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The Metabolic Architecture of Plant Cells

2002; Elsevier BV; Volume: 277; Issue: 46 Linguagem: Inglês

10.1074/jbc.m206366200

ISSN

1083-351X

Autores

Denis Rontein, Martine Dieuaide‐Noubhani, Érick J. Dufourc, Philippe Raymond, Dominique D. Rolin,

Tópico(s)

Plant tissue culture and regeneration

Resumo

The changes in the intermediary metabolism of plant cells were quantified according to growth conditions at three different stages of the growth cycle of tomato cell suspension. Eighteen fluxes of central metabolism were calculated from13C enrichments after near steady-state labeling by a metabolic model similar to that described in Dieuaide-Noubhani et al. (Dieuaide-Noubhani, M., Raffard, G., Canioni, P., Pradet, A., and Raymond, P. (1995) J. Biol. Chem. 270, 13147–13159), and 10 net fluxes were obtained directly from end-product accumulation rates. The absolute flux values of central metabolic pathways gradually slowed down with the decrease of glucose influx into the cells. However, the relative fluxes of glycolysis, the pentose-P pathway, and the tricarboxylic acid cycle remained unchanged during the culture cycle at 70, 28, and 40% of glucose influx, respectively, and the futile cycle of sucrose remained high at about 6-fold the glucose influx, independently from carbon nutritional conditions. This natural resistance to flux alterations is referred to as metabolic stability. The numerous anabolic pathways, including starch synthesis, hexose accumulation, biosynthesis of wall polysaccharides, and amino and organic acid biosynthesis were comparatively low and variable. The phosphoenolpyruvate carboxylase flux decreased 5-fold in absolute terms and 2-fold in relation to the glucose influx rate during the culture cycle. We conclude that anabolic fluxes constitute the flexible part of plant cell metabolism that can fluctuate in relation to cell demands for growth. The changes in the intermediary metabolism of plant cells were quantified according to growth conditions at three different stages of the growth cycle of tomato cell suspension. Eighteen fluxes of central metabolism were calculated from13C enrichments after near steady-state labeling by a metabolic model similar to that described in Dieuaide-Noubhani et al. (Dieuaide-Noubhani, M., Raffard, G., Canioni, P., Pradet, A., and Raymond, P. (1995) J. Biol. Chem. 270, 13147–13159), and 10 net fluxes were obtained directly from end-product accumulation rates. The absolute flux values of central metabolic pathways gradually slowed down with the decrease of glucose influx into the cells. However, the relative fluxes of glycolysis, the pentose-P pathway, and the tricarboxylic acid cycle remained unchanged during the culture cycle at 70, 28, and 40% of glucose influx, respectively, and the futile cycle of sucrose remained high at about 6-fold the glucose influx, independently from carbon nutritional conditions. This natural resistance to flux alterations is referred to as metabolic stability. The numerous anabolic pathways, including starch synthesis, hexose accumulation, biosynthesis of wall polysaccharides, and amino and organic acid biosynthesis were comparatively low and variable. The phosphoenolpyruvate carboxylase flux decreased 5-fold in absolute terms and 2-fold in relation to the glucose influx rate during the culture cycle. We conclude that anabolic fluxes constitute the flexible part of plant cell metabolism that can fluctuate in relation to cell demands for growth. Plants are able to grow under a wide range of environmental conditions (extreme temperatures, insufficient or excessive light, and shortage of water or mineral nutrients) and show a robust physiological homeostasis. To ensure this homeostasis, plant metabolism has to be very flexible (1Smirnoff N. Smirnoff N. Environment and Plant Metabolism: flexibility and acclimation. Bios Scientific Publishers, Oxford1995: 1-16Google Scholar). In all cells, the central carbon metabolism provides energy, cofactor regeneration, and building blocks for biomass and secondary metabolism. The flexibility of plant metabolism, which is probably an evolutionary adaptation to the variable environmental conditions plants normally experience, has been explained by the buffering effect of carbon storage and allocation (2ap Rees T. Hill S.A. Plant Cell Environ. 