus (6) Google Scholar, 2Kubis S.E. Rawsthorne S. Biochem. Soc. Trans. 2000; 28: 665-666Crossref PubMed Scopus (10) Google Scholar, 3Eastmond P.J. Rawsthorne S. Plant Physiol. 2000; 122: 767-774Crossref PubMed Scopus (152) Google Scholar, 4Eastmond P.J. Rawsthorne S. J. Exp. Bot. 1998; 49: 1105-1111Google Scholar, 5Dasilva P.M.F.R. Eastmond P.J. Hill L.M. Smith A.M. Rawsthorne S. Planta. 1997; 203: 480-487Crossref Scopus (86) Google Scholar, 6Eastmond P.J. Dennis D.T. Rawsthorne S. Plant Physiol. 1997; 114: 851-856Crossref PubMed Scopus (53) Google Scholar, 7Eastmond P. Kolacna L. Rawsthorne S. J. Exp. Bot. 1996; 47: 1763-1769Crossref Scopus (85) Google Scholar, 8Kang F. Rawsthorne S. Planta. 1996; 199: 321-327Crossref Scopus (71) Google Scholar, 9Kang F. Rawsthorne S. Plant J. 1994; 6: 795-805Crossref Scopus (159) Google Scholar, 10King S.P. Badger M.R. Furbank R.T. Austr. J. Plant Physiol. 1998; 25: 377-386Crossref Scopus (65) Google Scholar, 11King S.P. Lunn J.E. Badger M.R. Furbank R.T. Plant Physiol. 1997; 114: 31SCrossref Scopus (138) Google Scholar, 12King S.P. Lunn J.E. Furbank R.T. Plant Physiol. 1997; 114: 153-160Crossref PubMed Scopus (153) Google Scholar, 13Singh D.K. Malhotra S.P. Singh R. Indian J. Biochem. Biophys. 2000; 37: 51-58PubMed Google Scholar, 14Gupta R. Singh R. Indian J. Biochem. Biophys. 1997; 34: 288-295PubMed Google Scholar, 15Gupta R. Singh R. J. Biosci. 1996; 21: 819-826Crossref Scopus (8) Google Scholar, 16Gupta R. Singh R. J. Plant Biochem. Biotechnol. 1996; 5: 127-130Crossref Scopus (8) Google Scholar, 17Talwar G. Dua A. Singh R. Photosynthetica. 1996; 32: 221-229Google Scholar, 18Mehta M. Saharan M.R. Singh R. J. Plant Biochem. Biotechnol. 1995; 4: 11-16Crossref Scopus (4) Google Scholar, 19Singal H.R. Talwar G. Dua A. Singh R. J. Biosci. 1995; 20: 49-58Crossref Scopus (29) Google Scholar, 20Singal H.R. Sheoran I.S. Singh R. Plant Physiol. 1987; 83: 1043-1047Crossref PubMed Google Scholar, 21Bao X. Pollard M. Ohlrogge J.B. Plant Physiol. 1998; 118: 183-190Crossref PubMed Scopus (81) Google Scholar). 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 (11King S.P. Lunn J.E. Badger M.R. Furbank R.T. Plant Physiol. 1997; 114: 31SCrossref Scopus (138) Google Scholar, 12King S.P. Lunn J.E. Furbank R.T. Plant Physiol. 1997; 114: 153-160Crossref PubMed Scopus (153) Google Scholar, 22Schwender J. Ohlrogge J. Plant Physiol. 2002; 130: 347-361Crossref PubMed Scopus (156) Google Scholar, 23Hill L.M. Morley-Smith E.R. Rawsthorne S. Plant Physiol. 2003; 131Crossref PubMed Scopus (94) Google Scholar). 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. 24Thelen J.J. Ohlrogge J.B. Metab. Eng. 2002; 4: 12-21Crossref PubMed Scopus (324) Google Scholar). 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.Fatty acid synthesis has a high demand for reductant, and in other systems there is evidence that the supply of reductant can limit lipid accumulation (25Katz J. Landau B.R. Bartsch G.E. J. Biol. Chem. 1966; 241: 727-740Abstract Full Text PDF PubMed Google Scholar, 26Geer B.W. Lindel D.L. Lindel D.M. Biochem. Genet. 