©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Quantification of Compartmented Metabolic Fluxes in Maize Root Tips Using Isotope Distribution from C- or C-Labeled Glucose (*)

Martine Dieuaide-Noubhani (1)(§), Gérard Raffard (2), Paul Canioni (2), Alain Pradet (1), Philippe Raymond (1)(¶)

From the (1) Station de Physiologie Végétale, Institut National de la Recherche Agronomique, Centre de Recherches de Bordeaux, BP 81, 33883 Villenave d'Ornon Cedex, France and the (2) Résonance Magnétique des Systèmes Biologiques, UMR 9551 CNRS, Université de Bordeaux II, 146 rue Léo Saignat, 33076 Bordeaux, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
APPENDIX
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Metabolic pathways of the intermediate metabolism of maize root tips were identified and quantified after labeling to isotopic and metabolic steady state using glucose labeled on carbon-1, -2, or -6 with C or C. The specific radioactivity of amino acids and the C-specific enrichment of specific carbons of free glucose, sucrose, alanine and glutamate were measured and used to calculate metabolic fluxes. The non-triose pathways, including synthesis of polysaccharides, accumulation of free hexoses, and to a lesser extent starch synthesis, were found to consume 75% of the glucose entering the root tips. The cycle of synthesis and hydrolysis of sucrose was found to consume about 70% of the ATP produced by respiration. The comparison of the specific radioactivities of amino acids and phospholipid glycerol phosphate after labeling with [1-C] or [6-C]glucose revealed the operation of the pentose phosphate pathway. The transfer of label from [2-C]glucose to carbon-1 of starch glucosyl units confirmed the operation of this pathway and indicated that it is located in plastids. It was found to consume 32% of the hexose phosphates entering the triose pathways. The remaining 68% were consumed by glycolysis. The determination of the specific enrichment of carbohydrate carbons -1 and -6 after labeling with [1-C]glucose indicated that both the conversion of triose phosphates back to hexose phosphates and the transaldolase exchange contributed to this randomization. Of the triose phosphates produced by glycolysis and the pentose phosphate pathway, about 60% were found to be recycled to hexose phosphates, and 28% were directed to the tricarboxylic acid cycle. Of this 28%, two-thirds were found to be directed through the pyruvate kinase branch and one-third through the phosphoenolpyruvate branch. The latter essentially has an anaplerotic function since little malate was found to be converted to pyruvate (malic enzyme reaction).


INTRODUCTION

Non-photosynthetic cells of higher plants depend on the supply of a carbon substrate, usually sucrose provided by leaves or storage organs, for their metabolism. Sucrose is converted to sugar phosphates, which can be used for the biosynthesis of structural or storage carbohydrates, or degraded by respiration to produce ATP and intermediates for biosynthesis (1) . In recent years, specific aspects of the metabolism of non-green cells have been described semi-quantitatively by label distribution studies using classical techniques of radiolabeling or NMR methods for metabolite analysis. In most plant tissues studied, cycling between hexose phosphates and sucrose was indicated by the labeling of both glucose and fructose, and of the hexosyl moieties of sucrose, from labeled glucose or fructose (2, 3). The occurrence of a futile cycle between hexose phosphates and triose phosphates was indicated by the randomization of label between C-1 and C-6 observed in sucrose, starch, and hexoses (4, 5, 6) , and an activity of the pentose phosphate pathway was deduced from the different randomization from C-1 to C-6 than from C-6 to C-1 (4, 5, 7) . In addition, the limited randomization observed in starch indicated that, in non-green tissues, hexose phosphates rather than triose phosphates enter the plastid for starch synthesis (4, 5, 6) . Fluxes were calculated from specific yields or from randomization (5, 7) using simple models of glycolysis and the pentose phosphate pathway, which assumed complete recycling of fructose phosphate and a unidirectional transaldolase reaction. However, recycling may be partial, and the transaldolase reaction is reversible (8, 9) . These simple models may thus underestimate the flux into the pentose phosphate pathway and overestimate the conversion of triose phosphate to hexose phosphate. Fluxes of carbon input into the tricarboxylic acid cycle, including the partition of the glycolytic flux at the phosphoenolpyruvate (PEP)() branch point and the malic enzyme flux (10) , were determined in germinating embryos of lettuce. However, the situation in this material is unusual since fatty acids rather than carbohydrates are the source of acetyl-CoA entering the citrate synthase reaction (10) ; therefore, little is known of the relationship between glycolysis and the tricarboxylic acid cycle in the glucose-metabolizing tissue, which is the common situation in plants. Although most of the reactions occurring in plant intermediate metabolism have now been identified, our knowledge is still limited by the lack of quantitative determination of fluxes in extended networks.

Root tips of maize (Zea mays L.) are commonly used as a model for studies of energy metabolism in non-photosynthetic tissues. Their high metabolic activity makes them sensitive to limitations of oxygen or carbohydrate supply, which may determine the survival or death of the tissues. In an early work, based on the comparison of CO production from glucose labeled on C-1 or C-6, Gibbs and Beevers (11) detected no pentose phosphate pathway. As a result, it is sometimes assumed that the metabolism of the root tips of maize is very simple. The calculation of the rate of aerobic glycolysis from CO evolution presented by Hole et al.(12) requires CO to be produced after glycolysis only, as suggested in Ref. 11, and to be the only product of glucose oxidation. However, the pentose phosphate pathway is generally considered to account for 10-15% of the oxidation of carbohydrates in plant tissues (13). Since its main role is the supply of NADPH for biosynthesis, this pathway would be expected to be particularly active in the root tip, which is essentially a meristematic tissue. In aerobic root tips, the shortage of carbohydrate rapidly induces dramatic changes in metabolic activities, with a decrease in the rate of respiration (17) , a change in the carbon source of respiration from carbohydrates to lipids and proteins (18, 19) with an increase in the peroxisomal and mitochondrial -oxidation activities (20, 21) , and the induction of proteolytic enzymes (22) . Similar changes have been described in other plant material such as sycamore cells in culture (23) or asparagus spears (24). However, our knowledge of the metabolic network affected by these changes is incomplete and lacks quantitative data, thus precluding any prediction of their consequences and any comprehension of the mechanisms leading to cell acclimation, or failure, under stress conditions.

The aim of the present work was to identify and quantify the major metabolic fluxes of carbon metabolism of the maize root tip to gain a better understanding of its response to stress. We labeled to isotopic steady state with [C]- or [C]- labeled glucose and used metabolic pathway modeling to describe isotope distribution. This method has been applied successfully to animals (25-27) but has rarely been used for plants, and with monocompartmental models only (5, 7, 10) . We used NMR to determine the C enrichment of specific carbons of carbohydrates and amino acids. Using these data, we provide estimates for 20 metabolic fluxes, from sucrose turnover to inputs into the tricarboxylic acid cycle. We show definitely that the pentose phosphate pathway is active in maize root tips and that it is compartmented in the plastid; we suggest that the synthesis of pentans may account for identical CO evolution from [1-C] or [6-C]glucose. The use of carbon enrichments provided evidence for a contribution of the transaldolase reaction to the randomization of hexose phosphate carbons, which would not be detected from label exchange ratios. The model provided can be used to calculate fluxes from labeling data in intermediary metabolism in other plant systems.