1994; 17: 587-599Crossref Scopus (109) Google Scholar) and by the complexity of regulation or built-in redundancy owing to alternative enzymes and pathways for many processes (3Plaxton W.C. Ann. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 185-214Crossref PubMed Scopus (719) Google Scholar). For example, parallel glycolytic pathways are present in the cytosol and plastid; in the cytosol, the classic key sites for regulation of glycolysis at phosphofructokinase and pyruvate kinase (PK) 1The abbreviations used are: PK, pyruvate kinase; PEPC, phosphoenolpyruvate carboxylase; HPLC, high-performance liquid chromatography; SR, specific radioactivity; FW, fresh weight; DW, dry weight; PPP, pentose-phosphate pathway 1The abbreviations used are: PK, pyruvate kinase; PEPC, phosphoenolpyruvate carboxylase; HPLC, high-performance liquid chromatography; SR, specific radioactivity; FW, fresh weight; DW, dry weight; PPP, pentose-phosphate pathwaycan be bypassed by pyrophosphate:fructose-6-phosphate phosphotransferase and phosphoenolpyruvate carboxylase (PEPC) (3Plaxton W.C. Ann. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 185-214Crossref PubMed Scopus (719) Google Scholar).The development of genetic engineering, which introduced a new dimension to pathway knowledge by allowing precise modifications of specific enzymatic reactions in metabolic pathways, also highlighted the flexibility of plant primary metabolism (4Stitt M. Curr. Opin. Biotech. 1994; 5: 137-143Crossref Scopus (19) Google Scholar, 5Stitt M. Sonnewald U. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1995; 46: 341-368Crossref Scopus (204) Google Scholar, 6Herbers K. Sonnewald U. Tibtech. 1996; 14: 198-205Abstract Full Text PDF PubMed Scopus (23) Google Scholar). Pyrophosphate:fructose-6-phosphate phosphotransferase and cytosolic PK, which generally have been considered essential, were almost removed without any significant effect on growth or development (7Gottlob-McHugh S.G. Sangwan R.S. Blakeley S.D. Vanlerberghe G.C., Ko, K. Turpin D.H. Plaxton W.C. Miki B.L. Dennis D.T. Plant Physiol. 1992; 100: 820-825Crossref PubMed Scopus (45) Google Scholar, 8Hajirezaei M. Sonnewald U. Viola R. Carlisle S. Dennis D. Stitt M. Planta. 1994; 192: 16-30Google Scholar, 9Zrenner R. Krause K.-P. Apel P. Sonnewald U. Plant J. 1996; 9: 671-681Crossref PubMed Scopus (91) Google Scholar). In contrast, in yeast many genes encoding glycolytic enzymes (e.g. hexose kinase, phosphoglucose isomerase, phosphoglucose kinase, PK, and alcohol dehydrogenase) have been overexpressed many times, but in none of the mutants did the glycolytic flux differ substantially from the wild type flux (10Schaaff I. Heinisch J. Zimmermann F.K. Yeast. 1989; 5: 285-290Crossref PubMed Scopus (241) Google Scholar, 11Davies S.E.C. Brindle K.M. Biochemistry. 1992; 31: 4729-4735Crossref PubMed Scopus (96) Google Scholar). In plants, overexpression of the spinach choline monooxygenase gene in tobacco chloroplasts did not contribute to the accumulation of glycine betaine (12Nuccio M.L. Russel B.L. Nolte K., D. Rathinasabapathi B. Gaye D.A. Hanson A.D. Plant J. 1998; 16: 101-110Crossref Google Scholar). This inherent resistance to flux alteration by genetic manipulation, which was designed to enhance the yield in biosynthetic choline pathways, has also been named "rigidity" (13McNeil S.D. Rhodes D. Russell B.L. Nuccio M.L. Shachar-Hill Y. Hanson A.D. Plant Physiol. 2000; 124: 153-162Crossref PubMed Scopus (80) Google Scholar). The concept of "network rigidity" was initially proposed by Stephanopoulos and Vallino (14Stephanopoulos G. Vallino J.J. Science. 