Computational tools for isotopically instationary 13C labeling experiments under metabolic steady state conditions

(13)C metabolic flux analysis (MFA) has become an important and powerful tool for the quantitative analysis of metabolic networks in the framework of metabolic engineering. Isotopically instationary (13)C MFA under metabolic stationary conditions is a promising refinement of classical stationary MFA. It accounts for the experimental requirements of non-steady-state cultures as well as for the shortening of the experimental duration. This contribution extends all computational methods developed for classical stationary (13)C MFA to the instationary situation by using high-performance computing methods. The developed tools allow for the simulation of instationary carbon labeling experiments (CLEs), sensitivity calculation with respect to unknown parameters, fitting of the model to the measured data, statistical identifiability analysis and an optimal experimental design facility. To explore the potential of the new approach all these tools are applied to the central metabolism of Escherichia coli. The achieved results are compared to the outcome of the stationary counterpart, especially focusing on statistical properties. This demonstrates the specific strengths of the instationary method. A new ranking method is proposed making both an a priori and an a posteriori design of the sampling times available. It will be shown that although still not all fluxes are identifiable, the quality of flux estimates can be strongly improved in the instationary case. Moreover, statements about the size of some immeasurable pool sizes can be made.     

The 13C Chemical-Shift Index: A simple method for the identification of protein secondary structure using 13C chemical-shift data

Summary A simple technique for identifying protein secondary structures through the analysis of backbone ¹³C chemical shifts is described. It is based on the Chemical-Shift Index [Wishart et al. (1992) Biochemistry, 31, 1647–1651] which was originally developed for the analysis of ¹H chemical shifts. By extending the Chemical-Shift Index to include ¹³C, ¹³C and carbonyl ¹³C chemical shifts, it is now possible to use four independent chemical-shift measurements to identify and locate protein secondary structures. It is shown that by combining both ¹H and ¹³C chemical-shift indices to produce a consensus estimate of secondary structure, it is possible to achieve a predictive accuracy in excess of 92%. This suggests that the secondary structure of peptides and proteins can be accurately obtained from ¹H and ¹³C chemical shifts, without recourse to NOE measurements.Supplementary material is available in the form of a 10-page table (Table S1) describing the exact location of secondary structures in all 20 proteins as determined using the methods described in this paper. Requests for Table S1 should be directed to the authors.     

Contribution of flexible allocation priorities to herbivory tolerance in C4 perennial grasses: an evaluation with 13C labeling

The ability of plants to rapidly replace photosynthetic tissues following defoliation represents a resistance strategy referred to as herbivory tolerance. Rapid reprioritization of carbon allocation to regrowing shoots at the expense of roots following defoliation is a widely documented tolerance mechanism. An experiment was conducted in a controlled environment to test the hypothesis that herbivory-sensitive perennial grasses display less flexibility in reprioritizing carbon allocation in response to defoliation than do grasses possessing greater herbivory tolerance. An equivalent proportion of shoot biomass (60% dry weight) was removed from two C₄ perennial grasses recognized as herbivory-sensitive, Andropogon gerardii and Schizachyrium scoparium, and two C₄ perennial grasses recognized as herbivory-tolerant, Aristida purpurea and Bouteloua rigidiseta. Both defoliated and undefoliated plants were exposed to ¹³CO₂ for 30 min, five plants per species were harvested at 6, 72 and 168 h following labeling, and biomass was analyzed by isotope ratio mass spectrometry. The tallgrass, A. geraiddii, exhibited inflexible allocation priorities while the shortgrass, B. rigidiseta, exhibited flexible allocation priorities in response to defoliation which corresponded with their initial designations as herbivory-sensitive and herbivory-tolerant species, respectively. A. gerardii had the greatest percentage and concentration of ¹³C within roots and lowest percentage of ¹³C within regrowth of the four species evaluated. In contrast, B. rigidiseta had a greater percentage of ¹³C within regrowth than did A. gerardii, the greatest percentage of ¹³C within new leaves of defoliated plants, and the lowest concentration of ¹³C within roots follwing defoliation. Although both midgrasses, S. scoparium and A. purpurea, demonstrated flexible allocation priorities in response to defoliation, they were counter to those stated in the initial hypothesis. The concentration of ¹³C within new leaves of S. scoparium increased in response to a single defoliation while the percentage and concentration of ¹³C within roots was reduced. A. purpurea was the only species in which the percentate of ¹³C within new leaves decreased while the percentage of ¹³C within roots increased following defoliation. The most plausible alternative hypothesis to explain the inconsistency between the demonstrated responsiveness of allocation priorities to defoliation and the recognized herbivory resistance of S. scoparium and A. purpurea is that the relative ability of these species to avoid herbivory may make an equal or greater contribution to their overall herbivory resistance than does herbivory tolerance. Selective herbivory may contribute to S. scoparium's designation as a herbivorysensitive species even though it possesses flexible allocation priorities in response to defoliation. Alternatively, the recognized herbivory resistance of A. purpurea may be a consequence of infrequent and/or lenient herbivory associated with the expression of avoidance mechanisms, rather than the expression of tolerance mechanisms. A greater understanding of the relative contribution of tolerance and avoidance strategies of herbivory resistance are required to accurately interpret how herbivory influences plant function, competitive interactions, and species abundance in grazed communities.     