EXPERIMENTAL PROCEDURES

Materials

Maize seeds (Z. mays L. cv. DEA, Pioneer France Mas, France) were germinated for 3 days in the dark at 25 °C as described (18) . The 3-mm tips of primary roots were excised. Before labeling, they were starved of carbohydrates for 4 h in the medium described in Ref. 18 called ``medium A.'' This treatment depletes starch and fructose and decreases the pools of sucrose and glucose to 20 and 50% of their initial values, respectively (17, 18) . These tips, called ``prestarved root tips,'' were then transferred to medium A supplemented with 200 mM labeled glucose. This concentration of glucose is necessary to maintain their respiration rate (17) . For C labeling, triplicate samples of 30 prestarved root tips were incubated in 5-ml syringes containing 2 ml of medium A with 200 mM [1-C], [6-C], or [2-C]glucose (0.25-0.5 MBq/mmol) and bubbled with a N/O mixture (50/50, v/v). For NMR experiments, 500 or 1000 prestarved maize root tips were incubated in medium A (50 ml/1000 tips), supplemented with 200 mM [1-C]glucose. After incubation, the root tips were washed with abundant ice-cold water as described (28) to eliminate exogenous glucose and then frozen in liquid nitrogen. The CO produced was trapped by bubbling through a series of three tubes containing 2 ml of 2% KOH. At the end of incubation, the radioactivity of the KOH solutions was counted in a Packard scintillation counter.

Chemicals

Analytical-grade mineral salts and amyloglucosidase were purchased from Merck (Darmstadt, Germany), and HPLC grade solvents were from Prolabo (Paris). [1-C]Glucose (1.856 GBq/mmol), [2-C]glucose (1.7 GBq/mmol), and [6-C]glucose (0.263 GBq/mmol) were purchased from NEN (Paris) or Dositech (Paris), and [1-C]glucose (99% enrichment) was purchased from Commissariat l'Energie Atomique (Gif-sur-Yvette, France). Yeast hexokinase, Leuconostoc glucose-6-phosphate dehydrogenase, and Torula yeast 6-phosphogluconate dehydrogenase were obtained from Sigma.

Analysis of Metabolites

The extraction of soluble components was performed using boiling aqueous solutions of ethanol, as previously described (10) . The extract was concentrated by evaporation; water was added to make a volume of 1 or 2 ml, and this total extract was used for the preparation of lipids and water-soluble compounds. Lipids were extracted and saponified, and the fatty acids were extracted as described (29) . The radioactivity remaining in the ethanol-water phase was identified as glycerol phosphate by passing the ethanol-water phase through an anion exchange resin (Dowex 1-X8, Bio-Rad, formate). The amino acid fraction was separated by ion exchange chromatography using standard procedures (10) and analyzed by HPLC after derivatization with o-phthaldialdehyde as described (19) ; the specific radioactivity of each amino acid was determined as in Ref. 10. For HPLC analysis of sugars, the water-soluble extract was deionized using anion and cation exchange resins and analyzed as described (30) on a Aminex HPX-87C column (Bio-Rad). Each peak was collected and counted for determination of the specific radioactivity. The residue of ethanolic extraction was used for the analysis of starch. It was washed successively with 20% ethanol and water (1.5 ml each for 30 roots), and starch was converted to glucose and analyzed by HPLC as described (30) . Total protein was extracted and determined after mineralization as described (19) . For NMR analysis, glutamate was separated from the amino acid fraction as in Ref. 10, passed through a Chelex 100 column, dried, and dissolved in 350 µl of DO. The fraction containing the amino acids minus glutamate and aspartate was used for the H NMR analysis of alanine.

The specific radioactivity of C-1 of glucose (either free glucose or glucose from starch hydrolysis) was determined after decarboxylation. The reaction was performed, according to Ref. 31, in a Warburg vial containing 2 ml of 70 mM sodium phosphate, pH 7.7, 4 mM ATP, 3 mM NADP, 3.8 mM MgCl, 6.5 mM cysteine, 3 units of hexokinase, 5 units of glucose-6-phosphate dehydrogenase (in 20 µl of 0.2 M MgSO/glycerol (50/50, w/w)), and 3 units of 6-phosphogluconate dehydrogenase. The center well contained a filter paper with 0.2 ml of 2% (w/v) KOH for CO collection. The reaction was started by adding an extract volume containing 400 nmol of glucose, and the vial was stoppered immediately. After 2 h, 200 µl of 1 M HCl, kept in the sidearm of the Warburg vial, was mixed with the reaction medium to liberate CO. After 30 min, the filter paper was recovered, and the center well was rinsed three times with 200 µl of water; 500 µl of this solution was counted. The yield of the decarboxylation reaction, determined with standard [1-C]glucose, was 82 ± 2%.

NMR Spectroscopy

Extracts for NMR spectroscopy analysis were evaporated to dryness under vacuum (Speed Vac) and dissolved in 350 µl of DO. C NMR spectra were obtained at 100.6 MHz with a Bruker AM 400 spectrometer with a 5-mm reverse probe. Spectra were collected using the composite proton decoupling sequence, a 45° flip angle, a 4-s interpulse delay, and 16 K points. H NMR spectra were obtained at 400 MHz, using a 45° flip angle, a 6-s interpulse delay, and 4 K points. Except for the analysis of sugar fractions, water resonance was eliminated using a water presaturation sequence. Prior to Fourier transformation, C-free induction decays were zero-filled to improve digital resolution and multiplied by an exponential function to improve the signal-to-noise ratio (1.5 Hz line broadening). Peak assignment was done according to Refs. 32 and 33 and from spectra of pure compounds.

The absolute C enrichment of glutamate carbons C-2, C-3, and C-4, of alanine C-3, of and glucose C-1, and of sucrose glucosyl C-1 were determined from H NMR spectra, as the ratio of the area of the satellites (C-H coupling) to the total area of the multiplet. Deconvolution of the signals was used when necessary, as in Fig. 5 and Fig. 6. The relative enrichments of the glucose and sucrose carbons C-2 to C-6 were determined from C spectra, from the absolute enrichment of C-1 determined by H NMR, and the comparison of their peak areas to that of the same resonances from non-enriched sugars, the latter being equally labeled at all carbons by the 1.1% natural abundance. This method avoids the errors due to nuclear Overhauser and relaxation effects, which would occur in the determination of relative enrichments by direct comparison of peak areas in C spectra. The same method was used to determine the enrichment of alanine C-2 from the absolute enrichment of alanine C-3 and to confirm the enrichments of the glutamate carbons.


Figure 5: C and H (inset) NMR spectra of glucose from starch hydrolysate. Pre-starved maize root tips were incubated for 12 h with [1-C]glucose. Starch was extracted and hydrolyzed to glucose as described under ``Experimental Procedures.'' H and C spectra represent the accumulation of 250 and 1000 scans, respectively. Gi and Gi indicate the resonance of carbon i of and glucose, respectively. E is the C enrichment of glucose C-1, expressed in %.




Figure 6: C (top) and H (bottom) spectra of purified glutamate after 12 h of labeling with [1-C]glucose. Pre-starved maize root tips were incubated for 12 h with 200 mM [1-C]glucose. Glutamate was purified as described under ``Experimental Procedures.'' H and C spectra represent the accumulation of 128 and 10,000 scans, respectively. E is C enrichment expressed in %.



Calculations

The resolution of simultaneous algebraic equations was done using the solver DERIVE (Soft Warehouse, Inc.).