1991; 252: 1675-1681Crossref PubMed Scopus (457) Google Scholar) and was considered to result from mechanisms that stabilize flux ratios at branch points in metabolic networks. The enzymes bifurcating from a rigid node are activated by intermediates in the opposite branch. The interdependency of enzyme activities in competing branches stabilizes the ratio of branching fluxes and maintains flux distribution, which is optimal for growth. In view of carbon metabolism adaptations in response to environmental changes in plants, the following question can be raised: does the plant primary metabolism coordinate a relatively uniform constant distribution of building block metabolites and energy or does it support radically different flux distributions in response to different environmental stimuli?A variety of methods have been developed to quantify intracellular fluxes in animals (15Malloy C.R. Sherry A.D. Jeffrey M.H. J. Biol. Chem. 1988; 263: 6964-6971Abstract Full Text PDF PubMed Google Scholar, 16Bouzier A.-K. Goodwin R. de Gannes F.M. Valeins H. Voisin P. Canioni P. Merle M. J. Biol. Chem. 1998; 273: 27162-27169Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), microorganisms (17Marx A. de Graaf A.A. Wiechert W. Eggeling L. Sahm H. Biotechnol. Bioeng. 1996; 49: 111-129Crossref PubMed Scopus (349) Google Scholar), and plants (18Salon C. Raymond P. Pradet A. J. Biol. Chem. 1988; 263: 12278-12287Abstract Full Text PDF PubMed Google Scholar, 19Martin F. Boiffin V. Pfeffer P.E. Plant Physiol. 1998; 118: 627-635Crossref PubMed Scopus (56) Google Scholar, 20Dieuaide-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, 21Roscher A. Emsley L. Raymond P. Roby C. J. Biol. Chem. 1998; 273: 25053-25061Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 22Roscher A. Kruger N.J. Ratcliffe R.G. J. Biotech. 2000; 77: 81-102Crossref PubMed Scopus (100) Google Scholar, 23Hill S.A. ap Rees T. Planta. 1994; 192: 52-60Crossref Scopus (67) Google Scholar). Radio or stable isotopic tracers such as 14C or13C can be used according to three major methods (22Roscher A. Kruger N.J. Ratcliffe R.G. J. Biotech. 2000; 77: 81-102Crossref PubMed Scopus (100) Google Scholar). A commonly used method is to calculate a flux from the rate of end-product labeling and the specific radioactivity of the precursor (23Hill S.A. ap Rees T. Planta. 1994; 192: 52-60Crossref Scopus (67) Google Scholar). Alternatively, tissues are labeled to isotopic steady state, and intermediate labeling can be used for flux calculation using a linear equation, independently of time (18Salon C. Raymond P. Pradet A. J. Biol. Chem. 1988; 263: 12278-12287Abstract Full Text PDF PubMed Google Scholar, 20Dieuaide-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, 24Katz J. Rognstad R. Biochemistry. 1967; 6: 2227-2247Crossref PubMed Scopus (80) Google Scholar). Fluxes can also be measured directly by in vivo NMR using the techniques of magnetization transfers (21Roscher A. Emsley L. Raymond P. Roby C. J. Biol. Chem. 1998; 273: 25053-25061Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). In plants, major fluxes of carbohydrate metabolism have been estimated during starch breakdown by ripening bananas (23Hill S.A. ap Rees T. Planta. 1994; 192: 52-60Crossref Scopus (67) Google Scholar), during carbon starvation (20Dieuaide-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, 25Dieuaide-Noubhani M. Canioni P. Raymond P. Plant Physiol. 1997; 115: 1505-1513Crossref PubMed Scopus (44) Google Scholar), during hypoxia in corn root tips (21Roscher A. Emsley L. Raymond P. Roby C. J. Biol. Chem. 1998; 273: 25053-25061Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 26Edwards S. Nguyen B.-T., Do, B. Roberts J.K.M. Plant Physiol. 1998; 116: 1073-1081Crossref PubMed Scopus (72) Google Scholar), and in early germinating lettuce seeds (18Salon C. Raymond P. Pradet A. J. Biol. Chem. 1988; 263: 12278-12287Abstract Full Text PDF PubMed Google Scholar). Dieuaide-Noubhani et al. (20Dieuaide-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, 25Dieuaide-Noubhani M. Canioni P. Raymond P. Plant Physiol. 