1979; 17: 881-895Crossref PubMed Scopus (38) Google Scholar). Thus, determining the source of reductant for fatty acid synthesis in developing oil seeds is important, and in particular the contribution of NADPH made by the oxidative pentose phosphate pathway (OPPP) 1The abbreviations used are: OPPP, oxidative pentose phosphate pathway; C18, octadecanoic acid; C18:1, octadecenoic acid; C22, eicosanoic acid; C22:1, docosenoic acid; DHAP, dihydroxyacetone phosphate; Fru-6-P, fructose 6-phosphate; GAP, glyceraldehyde 3-phosphate; Glc-6-P, glucose 6-phosphate; GC, gas chromatography; MS, mass spectrometry; PEP, phosphoenol pyruvate; Rib-5-P, ribose-5-phosphate; Ru-5-P, ribulose 5-phosphate; Suc, sucrose; TAG, triacylglycerol; TA, transaldolase; TK, transketolase; Xu-5-P, xylulose 5-phosphate; PDH, pyruvate dehydrogenase complex.1The abbreviations used are: OPPP, oxidative pentose phosphate pathway; C18, octadecanoic acid; C18:1, octadecenoic acid; C22, eicosanoic acid; C22:1, docosenoic acid; DHAP, dihydroxyacetone phosphate; Fru-6-P, fructose 6-phosphate; GAP, glyceraldehyde 3-phosphate; Glc-6-P, glucose 6-phosphate; GC, gas chromatography; MS, mass spectrometry; PEP, phosphoenol pyruvate; Rib-5-P, ribose-5-phosphate; Ru-5-P, ribulose 5-phosphate; Suc, sucrose; TAG, triacylglycerol; TA, transaldolase; TK, transketolase; Xu-5-P, xylulose 5-phosphate; PDH, pyruvate dehydrogenase complex. to fatty acid synthesis is not known. Of the two reducing steps of fatty acid synthesis, in vitro data indicate that the first (3-ketoacyl-ACP reductase; EC 1.1.1.100) requires NADPH (27Sheldon P.S. Kekwick R.G.O. Smith C.G. Sidebottom C. Slabas A.R. Biochim. Biophys. Acta. 1992; 1120: 151-159Crossref PubMed Scopus (41) Google Scholar), whereas the second (enoyl-ACP reductase, EC 1.3.1.9) requires NADH (28Slabas A.R. Sidebottom C.M. Hellyer A. Kessel R.M.J. Tombs M.P. Biochim. Biophys. Acta. 1986; 877: 271-280Crossref Scopus (60) Google Scholar). NADH can be provided by the pyruvate dehydrogenase reaction in plastids, whereas it has long been thought that NADPH for reductive syntheses in nonphotosynthetic plastids is produced by the OPPP (29Neuhaus H.E. Emes M.J. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2000; 51: 111-140Crossref PubMed Scopus (459) Google Scholar). However, reductant could also be provided by steps in glycolysis (e.g. GAP-dehydrogenase; EC 1.2.1.13), by photosystems of green seeds, or by the import into the plastid of reducing equivalents generated in the mitochondria or cytosol. Thus, the OPPP represents one of several possible sources of reductant for oil synthesis in seeds, and the in vivo contribution of these alternatives has not been established.In recent years, it has become clear that measuring fluxes through the OPPP presents technical challenges and requires careful experimental design and interpretation. The effects of cycling among hexose, triose, and pentose pools via reversible reactions leads to label redistributions that must be quantitatively considered if one is to understand the sources of carbon and reductant (30Roscher A. Kruger N.J. Ratcliffe R.G. J. Biotechnol. 2000; 77: 81-102Crossref PubMed Scopus (100) Google Scholar). Understanding flux through metabolic networks that involve reversible, branching, and parallel pathways has been greatly aided by the development of steady state labeling methods using stable isotopes and isotopomer analysis (31Dieuaide-Noubhani M. Raffard G. Canioni P. Pradet A. Raymond P. J. Biol. Chem. 1995; 270: 13147-13159Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 32Edwards S. Nguyen B.T. Do B. Roberts J.K.M. Plant Physiol. 1998; 116: 1073-1081Crossref PubMed Scopus (72) Google Scholar, 33Fernie A.R. Roscher A. Ratcliffe R.G. Kruger N.J. Planta. 2001; 212: 250-263Crossref PubMed Scopus (195) Google Scholar, 34Rontein D. Dieuaide-Noubhani M. Dufourc E.J. Raymond P. Rolin D. J. Biol. Chem. 2002; 46: 43948-43960Abstract Full Text Full Text PDF Scopus (164) Google Scholar). Analysis of isotopomer distributions in intermediates and end-products of metabolism can provide information on the relative fluxes through alternative pathways and on flux ratios at branch points between pathways (see, for example, Refs. 35Szyperski T. Q. Rev. Biophys. 