RESULTS

Establishment of Isotopic and Metabolic Steady State Upon Incubation with Glucose

The time needed to reach isotopic and metabolic steady state was determined by following the evolution of CO, using [1-C]glucose as substrate. At isotopic steady state, specific radioactivities of intermediates of glycolysis and the tricarboxylic acid cycle are constant. Therefore, the rate of labeled CO evolution becomes constant. When root tips were not ``prestarved,'' the rate of labeled CO evolution increased continuously for more than 16 h, thus indicating that a longer incubation would be needed to reach steady state. When the endogenous pool of carbohydrates was depleted by a starvation pretreatment, the rate of labeled CO evolution became constant after 10 h (data not shown). The specific radioactivities of sucrose and fructose were already steady within 2 h (Fig. 1). At the same time, that of intracellular glucose was only about 60% of the final value; it then increased slowly to reach the steady-state value after 10 h. At that time, intracellular glucose and fructose had the same specific radioactivity, which was 90% that of the tracer glucose. The specific radioactivity of sucrose was twice that of glucose or fructose (Fig. 1). In C labeling experiments, an incubation time of 12 h was chosen. We verified on the NMR spectra that the enrichments of carbohydrates and of the amino acids glutamate and aspartate were the same at 12 and 18 h of incubation with [1-C]glucose, thus confirming the establishment of steady state at 12 h.


Figure 1: Specific radioactivities of glucose (), fructose (), and sucrose () during the incubation of maize root tips with [1-C]glucose. Excised maize root tips were pre-incubated for 4 h in the buffered mineral solution described under ``Experimental Procedures'' in the absence of glucose (prestarved tips) and then incubated with 200 mM [1-C]glucose (23.2 dpmnmol). The specific radioactivities of soluble sugars were determined after HPLC analysis as described under ``Experimental Procedures.'' Data presented are the mean of determinations on two samples in a representative experiment. Range was ±5% for glucose and fructose and ±10% for sucrose. Qualitatively similar data were obtained in four experiments with [1-C]glucose at different specific radioactivities.



Root tip growth continued during incubation in the presence of glucose. Average length increased from 3 to 10 mm, and average dry weight increased from 0.35 to 0.81 mg/tip in 12 h.

Setting Up the Metabolic Scheme

COand Amino Acid Labeling with [1-C] or [6-C]Glucose as Evidence for an Activity of the Pentose Phosphate Pathway-The lower enrichment of endogenous carbohydrates, compared with the precursor, might be explained by the loss of CO through the pentose phosphate pathway activity, the presence of an inactive pool of carbohydrates, and the gluconeogenesis of unlabeled carbohydrates from endogenous precursors. The hypothesis of the pentose phosphate pathway activity was tested by incubations with [1-C]glucose and [6-C]glucose. In agreement with earlier results (11) , no difference in the production of labeled CO was found between the two tracers (data not shown), indicating no pentose phosphate activity. However, as shown in Fig. 2 , the specific radioactivity of amino acids was 22-30% higher with [6-C]glucose than with [1-C]glucose. In the absence of the pentose phosphate pathway, this difference could be explained by the non-equilibrium of the triose phosphate isomerase reaction and the output of dihydroxyacetone phosphate (labeled with glucose C-1) to glycerol phosphate for lipid synthesis. In this case, the glycerol moiety of lipids would contain more label from [1-C] glucose than [6-C]glucose. However, the rate of incorporation of [1-C]glucose into lipid glycerol phosphate was only 70% of that of [6-C]glucose (Fig. 2D). This indicates that the specific radioactivity of dihydroxyacetone phosphate was 30% lower for [1-C]glucose than for [6-C]glucose. The observed similar differences in the specific radioactivities of dihydroxyacetone phosphate and alanine with [1-C]glucose or [6-C]glucose further indicated that the triose phosphate isomerase reaction was close to equilibrium. This disproves the hypothesis of non-equilibrium of the triose phosphates and favors the hypothesis of a flux through the oxidative branch of the pentose phosphate pathway. To account for the absence of difference in the production of labeled CO from both tracers, we assumed extra production of CO from [6-C]glucose in the decarboxylation of UDP-glucuronic acid to UDP-xylose for pentan synthesis; this non-triose pathway of carbohydrate metabolism is particularly active in elongating plant cells (13, 34) .


Figure 2: Specific radioactivities of amino acids and production of labeled glycerol phosphate during incubation of maize root tips with [1-C]glucose () or [6-C]glucose (). Prestarved maize root tips were incubated with 200 mM [1-C] or [6-C]glucose (30 ± 0.6 dpm/nmol). Specific radioactivities of glutamate (A), aspartate (B), and alanine (C) were determined after HPLC analysis. Glycerol phosphate (D) was purified as described under ``Experimental Procedures,'' and the incorporated radioactivity was counted. Data are means (±S.D.) of determinations on four samples from two independent experiments for the amino acids and on two samples for glycerol phosphate.



Labeling of Carbohydrates by Sucrose Turnover and Cycling between Hexose Phosphates and Triose Phosphates

Fig. 3 shows a typical C NMR spectrum of the ethanolic extract of maize root tips after 12 h of incubation with [1-C]glucose. Resonances assigned to glucose, sucrose, and fructose carbons are clearly visible. The spectrum shows numerous peaks of labeled intermediates related to the tricarboxylic acid cycle, including the amino acids glutamate, glutamine, aspartate, and alanine and the carboxylic acids citrate and malate. The highest amounts of C are at C-1 of glucose and C-1 of the glucosyl and fructosyl moieties of sucrose. The spectrum also shows appreciably more labeling at C-6 than at the other carbons of glucose and the sucrose moieties.


Figure 3: Proton-decoupled C NMR spectrum of the ethanolic extract of maize root tips after 12 h of labeling with [1-C]glucose. The spectrum represents the accumulation of 3,200 scans. InsetA, spectrum region (14-57 ppm) displaying amino acid resonances; B, spectrum region (66-83 ppm) displaying sugar resonances. Peak assignments: Mal, malate; Cit, citrate; Gi and Gi, Si and Si, resonances of carbon number i of and glucose and of the fructosyl and glucosyl moieties of sucrose, respectively; D, E, Q, and A indicate the resonances of carbon i of aspartate, glutamate, glutamine, and alanine, respectively.



We verified that the glucose present in the medium at the end of incubation was identical to the glucose supplied. Thus, glucose entering the maize root tips is labeled to 99% on C-1 and not labeled on the other carbons, which means that their C content is the natural enrichment, 1.1%. The enrichments of carbons of interest in selected metabolites are shown in . The label distributions observed in the glucosyl and fructosyl moieties of sucrose were similar, with averaged enrichments of 81 ± 1% and 14.5 ± 1.5% on C-1 and C-6, respectively (). Sucrose is a cytosolic compound that can be labeled on its glucosyl and fructosyl moieties when synthesized through the sucrose phosphate synthase reaction (35) . The substrates for this reaction come from the cytosolic glucose phosphate and fructose phosphate pools. Given this, the cytosolic hexose phosphates can be considered as a single pool where the C-1 or C-6 enrichments are the average enrichment of the corresponding carbons in the hexosyl moieties of sucrose (). The labeling of free glucose on C-6 suggests that intracellular glucose is formed from hexose phosphates, presumably via their incorporation into sucrose and subsequent hydrolysis of sucrose by an invertase (1, 2) , which produces free glucose and fructose. A C NMR spectrum of purified free fructose showed that its C-1 and C-6 were labeled (not shown), which is consistent with sucrose cycling through the sucrose phosphate synthase and invertase reactions. However, the hydrolysis of hexose phosphates by a phosphatase would have a similar effect to, and cannot be distinguished from, the sucrose phosphate synthase/invertase pathway of sucrose turnover. Intracellular free glucose showed less randomization than the hexosyl units of sucrose (), which is consistent with intracellular glucose being a mixture of entering precursor and recycled sucrose-glucosyl. The presence of residual extracellular glucose would increase this difference but has previously been shown to be negligible (28) .