1997; 115: 1505-1513Crossref PubMed Scopus (44) Google Scholar) identified and quantified 20 metabolic fluxes from sucrose turnover to input into the tricarboxylic acid cycle in maize root tips. Changes observed in the enrichments of intermediary metabolites during the first stages of sugar starvation indicated that sugar nutrition profoundly affects the metabolic network in excised corn root tips. Acetyl-CoA entering the tricarboxylic acid cycle is progressively supplied by lipids and proteins, and the anaplerotic flux through the PEPC vanishes long before carbohydrates are exhausted.The aim of the present work was to examine changes in fluxes of intermediary metabolism in heterotrophic plant cells in suspension cultures, in response to changes in the culture medium and growth stages during a growth cycle. Tomato cells were labeled to the isotopic steady state with [1-13C]glucose. 1The abbreviations used are: PK, pyruvate kinase; PEPC, phosphoenolpyruvate carboxylase; HPLC, high-performance liquid chromatography; SR, specific radioactivity; FW, fresh weight; DW, dry weight; PPP, pentose-phosphate pathwayH and13C NMR were used to determine 13C enrichments of specific carbons of carbohydrates and amino acids. Metabolic pathway modeling was used to interpret isotope distribution according to Ref.20Dieuaide-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. Using these data, 29 metabolic fluxes were quantified at 3 different physiological stages during the growth cycle of tomato cells. The results show the following: (i) That relative fluxes in the central metabolism (glycolysis, tricarboxylic acid cycle, and pentose-P pathway) and futile cycles (sucrose turnover and triose-P to hexose-P recycling) are high and stable. (ii) On the contrary, relative fluxes in anabolic pathways, such as the anaplerotic flux catalyzed by PEP carboxylase, are low and variable, constituting the flexible part of metabolism, which may fluctuate to fit precursor availability for the anabolic pathway in relation to cell needs.RESULTSCell Growth, Glucose Uptake, and Cellular Metabolite ContentThe growth rate of tomato cells in liquid culture was established by measuring fresh weight (FW), dry weight (DW), and cell number (Fig. 1, A andB). The growth curve profile was similar to that of most other plant cell cultures (35Kanabus J. Bressan R.A. Carpita N.C. Plant Physiol. 1986; 82: 363-368Crossref PubMed Google Scholar, 36Botha F.C. O'Kennedy M.M. Physiol. Plant. 1998; 102: 429-436Crossref Scopus (16) Google Scholar). Growth was exponential during the first 6 days after subculture. Fig. 1 A indicates that, after day 5, FW increased more by cell enlargement than by cell division. This was confirmed by the decrease in the rate of [3H]thymidine incorporation into DNA from day 4, and the disappearance of cyclin A2 and D3 mRNA at day 5, thus indicating the arrest of cell division (data not shown). In batch culture, most physical and chemical conditions change during the growth cycle. In our experiment, glucose disappeared from the medium in a sigmoid fashion (Fig. 1 A). The glucose influx rate (Vg) decreased in parallel, from 903 ± 45 at day 3 to 180 nmol.h−1.(106 cells)−1 at day 7.5 (Fig. 1 C). At day 6, nitrogen (NH 4+, 15 mm) and glucose (65 mm) were still available in the culture medium, whereas inorganic phosphate measured by the molybdate method was under 0.02 mm. The vacuolar phosphate was not detectable by in vivo 31P NMR, but the cytoplasmic Pi level was the same as on day 1 (data not shown). No Pi starvation symptom was observed. Growth (FW curve) slowed down after day 6 and stopped after day 10 when glucose was exhausted from the medium (Fig.1 A).During a culture cycle, 70% of the absorbed glucose was used to sustain respiration. The main end products of glucose metabolism accumulated during a growth cycle (i.e. soluble and insoluble sugars, organic acids, and proteins) are shown in Fig.2. Soluble sugars mainly accumulated at the end of the exponential phase between days 5 and 6 (Fig.2 A). At the end of the culture, the accumulation of cell wall polysaccharides, soluble sugars, and starch was, respectively, 1.7, 1.2, and 0.6 mmol of glucose equivalent per flask corresponding to 