1998; 31: 41-106Crossref PubMed Scopus (189) Google Scholar, 36Dauner M. Sauer U. Biotechnol. Prog. 2000; 16: 642-649Crossref PubMed Scopus (193) Google Scholar, 37Wittmann C. Heinzle E. Biotechnol. Bioeng. 1999; 62: 739-750Crossref PubMed Scopus (157) Google Scholar). With in vivo labeling, this approach yields quantitative information on systems unperturbed by cell disruption, mutations, or transgenic manipulation. The results of this approach can therefore distinguish the relative contributions of competing pathways and help guide rational engineering of metabolism.To take advantage of such methods, we have recently established culture conditions for developing B. napus embryos that mimic in planta growth and allow steady state labeling during storage product accumulation (22Schwender J. Ohlrogge J. Plant Physiol. 2002; 130: 347-361Crossref PubMed Scopus (156) Google Scholar). After feeding 13C-labeled carbon sources, the labeling pattern of various intermediates of central carbon metabolism are "imprinted" on seed oil and on the amino acids of seed protein; these can be measured by gas chromatography/mass spectrometry (GC/MS) and by NMR spectroscopy. Using these techniques, we deduced that the pyruvate that provides acetyl-CoA units for fatty acid is derived from Glc almost entirely by glycolytic cleavage (Embden-Meyerhof pathway) and that glycolysis rather than the OPPP accounts for most embryo hexose catabolism. Based on a preliminary analysis of labeling in fatty acids, we estimated that the net flux of Glc-6-P into OPPP is in the range of 5–10% of total influx of Glc-6-P. However, this preliminary estimate was based on making key assumptions about the reversibilities of transketolase (TK; EC 2.2.1.1) and transaldolase (TA; EC 2.2.1.2). In the present study, we have developed a quantitative model of glycolysis and OPPP and tested its ability to account for isotopomer labeling patterns and to yield reliable flux parameters in developing B. napus seeds.EXPERIMENTAL PROCEDURESChemicals—[U-13C6]Glc, [1,2-13C2]Glc, [1-13C]Glc, [6-13C]Glc, and [2-13C]Glc (all 99% 13C abundance) were purchased from Isotec (Miamisgurg, OH) and Omicron (South Bend, IN). Methoxyamine hydrochloride, α-amylase (EC 3.2.1.1) and Aspergillus niger amyloglucosidase (EC 3.2.1.3) were purchased from Sigma.Growth in the Presence of 13C-Labeled Sugars—Oilseed rape plants (B. napus L., cv. Reston) were grown as described before (22Schwender J. Ohlrogge J. Plant Physiol. 2002; 130: 347-361Crossref PubMed Scopus (156) Google Scholar). Siliques were harvested 20 days after flowering, and embryos were immediately dissected under aseptic conditions and transferred into culture medium (22Schwender J. Ohlrogge J. Plant Physiol. 2002; 130: 347-361Crossref PubMed Scopus (156) Google Scholar). In order to obtain fully labeled TAG and seed protein for analysis by GC/MS, five embryos were isolated at the early stage of oil accumulation (0.5–1 mg of fresh weight) and were grown for 14 days, each in 5 ml of growth medium under low light conditions (continuous light, 50 μmol m–2 s–1) under aseptic conditions. The growth medium contained Suc (80 mm), Glc (40 mm) and amino acids as carbon sources in concentrations that closely mimic in planta conditions during maximal oil synthesis (22Schwender J. Ohlrogge J. Plant Physiol. 