The labeling of the hexose phosphate C-6, as shown in sucrose (), may result from the resynthesis of fructose phosphate from triose phosphates by three ways. (i) The classical one is the reversal of glycolysis by aldolase and either fructose-1,6-bisphosphatase or phosphofructophosphotransferase. Phosphofructophosphotransferase is a particular enzyme present in the cytosol of higher plant cells, which catalyzes the reversible phosphorylation of fructose 6-phosphate using pyrophosphate as the phosphoryl donor (5) . (ii) The unidirectional transaldolase reaction of the pentose phosphate pathway incorporates the glucose C-1 present at the C-3 position of triose phosphates into the C-6 position of fructose phosphate (8, 9, 13) (preliminary calculations showed that a reversal of glycolysis sufficient to explain the high enrichment of hexose phosphate C-6 would produce a C-1 enrichment lower than the experimental one. The incorporation of free triose phosphate into the fructose phosphate resynthesized through the unidirectional transaldolase reaction of the pentose phosphate pathway was also insufficient to account for the enrichment of hexose phosphate C-6). (iii) The reversibility of the transaldolase reaction (8) may increase the labeling of hexose phosphate C-6 by exchange with triose phosphate C-3 without modifying the enrichment of hexose phosphate C-1. It has usually been ignored in plants because it does not affect calculations of the pentose phosphate pathway flux (9, 13) , but its occurrence was recently suggested in potato tubers and Vicia faba cotyledons (6) . All three pathways were considered for operation in the metabolic network (Fig. 4).


Figure 4: Pathways of carbohydrate metabolism in glucose-fed maize root tips. Metabolic pathways have been identified using the labeling of intermediates described in the text. Flux names are as defined under ``Appendix.'' Flux values are given in Table III.



The glucose carbons C-3 and C-4 were found to be slightly enriched to about 1 or 2% above natural enrichment (not shown), which suggested some resynthesis of pentose phosphates from fructose phosphate and triose phosphate through the non-oxidative branch of the pathway. This would imply that not only the transaldolase but also the transketolase reaction is reversible (9) . However, the very low enrichment of these carbons indicated that this flux would be a minor one. Since the reversal of this reaction was found to have little effect on the calculation of fluxes in glycolysis and the pentose phosphate pathway (9) , it was not included in the metabolic scheme. The triose phosphates used in the resynthesis of hexose phosphates might also be formed by gluconeogenesis from organic acids. In addition to glucose C-6, gluconeogenesis would also label glucose C-2 and C-5 from the labeled C-2 of oxalacetate (see below). The absence of any significant label on glucose and sucrose C-2 and C-5 (Fig. 3) indicated the absence of gluconeogenesis.

Plastidial Hexose Phosphates Import from the Cytosol, and Cycling in the Transaldolase Reaction

Starch was studied by H and C NMR after conversion to glucose. The enrichments of starch glucosyl C-1 and C-6 were 65 and 18-19%, respectively (Fig. 5, ). If starch were formed from triose phosphates, the enrichments of the C-1 and C-6 of its glucosyl units would be similar (4) . The higher enrichment of C-1, similar to that observed in other non-green plastids (4, 5, 6) , indicated that starch was formed essentially from hexose phosphates imported into the plastids. However, in the present case, we found more randomization in the glucosyl units of starch than in glucose or the hexosyl units of sucrose. Therefore, an exchange through the transaldolase reaction in the plastid, similar to that in the cytosol, was introduced into the metabolic scheme. Recycling of triose phosphates to hexose phosphates could account for this effect but was not included because recent evidence from enzyme studies indicates that fructose-1,6-bisphosphatase activity is absent (36) or has a low activity (37) in plastids of most starch-storing tissues. The different labeling of hexosyl units in the cytosol and in the plastids suggests that there is no significant efflux of hexose phosphates from the plastids to the cytosol. Labeling with [2-C]Glucose and Localization of the Pentose Phosphate Pathway-To obtain additional evidence for the occurrence of the pentose phosphate pathway, maize root tips were labeled with [2-C]glucose. A flux through this pathway, with recycling of triose phosphates to fructose 6-phosphate, would lead to the labeling of hexose phosphate C-1, which can be detected by the analysis of glucose, sucrose, or starch (6) . Free glucose and glucose produced from starch hydrolysis were purified, and the specific radioactivity of their C-1 was determined by decarboxylation. The specific radioactivity of glucose C-1 was only 0.38 dpmnmol compared to 1.65 ± 0.01 for starch glucosyl units; these values are equivalent to C enrichments of 2.6 and 11.1%, respectively (). The labeling of C-1 of either glucose or starch glucosyl confirmed the activity of the pentose phosphate pathway, and the higher labeling in starch than glucose indicated that this pathway was located essentially in the plastids. In fact, simulations using the metabolic model (see below) indicated that the labeling of cytosolic glucose C-1 could be accounted for by the reversibility of glycolysis from triose phosphates; therefore, cytosolic pentose phosphate pathway was not included in the metabolic scheme (Fig. 4).

Given a plastid location of the pentose phosphate pathway, the cytosolic and plastidial triose phosphates have to be in rapid exchange to explain the tracer dilution in alanine and tricarboxylic acid cycle intermediates in comparison with cytosolic hexose phosphates. Thus, triose phosphates were assumed to be close to equilibrium, and only one pool was considered (Fig. 4). This is in agreement with the high activity of the triose phosphate translocator found in vitro in non-green pea root plastids (38) .

Carbon Fluxes through the Tricarboxylic Acid Cycle

In animal and bacterial cells, alanine, glutamate, and aspartate are usually considered to be in equilibrium with the -oxoacids pyruvate, -oxoglutarate, and oxalacetate, respectively (39). This appears to be true in plant cells, where the labeling of these amino acids was found to reflect that of the corresponding organic acids (10) . We considered that, in maize root tips, where transaminase activities are high (19) , the C distribution in these amino acids also reflects the distribution in the corresponding -oxoacids. shows the C enrichment of the non-carboxylic carbons of these amino acids, as determined using H NMR (Fig. 6).

The C enrichment of alanine C-3 was 34.2%, and that of alanine C-2 was 3%. Glycolysis introduces the C-1 or C-6 of glucose into alanine C-3. In the absence of a diluting flux between hexose phosphates and pyruvate, the enrichment of alanine C-3 would be equal to the average of the hexose phosphate C-1 and C-6 enrichments (), i.e. (81 + 14.5)/2 = 48%. The dilution of alanine C-3 from 48 to 34.2% further confirms the activity of the pentose phosphate pathway. Alanine C-2 could be labeled from a labeled C organic acid, oxalacetate or malate, through the malic enzyme reaction or through the PEP carboxykinase and pyruvate kinase reactions. As previously observed (10) , these two pathways cannot be distinguished from each other using the present method. However, since PEP carboxykinase activity is low in maize root tips (16) , this flux was attributed to malic enzyme. Plant mitochondria differ from animal mitochondria in containing a malic enzyme activity, which converts malate to pyruvate (40) . Since the enrichment of alanine C-2 indicates that such an activity operates in the maize root tips, it was included in the model.

The enrichment of glutamate C-4 was identical to that of alanine C-3, thus indicating the absence of any flux of unlabeled carbon between pyruvate and glutamate. The C enrichments of glutamate C-2 and glutamate C-3 () were identical to each other and close to 25.5%; this value was lower than that for glutamate C-4, indicating the entry of a diluting flux of C compounds into the tricarboxylic acid cycle (26) . In glucose-fed tissues, PEP carboxylase activity is the most likely source of anaplerotic carbon (13, 41) . This reaction provides oxalacetate molecules labeled on C-3; label can be transferred to the C-2 position by the fumarase reaction. These carbons then provide the glutamate carbons C-2 and C-3. In the present case, the identical C enrichment of glutamate C-2 to glutamate C-3 indicated that the fumarase reaction was close to equilibrium. In addition, it also indicated the absence of the form of channelling, which converts oxoglutarate C-4 to oxalacetate C-3, which would give a higher labeling of glutamate C-2 than C-3 (27) ; however, the alternative form of channelling, which would convert oxoglutarate C-4 to oxalacetate C-2, could not be excluded because, in this case, the carbons 2 and 3 of glutamate would eventually become equally labeled (42).