Biosynthetically directed fractional 13C labeling facilitates identification of Phe and Tyr aromatic signals in proteins

Analysis of 2D [¹³C,¹H]-HSQC spectra of biosynthetic fractionally ¹³C labeled proteins is a reliable, straightforward means to obtain stereospecific assignments of Val and Leu methyl sites in proteins. Herein we show that the same fractionally labeled protein sample facilitates observation and identification of Phe and Tyr aromatic signals. This is the case, in part, because the fractional ¹³C labeling yields aromatic rings in which some of the ¹³C-¹³C J-couplings, present in uniformly labeled samples, are absent. Also, the number of homonuclear J-coupling partners differs for the -, - and -carbons. This enabled us to vary their signal intensities in distinctly different ways by appropriately setting the ¹³C constant-time period in 2D [¹³C,¹H]-HSQC spectra. We illustrate the application of this approach to an 18 kDa protein, c-VIAF, a modulator of apoptosis. In addition, we show that cancellation of the aromatic ¹³C CSA and ¹³C-¹H dipolar interactions can be fruitfully utilized in the case of the fractionally labeled sample to obtain high resolution ¹³C constant-time spectra with good sensitivity.     

Analysis of 13C labeling enrichment in microbial culture applying metabolic tracer experiments using gas chromatography-combustion-isotope ratio mass spectrometry

The applicability of gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) for the quantification of ¹³C enrichment of proteinogenic amino acids in metabolic tracer experiments was evaluated. Measurement of the ¹³C enrichment of proteinogenic amino acids from cell hydrolyzates of Corynebacterium glutamicum growing on different mixtures containing between 0.5 and 10% [1-¹³C]glucose shows the significance of kinetic isotope effects in metabolic flux studies at low degree of labeling. We developed a method to calculate the ¹³C enrichment. The approach to correct for these effects in metabolic flux studies using δ¹³C measurement by GC–C–IRMS uses two parallel experiments applying substrate with natural abundance and ¹³C-enriched tracer substrate, respectively. The fractional enrichment obtained in natural substrate is subtracted from that of the enriched one. Tracer studies with C. glutamicum resulted in a statistically identical relative fractional enrichment of ¹³C in proteinogenic amino acids over the whole range of applied concentrations of [1-¹³C]glucose. The current findings indicate a great potential of GC–C–IRMS for labeling quantification in ¹³C metabolic flux analysis with low labeling degree of tracer substrate directly in larger scale bioreactors.     