10, 8, and 4% of the initial glucose (Fig. 2, A andB). The organic acid content (0.1 mmol per flask at the end of the culture, Fig. 2 C) was a small fraction of accumulated carbon. Citrate and malate mainly accumulated after day 5 when cell growth started to decline (Fig. 2 C). Total proteins constantly increased during the cell growth cycle (Fig. 2 D). Their amino acid composition remained stable during the growth cycle. The carbon flux for each group of amino acids contributing to protein accumulation was calculated from the rate of protein accumulation (Fig.2 D) and the established pathway of amino acid biosynthesis (Fig. 3) (37Ireland R. Dennis D.T. Turpin D.H. Lefebvre D.D. Layzell D.B. Plant Metabolism. 2nd Ed. Addison Wesley Longman, England1997: 478-494Google Scholar, 38Bryan J.K. Stumpf P.K. Conn E.E. Biochemistry of Plants: A Comprehensive Treatise. 5. Academic Press Inc., New York1992: 403-452Google Scholar).Figure 2Changes in soluble sugars (A), insoluble compounds (B), organic acids (C), and total proteins (D), during the cell growth cycle. Sugars and organic acids were determined by enzymatic assay, proteins by Bio-Rad DC assay as described under "Experimental Procedures." Cell wall polysaccharides were determined from insoluble compounds after ethanolic extraction and starch digestion of the cells.Abscissa, days in culture after transfer to fresh medium.Vertical bars represent the S.D. of the mean from four samples in two independent experiments.View Large Image Figure ViewerDownload (PPT)Figure 3Biosynthetic pathways of amino acid groups connected to central metabolism and amino acid compositions of total proteins in tomato cells. Amino acid biosynthesis from 2-OG (Vglu), OAA (Vasp), pyruvate + acetyl-CoA (Vala), PEP (Vpep), and erythrose-P (Very) are shown. The numbers represent the amino acid percentage for each metabolic group in total proteins. The sum of the amino acid percentages is superior to 100% because some amino acids belong to several groups. Amino acids are determined after acid hydrolysis and HPLC analysis under "Experimental Procedures." Results are the mean of two experiments in each of the three physiological conditions as described under "Experimental Procedures."View Large Image Figure ViewerDownload (PPT)The fluxes to fatty acid and nucleic acid synthesis were minor and not included in the model. In tomato cell suspensions, nucleic acids (DNA-RNA) were estimated to be 1 μg.(106cells)−1, which corresponds to a relative flux of pentose-P into the nucleic acids equal to only 0.1% of the glucose influx.Carbohydrate and Amino Acid Enrichments Measured by 1The abbreviations used are: PK, pyruvate kinase; PEPC, phosphoenolpyruvate carboxylase; HPLC, high-performance liquid chromatography; SR, specific radioactivity; FW, fresh weight; DW, dry weight; PPP, pentose-phosphate pathwayH and 13C NMRThe time needed to reach the isotopic and metabolic steady state was determined as in a previous study (20Dieuaide-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) by monitoring the evolution of 14CO2 and the specific radioactivity of cellular soluble sugars, starch, Ala, and Glu. The soluble sugars and amino acids were near isotopic steady state after 3 and 4 days, respectively (data not shown).Typical 13C and 1The abbreviations used are: PK, pyruvate kinase; PEPC, phosphoenolpyruvate carboxylase; HPLC, high-performance liquid chromatography; SR, specific radioactivity; FW, fresh weight; DW, dry weight; PPP, pentose-phosphate pathwayH NMR spectra of purified glucose, sucrose, and starch from tomato cells after 5 days of culture with [1-13C]glucose are shown in Fig.4. The resonance assigned to glucose, fructose, and sucrose are clearly visible. The highest amounts of13C were at C-1 of α and β free glucose (Fig.4 A), C-1 of glucosyl and fructosyl moieties of sucrose (Fig.4 B), and C-1 of α and β glucose deriving from starch hydrolysis (Fig. 4 C). The spectra also showed appreciably more labeling at C-6 than at the other carbons (except C-1) of these hexose residues. The carbon enrichment values of these sugars are presented in Table I.Figure 413 C and 1H (inset) NMR spectra of glucose (A), sucrose (B), and glucose from starch hydrolysate (C). Tomato cells were fed with [1-13C]glucose for 5 days. Cellular sugars were purified by HPLC as described under "Experimental Procedures." Starch was extracted and hydrolyzed to glucose. The terms Giα andGiβ indicate the resonance of carbon i of α and β glucose. The terms Sig and Sif indicate the resonance of carbon i of glucosyl and fructosyl moieties. 