Determination of Fluxes

The metabolic network is shown in Fig. 4, and flux parameters are defined under ``Appendix'' and shown in I. V(G) (the rate of glucose uptake), SS (the flux to soluble sugar accumulation), St (the rate of starch accumulation), Lip (the rate of fatty acid synthesis), and V(CO) (the rate of CO evolution) were determined directly (I, Fig. 7 ).


Figure 7: Changes in soluble sugars (glucose (), sucrose (), and fructose ()) in excised maize root tips. Maize root tips were pre-starved for 4 h and, at the time indicated by an arrow, tranferred to an incubation medium containing 200 mM glucose. The data are the mean of determinations on four samples from two independent experiments.



Other fluxes of glucose metabolism were calculated using mathematical models of tracer distribution at metabolic and isotopic steady states for the metabolic network shown in Fig. 4. Equations were written according to Katz and Rognstad (9) . The flux variables and equations are presented under ``Appendix.'' The relative fluxes have been determined in three steps as described under ``Appendix'': (i) estimation of the activity of ``malic enzyme'' relative to that of PEP carboxylase (V) and determination of the enrichment of PEP C-3 (T3), (ii) estimation of the carbon fluxes into the tricarboxylic acid cycle through the PEPC and citrate synthase reactions relative to each other (V and V), and (iii) estimation of the fluxes of sugar metabolism. Flux values are given in I.

Malic Enzyme Flux

As shown in Fig. 4, pyruvate is formed either from PEP by pyruvate kinase or from malate by malic enzyme. A model limited to this set of three compounds was used to calculate both V relative to V + V and T3, as described under ``Appendix.'' The value of V = 0.08 indicated that the malic enzyme flux only provided 8% of the pyruvate entering the tricarboxylic acid cycle. The effect of the malic enzyme flux was to enrich pyruvate C-2 (P2) and dilute pyruvate C-3 (P3) relative to the triose phosphate carbons T2 and T3. The value of T2 was the natural enrichment 1.1%; that of T3 was 35.0 ± 0.6%, i.e. not very different from P3 (34.2 ± 0.5%, ). The value of V was normalized to V(G), using the value of TCA, the flux entering the tricarboxylic acid cycle (see below), giving Mal = 0.01 (I).

PEP Carboxylase and Citrate Synthase Fluxes

In the absence of the PEPC flux, the enrichment of the glutamate carbons C-2 and C-3 would be equal to that of glutamate C-4 (25) , i.e. 33.8 ± 0.6% (); the observed values, however, are 25.4 ± 0.4% (), thus indicating the operation of the PEP carboxylase reaction. This reaction produces oxalacetate whose C-3 is T3, enriched to 35 ± 0.6% (see above), and C-2 is T2 (1.1%). After randomization through the fumarase reaction, the mean enrichment of these oxalacetate carbons is 18%. This flux mixes with that from the tricarboxylic acid cycle, giving oxalacetate carbons O2 and O3 identical to the glutamate carbons C-3 and C-2. The ratio of the carbon flux through PEP carboxylase to that through citrate synthase was calculated from these values, according to Ref. 10 (see ``Appendix''), giving V/V = 1.03 ± 0.14. This value indicates that 52 ± 7% of the carbon entered the tricarboxylic acid cycle as oxalacetate and 48% as acetyl-CoA. Since one triose phosphate molecule brings four carbons when entering the tricarboxylic acid cycle through the PEPC reaction and only two carbons through the citrate synthase reaction, it was calculated that, of the triose phosphates entering the tricarboxylic acid cycle, 35 ± 5% went through the PEPC reaction and 65% through the citrate synthase reaction. The corresponding values normalized to V(G) were PEPC = 0.06 and CS = 0.12 (I).

Calculation of glutamate C-4 enrichment from the multiplets of the C spectra (Fig. 6) by the method of Malloy et al.(26) was in agreement with our direct determination from the H NMR spectrum. However, fluxes entering the tricarboxylic acid cycle could not be determined reliably because, in the range of low enrichments obtained in the present experiment, small errors in the relative size of doublets and triplets of glutamate resulted in large errors on calculated carbon fluxes (results not shown).

Glycolysis and the Pentose Phosphate Pathway

The equations describing the labeling of free glucose, cytosolic and plastidial hexose phosphates, and triose phosphates at isotopic steady state are given under ``Appendix.'' In the case of [1-C]glucose labeling, only the equations describing the hexose C-1 and C-6 and the triose phosphate C-3 were used.

The relative flux of sucrose cycling, Suc, was determined graphically from a plot of Equations 1 and 6 in which the parameters H1 and H6 were given the two extreme experimental values of the enrichment of sucrose C-1 (79 and 81%) and sucrose C-6 (13 and 14%), respectively (Fig. 8). The estimated value of Suc was 3.1 ± 0.3 times the flux of glucose entering maize root tips. The cytosolic fluxes of hexose phosphate resynthesis (TFc) and of triose phosphate exchange through the cytosolic transaldolase reaction (Tlc) were determined from Equations 7 and 12, using the Suc values determined above; they were TFc = 0.38 ± 0.03 and Tlc = 0.25 ± 0.1.


Figure 8: Graphic determination of the rate of sucrose synthesis and degradation (Suc). Graphic representation of Equation 1, Suc H1 + S1 = (1 + Suc) Gl1 (``Appendix''), shows Gl1 as a function of Suc for the two extreme values of the enrichment of cytosolic hexose phosphate C-1 (H1 = 0.79 and 0.81). Graphic representation of Equation 6, Suc H6 + S6 = (1 + Suc) Gl6, shows Gl6 as a function of Suc for the two extreme values of the enrichment of cytosolic hexose phosphate C-6 (H6 = 0.13 and 0.14). For each equation, an area was delimited to take into account the variation of Gl1 from 0.83 and 0.84 and that of Gl6 from 0.09 and 0.1. The enrichment values used here were taken from Table I and corrected for the 1.1% natural abundance. The overlap region corresponds to the interval of variation of Suc, 2.8-3.4.



The Equations 13, 18, 21, 23, 25, and 26 were then solved simultaneously to determine the relative fluxes Pl, P, A, Tlp, Pol, and Pent. The results (I) indicate that 22 ± 2% of the glucose entering the cell goes to plastids (flux Pl). These hexose phosphates are essentially metabolized through the pentose phosphate pathway (p = 0.27 ± 0.03) and converted to triose phosphates; the flux of fructose phosphate found to be recycled to glucose phosphate was A = 0.06 ± 0.02, which is 22% of the flux entering the pentose phosphate pathway. Most of the triose phosphates produced in plastids were found to be exported to the cytosol. A small part may remain in the plastid and be oxidized by the plastidial pyruvate dehydrogenase, thus contributing to fatty acid synthesis (43) . The relative glycolytic fluxes from hexose phosphates to triose phosphates, FT, and from triose phosphates to PEP, Glyco, were estimated to be 0.42 and 0.21, respectively. The plastidial transaldolase flux was calculated with a large error (Tlp = 0.04 ± 0.04). Absolute values of fluxes (I) were calculated using the relative values and the rate of glucose influx into maize root tips (V(G) = 215 nmolhtip).