2002; 130: 347-361Crossref PubMed Scopus (156) Google Scholar). For different labeling experiments, part of the Glc or Suc was replaced by 13C-labeled sugars. A 1:10 isotopic dilution of 13C-labeled Glc was achieved by a mixture of, for example, [1,2-13C2]Glc/Glc/Suc (10:10:80) (mol % hexose units) or, in the case of uniformly 13C-labeled sugars, by a mixture of [U-13C6]Glc/Glc/[U-13C12]Suc/Suc (2:18:8:72) (mol % hexose units). Experiments with Glc labeled in different positions were also performed using, for example, [1-13C]Glc/[1,2-13C2]Glc/Suc (10:10:80) (mol % hexose units).In some experiments aimed at analysis of intermediates and starch, embryos were labeled for 3 days. In one such experiment, embryos were cultured with [U-13C6]Glc/Glc/[U-13C12]Suc/Suc (2:18:8:72) (mol % hexose units), and after 3 days labeled Suc, free amino acids, and starch were extracted and analyzed by GC/MS methods. In other experiments aimed at labeling free Suc and starch, 50 embryos in the early stage of oil accumulation (2–3 mg fresh weight) were grown for 3 days in 20 ml of growth medium with either [1-13C]Glc or [6-13C]Glc (99% 13C enrichment, 20 mm). Since Suc labeled at C-1 or C-6 of hexose units was not available, Suc in the growth medium was substituted by its analog palatinose (6-O-α-d-glucopyranosyl-d-fructofuranose, 80 mm), which is not taken up or metabolized in plants but which appears to have similar signal functions to Suc (38Fernie A.R. Roessner U. Geigenberger P. Plant Physiol. 2001; 125: 1967-1977Crossref PubMed Scopus (69) Google Scholar). Therefore, in these experiments, the main carbohydrate carbon source was the labeled Glc, and the starch and seed oil in the embryos were substantially labeled. These experiments yielded 1–10 mg of free Suc, Glc (from starch), and seed oil for analysis by NMR spectroscopy. To ensure that the palatinose in the growth medium has no major artificial influence on the results, analogous experiments using [1-13C]Glc with unlabeled sucrose were also performed, which confirmed the experimental results with palatinose although with inferior accuracy due to the isotopic dilution of label from the unlabeled sucrose.Extraction of Lipids and Proteins—Labeled embryos were ground, and lipids were extracted with hexane/diethylether (1:1, v/v); proteins were extracted in a buffer containing sodium phosphate, pH 7.5 (10 mm), and NaCl (500 mm) as described by Schwender and Ohlrogge (22Schwender J. Ohlrogge J. Plant Physiol. 2002; 130: 347-361Crossref PubMed Scopus (156) Google Scholar). Extracted soluble proteins were precipitated by the addition of one-tenth volume of 50% trichloroacetic acid.Extraction of Sucrose—Embryos labeled for 3 days were ground in a glass homogenizer in methanol/H2O (1:1) (v/v) and extracted three times at 50 °C. The combined extracts were separated into a water-soluble and a lipid fraction by adding chloroform to a final ratio close to CHCl3/methanol/H2O (8:4:3) (39Folch J. Lees M. Sloane-Stanley G.H.S. J. Biol. Chem. 1951; 226: 497-509Abstract Full Text PDF Google Scholar). The aqueous phase containing mainly Suc was freeze-dried and dissolved in D2O for NMR analysis.Starch Degradation—After extraction of lipids and water/methanol-soluble compounds, the cell residue (equivalent to 50–100 mg fresh weight tissue) was washed three times with 5 ml of 80% (v/v) aqueous methanol and dried under vacuum. After the addition of 1 ml of H2O and sealing and heating at 110 °C for 1 h, starch was degraded to Glc by the addition of 1 ml of 0.1 m acetate buffer (pH 4.8), 20 units α-amylase, and 20 units amyloglucosidase with heating to 55 °C for 3 h. Proteins were precipitated by the addition of 1 volume of ethanol, sealing and heating to 100 °C for 5 min, and centrifugation. The supernatant was reduced in volume by evaporation under nitrogen, freeze-dried, and dissolved in D2O for 13C NMR spectroscopy or derivatized for GC/MS analysis.Measurement of Glucose Labeling—For analysis by GC/MS, Glc was derivatized to Glc methoxime penta-acetate. 