Classification of amino acid spin systems using PFG HCC(CO)NH-TOCSY with constant-time aliphatic 13C frequency labeling

Summary We have developed a useful strategy for identifying amino acid spin systems and side-chain carbon resonance assignments in small ¹⁵N-, ¹³C-enriched proteins. Multidimensional constant-time pulsed field gradient (PFG) HCC(CO)NH-TOCSY experiments provide side-chain resonance frequency information and establish connectivities between sequential amino acid spin systems. In PFG HCC(CO)NH-TOCSY experiments recorded with a properly tuned constant-time period for frequency labeling of aliphatic ¹³C resonances, phases of cross peaks provide information that is useful for identifying spin system types. When combined with ¹³C chemical shift information, these patterns allow identification of the following spin system types: Gly, Ala, Thr, Val, Leu, Ile, Lys, Arg, Pro, long-type (i.e., Gln, Glu and Met), Ser, and AMX-type (i.e., Asp, Asn, Cys, His, Phe, Trp and Tyr).     

Improved labeling strategy for 13C relaxation measurements of methyl groups in proteins

Selective incorporation of ¹³C into the methyl groupsof protein side chains is described as a means for simplifying themeasurement and interpretation of ¹³C relaxation parameters.High incorporation (>90%) is accomplished by using pyruvate(3-¹³C, 99%) as the sole carbon source in the growthmedia for protein overexpression in E. coli. This improved labeling schemeincreases the sensitivity of the relaxation experiments by approximatelyfivefold when compared to randomly fractionally ¹³C-labeledprotein, allowing high-quality measurements on relatively dilute (<1 mM)protein samples at a relatively low cost.     

A13C NMR study of the hinge region of a mouse monoclonal antibody

Summary A¹³C NMR study is reported of the hinge region of an intact mouse monoclonal antibody with a molecular weight of 150 K. Cys, Ile, and Pro analogs of the antibody labeled with¹³C at the carbonyl carbon were prepared by growing hybridoma cells in the serum-free media. Resonance assignments have been performed as described previously [Kato, K., Matsunaga, C., Igarashi, T., Kim, H., Odaka, A., Shimada, I. and Arata, Y. (1991)Biochemistry,30, 270–278]. The spectral data obtained show that¹³C NMR can give detailed information about the structure of the hinge region of the intact antibody molecule. Prospects for the future role of¹³C NMR in the structural analyses of larger proteins are briefly discussed.Dedicated to the memory of Professor V.F. Bystrov     

Synthesis of methyl 4'-O-methyl-13C12-beta-D-cellobioside from 13C6-D-glucose. Part 1: reaction optimization and synthesis

A high yielding synthetic route for methyl 4'-O-methyl-beta-D-cellobioside starting from d-glucose was established. The reaction conditions optimized with nonlabeled materials were used for the synthesis of methyl 4'-O-methyl-13C12-beta-D-cellobioside, a compound having more than 99% 13C enrichment at each of the twelve pyranose carbon atoms. The labeled compound is required to study the hydrogen bond network of cellodextrins and cellulose by CPMAS NMR experiments.     

Efficient 13C/15N double labeling of the avirulence protein AVR4 in a methanol-utilizing strain (Mut+) of Pichia pastoris

Cost effective ¹³C/¹⁵N-isotope labeling of the avirulence protein AVR4 (10 kDa) of the fungal tomato pathogen Cladosporium fulvum was achieved with the methylotrophic yeast Pichia pastoris in a fermentor. The ¹³C/¹⁵N-labeled AVR4 protein accumulated to 30 mg/L within 48 h in an initial fermentation volume of only 300 mL, while prolonged optimized overexpressions yielded 126 mg/L. These protein yields were 24-fold higher in a fermentor than in flask cultures. In order to achieve these protein expression levels, we used the methanol-utilizing strain (Mut⁺) of Pichia pastoris which has a high growth rate while growing on methanol as the only carbon source. In contrast, the methanol-sensitive strain (Mut^S) could intrinsically yield comparable protein expression levels, but at the expense of additional carbon sources. Although both strains are generally used for heterologous protein expression, we show that the costs for ¹³C-isotope labeling can be substantially reduced using the Mut⁺ strain compared to the Mut^S strain, as no ¹³C₃-glycerol is required during the methanol-induction phase. Finally, nitrogen limitations were precluded for ¹⁵N-labeling by an optimal supply of 10 g/L (¹⁵NH₄)₂SO₄ every 24 h.     