Validation of the Model

To validate the model, the general equations given in Annex were used to calculate the expected C-1 enrichments of free glucose and the plastidial hexose phosphates after labeling with [2-C]glucose, using the flux values of I and C-2 enrichment equal to 98% above natural abundance. The calculated enrichments of free glucose C-1 and starch glucosyl C-1 were Gl1 = 2.5 ± 0.5% and G1 = 15.3 ± 2.5%, respectively, to be compared with the experimental values 2.6 ± 0.2% and 11.1 ± 0.1% (). Since G1 is very sensitive to the flux values A and Pl, which were determined with large relative errors, we conclude that the model correctly accounts for the experimental data.

The fluxes Pent and Pol correspond to the synthesis of polysaccharides, which are either incorporated into cell walls or secreted by the root tip. The sum of the calculated fluxes, 121 nmol hexosehtip, would be equivalent to a weight increase of 19 µghtip. The residue of the ethanol/water extraction includes cellulose, which represents 47% of the structural carbohydrate in young maize plants (44) ; the increase in the dry weight of this residue was found to be 8 µghtip. The values of Pent and Pol calculated by the model are therefore consistent with this experimental value. The PEPC flux of 13 nmolhtip would provide carbon for the synthesis of 26 nmolhtip of amino acids of the aspartate and glutamate families; other amino acids do not require this pathway. The measured increase in total protein, 6 ± 2 µghtip, corresponds to about 60 ± 20 nmolhtip of amino acids, which also is in reasonable agreement with the estimation of PEPC. The relative net rate of CO evolution calculated from Equation 25 and the mean value of fluxes shown in I is 0.182, which is close to the experimental value, V(CO) = 0.18.


DISCUSSION

Labeling to isotopic steady state is a classical method for identifying metabolic pathways and quantifying metabolic fluxes (25) . The present work relied mainly on labeling with [1-C]glucose and H and C NMR analysis of extracts for determination of C enrichments. Additional results were obtained using [C]glucose labeled on specific carbons. The metabolism of glucose studied in the present work is likely to reflect the metabolism of carbohydrates in this organ in vivo for the following reasons. (i) The concentration of glucose used here (200 mM) appears to be close to that seen by the root tip cells since it is intermediate between the sugar concentration in the phloem, about 0.5 M(34) , and that in the root tip cells, about 80 mM(17, 18) ; moreover, it has been shown that this glucose concentration is necessary to sustain the normal respiration rate of the root tips (17) and to avoid the increase in proteolytic activities, which results from limited sugar supply (22) . (ii) Glucose is the major carbohydrate in root tip cells (17, 18) . (iii) Although the carbon starvation pretreatment used here may appear artificial, it probably mimics a situation that commonly occurs in normal plant life (Refs. 18 and 20 and references therein). (iv) Finally, under the conditions used, the excised root tips continued growing at a high rate.

The major pathways of carbohydrate metabolism in maize root tips were described quantitatively from the measurement of enrichment of specific carbons in carbohydrates and in the amino acids glutamate and alanine. No fit could be obtained with the simplest models, and a relatively complicated metabolic scheme (Fig. 4) was necessary to account for the labeling data. The pathways described include sucrose cycling, different steps of glycolysis, the pentose phosphate pathway, located in the plastids, and the entries into the tricarboxylic acid cycle. The validity of the model was confirmed by independent experiments, including transfer of glucose C-2 to the C-1 position, and measurement of the increases in insoluble polysaccharides and total proteins, which were in agreement with the calculated fluxes, thus suggesting that the model adequately describes the metabolism of the root tip. This may be surprising in view of the morphologic heterogeneity of the root tip. Conversely, this may indicate that the metabolic pathways here described occur in most of the dividing and elongating cells of the root tip. Differences between cells may occur in pathways not studied here, or else cells with a significantly different metabolism may be insufficiently numerous to disturb the results.

It was surprising that no difference was found in the production of labeled CO from [1-C] or [6-C]glucose (Ref. 11 and this study) despite the high activity of the pentose phosphate pathway found in the present work. It has been pointed out that pentan synthesis might affect the interpretation of CO labeling data (13) , but the flux in this pathway had not been determined before. Since a high activity of pentan synthesis would be expected in a growing tissue for cell wall formation, it was included in the model to balance the production of labeled CO by the pentose phosphate pathway. This non-triose pathway, flux Pent, was found to consume 19% of the glucose entering the root tip cells.

More generally, the non-triose pathways, which include both the formation of polysaccharides represented by the sum Pol + Pent (I), and the accumulation of soluble carbohydrates consumed as much as 74% of the glucose entering the root tips. On the other hand, the synthesis of starch and lipids represented minor fluxes. The low contribution of starch to the metabolism of the root tip is striking compared with most other plant tissues.

The presence of a pentose phosphate pathway was deduced from the dilution of the triose phosphate enrichment reflected in alanine and other amino acids, given that the triose phosphate isomerase was shown to be close to equilibrium. Its presence was confirmed, and its plastid location was established, by the transfer of label from C-2 of [2-C]glucose to the C-1 position of starch glucosyl. Indeed, a simulation of the latter experiment indicated that the cytosolic resynthesis of hexose phosphates from triose phosphates (TFc) was sufficient to account for the labeling of cytosolic glucose C-1 from [2-C]glucose, thus leaving no evidence for any significant activity of the oxidative pentose phosphate pathway in the cytosol. The oxidative pentose phosphate pathway (P) metabolized 27% of the glucose entering the maize root tips, and 38% of the hexose phosphates metabolized in triose phosphate pathways. A similarly high activity of the oxidative pentose phosphate pathway was found in cells of Chenopodium rubrum(5) . In isolated pea chloroplasts, the activities of the two dehydrogenases of the pentose phosphate pathway were found to be in excess of the flux of glucose consumption by respiration (48) .

To account for the low enrichment of triose phosphates and the high enrichment of starch glucosyl units, we had to consider that only some of the fructose phosphates produced by the oxidative pentose phosphate pathway were recycled. This flux, A = 0.06 ± 0.02, was 22% of the flux entering the pentose phosphate pathway instead of 66% in the case of complete recycling shown in usual models (5, 7) . This metabolic feature implies that two distinct fructose phosphate pools are present inside the plastids (Fig. 4).

There is overwhelming evidence that in addition to a complete glycolytic pathway in the cytosol, higher plant cells also contain most, if not all, of the enzymes of the glycolytic pathway in the plastids. In all the tissues examined so far, plastids contain the enzymes necessary for the conversion of hexose phosphates to triose phosphates (38, 46, 47) . The reason why only one glycolytic pathway was shown in the metabolic scheme (Fig. 4) is that it was not possible to decide, from the experimental data, how much of the total glycolytic flux was located in the cytosol and how much in the plastid. The different possibilities examined (not shown) induced modifications on the fluxes of transaldolase (Tlc and Tlp) and of the cytosolic triose phosphate to hexose phosphate conversion (TFc) but were found to have little effect on the other fluxes.

There is ample evidence that glucose 6-phosphate can be transported into non-green plastids (38, 43) . Studies of the route of starch synthesis using either enzyme activities (36, 38) or label distribution (4, 5) indicate that starch glucosyl units arise from glucose phosphate imported from the cytosol. Our results essentially confirm this view. However, the high redistribution found here between the C-1 and C-6 positions in starch glucosyl units compared with sucrose was not found in other materials. We attributed this difference to a plastidial transaldolase activity because relatively high transaldolase and transketolase activities have been found in amyloplasts of sycamore cells (46) and plastids of pea roots (49) . The alternative hypothesis, resynthesis of hexose phosphates from triose phosphates, appears less likely because of the absence, or low activity, of fructose-1,6-bisphosphatase in most non-green plastids (36, 37) .