1 ml of methoxyamine hydrochloride in pyridine (20 mg/ml) was added to 50–100 μg of Glc and heated to 50 °C for 1 h. After cooling to room temperature, 1 ml of acetic acid anhydride was added, and the sample was again heated to 50 °C for 1 h. Finally, the derivative was extracted with toluene after adding 1 volume of H2O to the reaction. The ions m/z 360, m/z 289, and m/z 89 (C15H22O9N (Glc(1–6)), 2Carbon atoms in different molecules are denoted as subscripts. For example, Glc(1–3) refers to the part of the molecule comprising carbons 1, 2, and 3 of Glc, and pyruvate(1–2) refers to carbons 1 and 2 of pyruvate. C12H17O9N (Glc(3–6)), and C3H7O2N (Glc(1–2)), respectively) were monitored by GC/MS.Measurement of Lipid Labeling—For analysis by GC/MS or 13C NMR, the lipid fraction consisting mainly of TAG was hydrogenated (40Bao X.M. Focke M. Pollard M. Ohlrogge J. Plant J. 2000; 22: 39-50Crossref PubMed Google Scholar). For analysis of fatty acids and glycerol by GC/MS, lipids were transesterified by heating to 90 °C in 5% (w/v) HCl in methanol for 1 h. After cooling to room temperature, 1 volume of H2O was added, and fatty acid methyl esters were extracted with hexane (41Browse J. McCourt P. Somerville C. Anal. Biochem. 1986; 152: 141-145Crossref PubMed Scopus (395) Google Scholar). The aqueous phase was freeze-dried, and the residue, containing glycerol, was derivatized with trifluoroacetic acid anhydride for 1 h at room temperature to obtain glycerol trifluoroacetate. Residual derivatization reagent was removed with a stream of nitrogen, and the derivatives were dissolved in toluene.Measurement of Label in Amino Acids of Storage Proteins—Proteins were hydrolyzed in 6 n HCl for 24 h at 100 °C. HCl was evaporated at 50 °C under a stream of nitrogen. Amino acids were dissolved in 0.1 n HCl and loaded on an H+ exchange column (AG 50W-X4; Bio-Rad). After washing with 5 volumes of H2O, amino acids were eluted with 2 n NH4OH. After most of the NH4OH was removed under a stream of nitrogen, the sample was lyophilized and then derivatized to their N,O-tert-butyldimethylsilyl derivatives by adding 100 μl of N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide/acetonitrile (1:1) to 100 μg of amino acids and heating at 120 °C for 1 h (36Dauner M. Sauer U. Biotechnol. Prog. 2000; 16: 642-649Crossref PubMed Scopus (193) Google Scholar, 42Das Neves H.J.C. Vasconcelos A.M.P. J. Chromatogr. 1987; 392: 249-258Crossref PubMed Scopus (141) Google Scholar). The identities of different fragments of the TBDMS amino acid derivatives in mass spectra were derived from the literature (36Dauner M. Sauer U. Biotechnol. Prog. 2000; 16: 642-649Crossref PubMed Scopus (193) Google Scholar, 42Das Neves H.J.C. Vasconcelos A.M.P. J. Chromatogr. 1987; 392: 249-258Crossref PubMed Scopus (141) Google Scholar).GC Conditions—One microliter of each derivatized sample (100–500 ng/μl) was analyzed with a HP 5890 II (Hewlett-Packard) gas chromatograph/mass spectrometer (HP 5972 quadrupole MS). Carrier gas was helium at 1 ml/min. For fatty acid methyl esters, a DB23 column (30 m × 0.25 mm) was used (J&W Scientific, Folsom, CA). For N,O-tert-butyldimethylsilyl derivatives of amino acids, Glc methoxime penta-acetate, and glycerol trifluoroacetate, a 30 m × 0.25-mm DB1 column was used (J&W Scientific). The GC conditions for fatty acid methyl esters and N,O(S)-tert-butyldimethylsilyl derivatives of amino acids were as previously described (22Schwender J. Ohlrogge J. Plant Physiol. 