1H and 13C spectra represent the accumulation of 128 and 256 scans, respectively.View Large Image Figure ViewerDownload (PPT)Table ISteady-state enrichments of carbohydrates of cultured tomato cells with [1-13C]glucose during exponential phase (day 5), arrest of cell division (d 6), and pre-stationary phase (day 7.5)13C enrichmentGlucoseSucrose glucosylStarch glucosyl%Day 5C-161.3 ± 0.3a56.6 ± 1c48.5 ± 0.5cC-616.1 ± 0.5b17 ± 1.3d17.1 ± 0.8dDay 6C-161.2 ± 0.3a56 ± 1c50 ± 1.5fC-616.6 ± 0.5b18.2 ± 1.5d17.8 ± 1.8dDay 7.5C-161.6 ± 0.3a56.1 ± 1.2c53.4 ± 1.1hC-618.6 ± 0.6g18.6 ± 1.8d20 ± 1.4iThe enrichments (in percent) were determined from 1The abbreviations used are: PK, pyruvate kinase; PEPC, phosphoenolpyruvate carboxylase; HPLC, high-performance liquid chromatography; SR, specific radioactivity; FW, fresh weight; DW, dry weight; PPP, pentose-phosphate pathwayH and13C spectra as described under "Experimental Procedures." Results are given as mean ± S.D. (n = 2) taking into account the error associated with the integration measurements. Statistical significance was evaluated by using Fisher-Student's test analysis as appropriate (p = 0.05); the results are indicated by subscript letter for significant different value. Open table in a new tab 1The abbreviations used are: PK, pyruvate kinase; PEPC, phosphoenolpyruvate carboxylase; HPLC, high-performance liquid chromatography; SR, specific radioactivity; FW, fresh weight; DW, dry weight; PPP, pentose-phosphate pathwayH and 13C NMR spectra of Glu and Ala are presented in Fig. 5, and enrichments are shown in Table II. Glu, C-2, and C-3 have similar enrichment values at about 75% of the C-4 enrichment. The13C enrichment of Ala C-3 was close to that of Glu C-4. Ala C-2 enrichment was just above background.Figure 51 H (top andinset) and 13C-NMR spectra of Glu (A) and Ala (B). Tomato cells were fed with [1-13C]glucose for 5 days. Amino acids were purified by cation exchange resin as described under "Experimental Procedures." The terms Cig indicate the resonance of carbon i of Glu and Cia the resonance of carboni of Ala. 1H and 13C spectra represent the accumulation of 128 and 1800 scans, respectively.View Large Image Figure ViewerDownload (PPT)Table IISteady-state enrichments of amino acids during exponential phase (day 5), arrest of cell division (day 6), and pre-stationary phase (day 7.5)13C enrichmentAlanineGlutamate%Day 5C-22.3 ± 0.4j24.2 ± 0.3lC-330.4 ± 0.2k22.4 ± 0.4mC-430.5 ± 0.2kDay 6C-23 ± 1j25 ± 0.4nC-330.6 ± 0.2k23.4 ± 0.4oC-430.8 ± 0.6kDay 7.5C-22.1 ± 0.4j27.2 ± 0.4qC-331 ± 0.2p25.8 ± 0.4rC-431.6 ± 0.2sThe enrichments (in percent) were determined from 1The abbreviations used are: PK, pyruvate kinase; PEPC, phosphoenolpyruvate carboxylase; HPLC, high-performance liquid chromatography; SR, specific radioactivity; FW, fresh weight; DW, dry weight; PPP, pentose-phosphate pathwayH and13C spectra as described under "Experimental Procedures." Results are given as mean ± S.D. (n = 2) taking into account the error associated with the integration measurements. Statistical significance was evaluated by using the Fisher-Student test analysis as appropriate (p = 0.05); the results are indicated by subscript letter for significant different values. Open table in a new tab Modeling the Metabolic NetworkThe metabolic scheme that accounts for tracer distribution is essentially similar to that developed in Ref. 20Dieuaide-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, with some modifications described below and shown in Fig.6.Figure 6Pathways of carbohydrate metabolism in tomato