The glycolytic flux of hexose phosphate to triose phosphates, FT = 0.42, was 1.6 times the flux into the pentose phosphate pathway (P). The unidirectional flux of triose phosphate production, not including transaldolase activities, was 0.63, of which 68% was from glycolysis and 32% from the pentose phosphate pathway. 60% of the triose phosphates were found to be recycled to hexose phosphates (TFc). Because of transaldolase activity, triose phosphate recycling may have been overestimated when calculated from randomization of C-1 to C-6 (5, 7) . Here, the use of enrichments allowed this reaction to be distinguished from transaldolase exchange, resulting in a more accurate value. 28% of the triose phosphates were found to go to the tricarboxylic acid cycle (TCA), and the remainder was recycled in the pentose phosphate pathway or used for the synthesis of fatty acid (Lip).

The carbon flux to the tricarboxylic acid cycle was found to be divided at the PEP branch point, with 33% going to PEP carboxylase and 67% to the pyruvate kinase reaction. Only 8% of pyruvate was found to derive from C compounds, thus suggesting that most of the PEP carboxylase flux was used for biosynthesis from tricarboxylic acid cycle intermediates, presumably the biosynthesis of amino acids of the aspartate and glutamate families. In another quantitative study of the malic enzyme flux (10) , it was also found that this flux was a minor one. It might become higher when C compounds, such as malate or asparagine, are being used as respiratory substrates (19) .

The similar enrichment of alanine C-3 and glutamate C-4 () is consistent with the view that glycolysis is the sole source of acetyl-CoA for the tricarboxylic acid cycle in glucose-fed tissues. However, since lipids and proteins were found to be labeled during the long incubation time needed to reach isotopic steady state (result not shown), this result is not a proof that the turnover of fatty acids and amino acids provides negligible amounts of acetyl-CoA to the tricarboxylic acid cycle.

The contribution of the different pathways to the total flux of CO was calculated from Equation 25 (``Appendix'') and flux values from I. The tricarboxylic acid cycle, including the pyruvate dehydrogenase reaction, contributed 53%, the pentose phosphate pathway 24%, and pentan synthesis 17% of total CO evolved. Thus, the estimation of the rate of glycolysis from the rate of CO evolution would overestimate the pyruvate kinase flux by about 50% (compare V(CO) = 0.18 and CS = 0.12) but would underestimate the fructose phosphate to triose phosphate flux by a factor of 2.3 (compare V(CO) with FT = 0.42 in I).

The major flux determined in the intermediate metabolism of the maize root tips was that of synthesis and degradation of sucrose. This futile cycle has been described recently in a number of plant tissues (2, 3, 7, 35) but does not appear to occur in the more mature portion of the maize root (45) . The cost of synthesis and degradation of one sucrose molecule is 2 ATP, as far as pyrophosphate is used as ATP equivalent (7). Therefore, 3.1 mol of ATP are consumed in sucrose cycling for each hexose entering the tissues. The relative flux of ATP regeneration was calculated from the carbon flux through glycolysis (Glyco = 0.21 produces 0.42 ATP and 0.42 NADH) and the different steps of pyruvate oxidation in the mitochondria (with CS = 0.12, PEPC = 0.06, and the maximum value ATP/O = 3). Assuming that the oxidation of cytosolic NADH gives 3 ATP after production of malate, which is then oxidized in the mitochondria, mitochondrial respiration was calculated to produce a maximum of 4.5 ATP per hexose molecule entering the root tip. Thus, the turnover of sucrose consumes 69% of the ATP produced by mitochondrial respiration. An even higher figure was obtained for banana tissue (7) , which is consistent with the absence of biosynthetic activities linked with growth in that material. This futile cycle could be involved in the regulation of the concentrations of sucrose and hexose, which determine the water status of these cells.

The present work describes intermediate metabolism in growing higher plant tissues fed with non-limiting amounts of carbohydrate. However, there is increasing evidence that the supply of carbohydrates fluctuates and controls the activity of plant cells. Some of the metabolic effects of sugar starvation have been described (17, 18, 19, 20, 21, 22, 23, 24) , and it has been established that the expression of some genes is under metabolic control by sugars or their sugar metabolites (50) . The present study provides the basis for a study of the metabolic response of plant cells to varying carbon availability or other stresses.


APPENDIX

Malic Enzyme Flux and PEP C-3 Enrichment

Pyruvate is formed either from PEP by pyruvate kinase (flux V) or from malate by malic enzyme (flux V) and is degraded to acetyl-CoA by pyruvate dehydrogenase; pyruvate outflow to alanine for protein synthesis has been neglected. A sub-model limited to this set of reactions was used. The sum of pyruvate formation fluxes was normalized to 1 so that at metabolic steady state, V + V = 1. T2 and T3, O2 and O3, and P2 and P3 represent the enrichments of carbons 2 and 3 of PEP, oxalacetate, and pyruvate (or alanine), respectively. At isotopic steady state, V.T2 + V.O2 = P2 and V.T3 + V.O3 = P3.

Specific enrichments were deduced from the data in . PEP C-2 (T2), which derives from C-2 or C-5 of the hexose phosphate, was not labeled and was assumed to have the natural enrichment, i.e. T2 = 1.1%. Malate C-2 and C-3 are identical to the C-3 and C-2 of glutamate, respectively, i.e. O2 = O3 = 25.4 ± 0.4%. P2 and P3 are 3 and 34.2 ± 0.5%, respectively. Solving the three equations with these values gave V = 0.08 and T3 = 35 ± 0.6%. V was then normalized to V(G) to give Mal = 0.01 as described in the text (I).

PEPC and CS Reactions

The relative fluxes through the PEPC (PEPC) and the citrate synthase (CS) reactions were determined from the corresponding values V and V, calculated using the model of Salon et al.(10) , which uses specific enrichments to estimate the carbons fluxes entering the tricarboxylic acid cycle. The flux parameters were set as follows. (i) Since no isocitrate lyase activity (20) and no gluconeogenesis (this article) were detected, carbon input from the glyoxylic cycle and output to gluconeogenesis were set to 0. (ii) The only source of acetyl-CoA was pyruvate (i.e. no contribution from fatty acids). (iii) In the absence of an experimental value, we assumed that the distribution of the anaplerotic oxalacetate flux resulting from the PEPC activity was divided equally to glutamate- and aspartate-derived amino acids; this corresponds to carbon fluxes of 45% to aspartate and 55% to glutamate. (iv) To account for the complete randomization of oxalacetate C-2 and C-3, the apparent fumarase rate was set to 5 (see Ref. 10). The enrichments of PEP carbons were T1 = 1.1% and, as determined above, T2 = 1.1% and T3 = 35 ± 0.6%; the enrichments of oxalacetate carbons C-2 and C-3 were 25.3 ± 0.4%, determined from the values of glutamate C-3 and C-2 ().

Fluxes of Glucose Metabolism

The flux parameters, in hexose equivalents, are as follows. For cytosol: 1) V(G) (flux of glucose inflow into the maize root tips, normalized to 1 for calculation of relative fluxes, i.e.V(G) = 1); 2) Suc (flux of synthesis and degradation of sucrose via sucrose phosphate synthase and invertase); 3) SS (accumulation of soluble carbohydrates in the maize root tip); 4) Pent (synthesis of pentans with CO production from glucose C-6); 5) Pol (hexose phosphate output for polysaccharide synthesis); 6) TFc (resynthesis of hexose phosphates from triose phosphates); 7) FT (glycolytic degradation of hexose phosphates to triose phosphates in the cytosol); 8) Tlc (exchange of the C-4 to C-6 moiety of cytosolic fructose phosphate and free triose phosphates through the transaldolase reaction); 9) Pl (hexose phosphate transport from cytosol to plastid).