2002; 130: 347-361Crossref PubMed Scopus (156) Google Scholar). For glycerol trifluoroacetate, the injector temperature was 250 °C. Initial temperature was 60 °C for 2 min, increased to 240 °C at 20 °C/min and a final temperature at 240 °C for 10 min. Data were analyzed by the Chem Station Program (HP G1043C, Hewlett-Packard).Measurement of Fractional Labeling by Mass Spectrometry—In mass spectra of labeled compounds, selected molecular fragments were monitored. Single ion monitoring was generally used with >20-ms acquisition time for each ion. The mass spectra of each ion were integrated over the entire chromatographic peak to avoid the influence of possible isotope fractionation during GC separation. Background correction was performed with mass spectra taken just before each chromatographic peak. Reproducibility of isotope ratios was checked with unlabeled reference substances over a concentration range of 2 orders of magnitude. The ion clusters were corrected for natural isotope abundance in heteroatoms and in derivative residues as well as in the labeled molecule (43Lee W.N.P. Byerley L.O. Bergner E.A. Edmond J. Biol. Mass Spectrom. 1991; 20: 451-458Crossref PubMed Scopus (214) Google Scholar). The molar abundances of molecule fragments containing i labeled carbons are referred to as m i. The identity of ions was checked by comparison of the measured mass distribution of a fragment of unlabeled compounds with the theoretical distribution, as derived from the elemental composition and natural isotope abundances (43Lee W.N.P. Byerley L.O. Bergner E.A. Edmond J. Biol. Mass Spectrom. 1991; 20: 451-458Crossref PubMed Scopus (214) Google Scholar). Only fragments that were in good agreement with the theoretical mass distribution were used for measurements. In the case of TBDMS-amino acids and Glc methoxime penta-acetate, the ion purity was also verified by derivatization of 13C-labeled amino acids (hydrolysis of U-13C-labeled protein, 99% 13C; Isotec) and Glc ([1-13C]Glc, [6-13C]Glc, [1,2-13C2]Glc, and [U-13C6]Glc), respectively, which leads to mass shifts of the isotopomer clusters defined by the presence of one or more 13C-labeled carbon atoms in the monitored fragment. The fragmentation of glycerol trifluoroacetate during MS analysis was established by analogy to glycerol triacetate (44Neese R.A. Benowitz N.L. Hoh R. Faix D. LaBua A. Pun K. Hellerstein M.K. Am. J. Physiol. 1994; 267: E1023-E1034PubMed Google Scholar). The fragment m/z 158 contains glycerol(1–3). In the mass spectra of saturated fatty acid methyl esters, the ion m/z 74 can be used to measured labeling in C18(1–2) (22Schwender J. Ohlrogge J. Plant Physiol. 2002; 130: 347-361Crossref PubMed Scopus (156) Google Scholar). Since in the extracted TAG, C18:1 dominated over C18 and since fatty acids were hydrogenated before GC/MS analysis, the measured C18(1–2) represents mainly C18:1(1–2).Comparision of Measured and Simulated Labeling in Glucose by Least Squares Fitting—Embryos were labeled for 14 days with [U-13C12]Suc/[U-13C6]Glc (each diluted 1:10 with unlabeled sugar). Labeling in the glucosyl units of starch was measured by GC/MS. The fractional 13C enrichment was measured in the fragments Glc(1–2), Glc(3–6), and Glc(1–6). The measured mass isotopomers 3The molar abundances of molecule fragments containing i labeled carbons are referred to as m i. m 1 and m 2 of Glc(1–2), m 