cells. Metabolic pathways have been identified by using the labeling of intermediates as described in the text. Flux names are defined under "Appendix." Flux values are given in TableIV.View Large Image Figure ViewerDownload (PPT)Sugar MetabolismThe label distributions observed in the glucosyl and fructosyl moieties of sucrose were similar. It was therefore assumed that the cytosolic hexose-P constituted a single pool (Fig. 6). The labeling of intracellular free glucose C-6 suggested that part of intracellular glucose was formed from hexose-P via a sucrose cycle (Vi) (Fig. 6). According to Ref. 20Dieuaide-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, the C-6 enrichment of the hexose-P pool can be explained by both triose-P recycling and exchange through the cytosolic transaldolase reaction (Vtald) (Fig. 6). The absence of detectable label in free glucose C-2 and C-5 suggests the absence of gluconeogenesis (Fig. 6). The fact that the total enrichment (C-1 + C-6) of free glucose, sucrose, and starch glucosyl was gradually lower than that of the glucose precursor (92%) was accounted for by the pentose-P pathway (Vppp), the only possibility in the absence of gluconeogenesis (Fig. 6). The similar enrichments of starch glucosyl C-6, and sucrose glucosyl C-6 (Table I) indicated that starch was essentially formed from hexose-P imported from the cytosol with no further exchange of hexose-P C-6 with triose-P C-1. This was not the case in corn root tips (20Dieuaide-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) and suggested an exchange between the plastidial and cytosolic hexose-P. This is shown in the present model as a reverse flux (Vhpc) of hexose-P from plastids to cytosol (Fig. 6). The hexose-P exchange between cytosol and plastids could be performed either by a hexose-P translocator of the plastid double membrane, as suggested recently (39Flugge U.I. Curr. Opin. Plant. Biol. 1998; 1: 201-206Crossref PubMed Scopus (47) Google Scholar), or by a glucose translocator (40Weber A. Servaites J.C. Geiger D.R. Kofler H. Hille D. Groner F. Hebbeker U. Flugge U.I. Plant Cell. 2000; 12: 787-802Crossref PubMed Scopus (178) Google Scholar). The lower enrichment of the starch glucosyl C-1 compared with cytosolic hexose-P C-1 (Table I) indicated the occurrence of a flux through the plastidial PPP, with recycling of C-1 unlabeled hexose-P inside plastids. As in Ref. 20Dieuaide-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, the cytosolic and plastidial triose-P were assumed to be in rapid exchange, and only one pool was considered.Anaplerotic and Respiratory Carbon Fluxes through the Tricarboxylic Acid CycleThe glycolytic flux (Vgly) measured on the linear pathway between triose-P and PEP is split between the oxidative flux through PK, the anaplerotic flux through PEPC, which is the main source of four carbon compounds in the tricarboxylic acid cycle, and amino acid biosynthesis using PEP as precursor (Fig. 6). According to (18Salon C. Raymond P. Pradet A. J. Biol. Chem. 1988; 263: 12278-12287Abstract Full Text PDF PubMed Google Scholar, 20Dieuaide-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), the anaplerotic flux leads to the dilution of C-2 and C-3 enrichments compared with C-4 enrichment of Glu, thus allowing the quantification of the relative flux through the oxidativeversus anaplerotic pathways (Table II). The identical enrichment of Glu C-4 and Ala C-3 (Table II) indicated that there was no diluting flux between pyruvate and Glu, i.e. glycolysis was the only source of acetyl CoA. The low enrichment of C-2 Ala showed the occurrence of a relatively small flux from malate to Ala through the malic enzyme reaction (Fig. 6).Amino Acid FluxesOur model also includes the fluxes of amino acid biosynthesis calculated from the rate of amino acid accumulation into total proteins (Fig. 2 D). During the culture cycle, the amino acid composition of total protein remained constant (Fig. 3). Table III presents the conversion factor (F) used to calculate the fluxes (Vx)

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