For plastids: 10) St (synthesis of starch); 11) P (flux of hexose phosphates into the pentose phosphate pathway). The model indicates that the triose phosphates used by aldolase (flux P/6) are taken from the triose phosphate pool and can thus incorporate label in hexose phosphate C-6, in agreement with the proposal by Viola et al.(6) (see also Ref. 25). 12) A (flux of the fructose phosphates produced from pentose phosphates, back to the glycolytic hexose phosphate pool). The model has to account for the loss of enrichments from the cellular hexose phosphates, for the different redistribution of label in the hexose units, and for the enrichment of the starch glucosyl C-1 after incubation with [2-C]glucose (C-1 = 11.1%). Flux A had to be included because no fit of the experimental data was found when the model assumed that three pentose phosphates were either recycled to two fructose 6-phosphates and 1 triose-phosphate (as in most models) or all degraded to triose phosphates. In the first case, the flux of triose phosphates to PEP and the tricarboxylic acid cycle was estimated to be 0; this hypothesis was rejected because it does not account for the labeling of the amino acids aspartate and glutamate. In the second case, the simulation of a [2-C]glucose labeling resulted in an enrichment of the plastidial hexose phosphate C-1 of only 4.5%, whereas the experimental value is equivalent to 11.1% (). Flux A derives from a particular pool of fructose phosphate originating from the pentose phosphate pathway, which is not in equilibrium with the glycolytic fructose 6-phosphate. The flux of hexose phosphates formed in the pentose phosphate pathway and immediately degraded to triose phosphates is 2P/3-A. 13) Tlp (exchange of the C-4 to C-6 moiety of plastidial fructose phosphate with free triose phosphate through the transaldolase reaction); 14) Glyco (total glycolytic flux of triose phosphates to both tricarboxylic acid cycle and fatty acid synthesis); 15) TCA (flux of PEP into the tricarboxylic acid cycle by both the PEP carboxylase and the pyruvate kinase reactions); 16) Lip (flux of PEP to fatty acid biosynthesis).

The enrichment of labeled carbons in the different compounds are designated by 1 or 2 letters for the compound and a number, which indicates the carbon position in the molecule: S designates external glucose; Gl designates intracellular free glucose; H, cytosolic hexose phosphate; G, starch hexosyl units and plastidial hexose phosphates; T, triose phosphates; P, pyruvate and alanine; O, oxalacetate. Glucose influx into root tips was 1.

Equations of Hexose Metabolism

Isotopic steady state equations for free glucose are as follows.

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

Equations for cytosolic hexose phosphates are as follows.

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

Equations for plastidial hexose phosphates are as follows.

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

Equations for triose phosphates are as follows.

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

Metabolic steady state equations are as follows.

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

TCA = Glyco - Lip. This equation applies to the PEPC/CS ratio and the distribution of the PEPC carbon flux to amino acid synthesis described above.

The rates of CO evolution from [1-C]glucose and [6-C]glucose are equal; then, from Equation 25,

On-line formulae not verified for accuracy

where the symbols defined above are used for carbons labeled from [1-C]glucose, with a (`) added for carbons labeled from [6-C]glucose; V and V are the fluxes of citrate synthase and PEP carboxylase normalized to TCA = 1 as in Ref. 10. TCA is the glycolytic carbon input into the tricarboxylic acid cycle. Equation 26 was formulated as carbon enrichment rather than C-specific activities. After labeling with [1-C]glucose, enrichment values of cytosolic hexose phosphate C-6 (H6) and plastidial hexosyl unit C-1 (G1) were taken from , and that of triose phosphate C-3 (T3) was calculated from P3 and V as described above. After labeling with [6-C]glucose, the enrichments of supplied glucose C-1 and C-6 were S`1 = 0, S`6 = 0.98; that of triose phosphate C-3 was T`3 = 0.49, which assumes negligible dilution by the pentose phosphate pathway. The enrichments of cytosolic hexose phosphates were calculated from Equations 1, 6, 7, and 12, which gave H`6 = 0.76 and H`1 = 0.12; since the ratios G1/H1 and G`1/H`1 must be equal, then G`1 = 0.1. The values of O4 and O`4 depend on the enrichment of P3 and of the CO fixed by PEP carboxylase; the enrichment of CO was not determined but was found to be identical with both [1-C] and [6-C]glucose. Given this, the model of Ref. 10 shows that, for CO enrichments varying between 0.05 and 0.2, the difference O4-O`4 remains in the range -0.036 ± 0.002; this mean value was used.

  
Table: Steady state C enrichments of carbohydrate and amino acid carbons after incubation of maize root tips with [1-C]glucose

The enrichments (in %) were determined from H and C spectra as described under ``Experimental Procedures.'' Results are given as mean ± S.D. (n = 3).


  
Table: Steady state-specific radioactivities of soluble carbohydrates (glucose, sucrose, and starch) and of C-1 of glucose and starch after incubation of maize root tips with [2-C]glucose

Maize root tips were incubated for 12 h with [2-C]glucose (16 ± 0.2 dpmnmol); the specific radioactivities of glucose, sucrose, and of starch glucosyl were determined as described under ``Experimental Procedures.'' Specific radioactivities of C-1 of free glucose and of starch glucosyl were determined by enzymatic decarboxylation and counting the radioactivity incorporated in CO. Equivalent enrichment of C-1 was calculated from the equation: E(%) = specific radioactivity of C-1 * 98/16 + 1.1 where 98 is the enrichment (%) of labeled glucose above natural abundance in an experiment where maize root tips would be incubated with [2-C]glucose (99% isotopic enrichment), and 16 (dpmnmol) is the actual specific radioactivity of the labeled substrate. Results are the mean (±S.D.) of three determinations from two independent experiments. ND, not determined.


  
Table: Relative and absolute values of metabolic fluxes in maize root tips incubated with 200 mM glucose

Relative fluxes were calculated by normalizing rates in hexose units to the rate of glucose influx (215 nmolhtip). Absolute fluxes are given in nmol hexose equivalenthtip. Some fluxes were determined directly. The rate of glucose uptake V(G) was 215 nmolhtip, and was normalized to 1, i.e. V(G) = 1. The rate soluble sugar accumulation, SS, was determined from the change in glucose and fructose levels, which were increasing linearly around 12 h at a rate of 39 nmolhtip, whereas sucrose had reached a plateau after 5 h (Fig. 7). The mean rate of starch accumulation over 12 h, St, was only 3 nmol of hexose equivalenthtip. The accumulation of fatty acids, Lip, was estimated from the increase in dry weight (0.038 mghtip), assuming that the lipid content per mass unit (18) remained constant; Lip was 7 nmol of hexose equivalenthtip. The rate of CO evolution, V(CO ), was 230 nmolhtip (18). Flux values calculated from the model based upon carbon enrichment studies are shown as the mean and range of separate simulations using extreme values of enrichments (see Suc determination in Fig. 8).



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Farmacologie, Campus Gasthulsberg, Katholleke Universiteit Leuven, Herestraat 49, B-3000 Leuven, Belgium.

To whom correspondence should be addressed. Tel.: 33-56-84-32-47; Fax: 33-56-84-32-45.

The abbreviations used are: PEP, phosphoenolpyruvate; TCA, the flux entering the tricarboxylic acid cycle; HPLC, high pressure liquid chromatography; PEPC, PEP carboxylase; CS, citrate synthase.


ACKNOWLEDGEMENTS

We thank Dr Bryan Collis (School of Biological Sciences, Bangor, UK) and Ann Collis for improving the English of the manuscript and Dr Blanc (Université de Bordeaux II, UFR MISS) for the software DERIVE.


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