1 to m 4 of Glc(3–6), and m 1 to m 6 of Glc(1–6) were compared with values predicted by the computer model. For each mass isotopomer i, the difference between measurement and prediction (Δi) was calculated. The sum of squared differences (ΣΔi2) was calculated as a measure for the similarity between measured and predicted mass isotopomers. By variation of the model parameters X, V TK, V TA, and V TPC, minima for ΣΔi2 were determined as shown in Figs. 3 and 4.Fig. 4Optimized flux parameter values and obtaining a global solution by experimental over determination. Embryos were labeled for 14 days with [U-13C12]sucrose/[U-13C6]Glc. Glc obtained by starch hydrolysis was derivatized and analyzed by GC/MS (see "Experimental Procedures"). The measured fractional enrichment in fragments Glc(1–2), Glc(3–6), and Glc(1–6) was compared with the fractional enrichments predicted by the model. The sum of squared differences (ΣΔi2; see "Experimental Procedures") was calculated for a range of values in the V TK/V TA plane, with V TPC = 1.0. A, contour map showing lines of equal ΣΔi2 in the V TK/V TA plane. These contour lines were calculated using V TPC = 1 and X = 0.12. The contour line of ΣΔi2 = 2 × 10–4 surrounds an area (shaded) that corresponds to the limits of confidence that we place around the combination of V TK and V TA values (black point), which gives the best fit to the experimental data (where ΣΔi2 has its minimum). Depending on the value of X, the best fit combination of V TK and V TA changes (dashed arrow). Two such additional points are shown that correspond to V TK/V TA values that yield minima in ΣΔi2 when X = 0.05 and X = 0 (open circles). B shows the results of optimization of the fit for V TA and X for the same experiment and for A. In this case, there is no single minimum for ΣΔi2 but instead a set of points indicated by the dashed line with a confidence area again based on a 2 × 10–4 threshold for ΣΔi2 shaded in gray. The results of optimizing the fit for V TA and X for an experiment with [1,2-13C2]Glc/[1-13C]Glc (1:1) are also shown in B. In this case, mass spectroscopic data from C18:1(1–2) showing a m 1/m 2 ratio of 1.24 ± 0.04 (n = 3 independent experiments) was measured. A second dashed line shows the set of V TA/X points for which the simulated m 1/m 2 ratio matches the experimental value best. The area where m 1/m 2 = 1.24 ± 0.04 is indicated in gray. The two sets of optimized values for X and V TA for the two experiments intersect at X = 0.12, V TA = 0.01. This point defines a single overall optimum for the parameters X, V TK, V TA, and V TPC. The resulting confidence interval for X is indicated by a double-headed arrow along the x axis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)NMR Analysis—NMR analyses of aqueous extracts (containing predominantly Suc) of Glc (isolated from starch) and of storage lipids (mainly triacylglycerols) were performed with a Varian VXR 500 MHz spectrometer equipped with a 5-mm 13C-1H switchable probe. 1H and 13C NMR spectra were measured with a 90° pulse angle, 1H waltz decoupling during acquisition only (for 13C spectra), and full relaxation (recycle times = 60 s). Data processing included zero filling and multiplication of the free induction decays by an exponential function to improve the signal-to-noise ratio. NMR peak assignment for Glc, Suc, and TAG was performed using literature values (45Shachar-Hill Y. Pfeffer P.E. Nuclear Magnetic Resonance in Plant Biology. American Society of Plant Physiologists, Rockville, MD1996: 196-250Google Scholar) and by comparison with
Referência(s)