Respiration from C3 plant green manure added to a C4 plant carbon dominated soil

Application of tree leaves (C₃ plants) on maize (Zea mays L.) (C₄ plant) fields is an agroforestry management technology to restore or maintain soil fertility. The rate at which the tree leaves decompose is crucial for the nutrient supply to the crop. We studied the in situ decomposition of Sesbania sesban (L.) Merr. leaves or C₃ sugar for 4 – 8 days after application to a maize field in Kenya. By using the difference of around 10‰ in natural abundance of ¹³C between the endogenous soil C (mainly C₄) and the applied C (C₃), we could calculate the contributions of the two C sources to soil respiration. The δ¹³C value of the basal respiration was from –15.9 to –16.7‰. The microbial response to the additions of leaves and sugar to this tropical soil was immediate. Application of sesbania leaves gave an initial peak in respiration rates that lasted from one to less than 6 days, after which it levelled off and remained about 2 – 3 times higher (230–270 mg C m^-2 h^-1) than the control respiration rates throughout the rest of the experiment (5 – 8 days). In the sugar treatment, there was no initial peak in respiration rate. The respiration rate was 170 mg C m^-2 h^-1 after 4 days. At the end of the experiments, after 4–8 days, as much as 14–17% of the added C had been respired and about 60% of the total respiration was from the added sesbania leaves or C₃ sugar. This non-destructive method allows repeated measurements of the actual rate of C mineralisation and facilitates decomposition studies with high temporal resolution in the field. This revised version was published online in August 2006 with corrections to the Cover Date.     

Interspecific variation in bulk tissue, fatty acid and monosaccharide delta(13)C values of leaves from a mesotrophic grassland plant community

The leaves of 37 grass, herb, shrub and tree species were collected from a mesotrophic grassland to assess natural variability in bulk, fatty acid and monosaccharide delta(13)C values of leaves from one plant community. The leaf tissue mean bulk delta(13)C value was -29.3 per thousand. No significant differences between tissue bulk delta(13)C values with life form were determined (P=0.40). On average, C(16:0), C(18:2) and C(18:3) constituted 89% of leaf tissue total fatty acids, whose delta(13)C values were depleted compared to whole leaf tissues. A general interspecific (between different species) trend for fatty acids delta(13)C values was observed, i.e. delta(13)C(16:0)delta(13)C(xylose)>delta(13)C(glucose)>delta(13)C(galactose), was consistently observed. Therefore, we have shown (i) diversity in compound-specific delta(13)C values contributing to leaf bulk delta(13)C values; (ii) interspecific variability between bulk and compound-specific delta(13)C values of leaves of individual grassland species, and (iii) trends between individual fatty acid and monosaccharide delta(13)C values common to leaves of all species within one plant community.     

Measurement of rhizosphere respiration and organic matter decomposition using natural 13C

Due to the limitations in methodology it has been a difficult task to measure rhizosphere respiration and original soil carbon decomposition under the influence of living roots. ¹⁴C-labeling has been widely used for this purpose in spite of numerous problems associated with the labeling method. In this paper, a natural ¹³C method was used to measure rhizosphere respiration and original soil carbon decomposition in a short-term growth chamber experiment. The main objective of the experiment was to validate a key assumption of this method: the ¹³C value of the roots represents the ¹³C value of the rhizosphere respired CO₂. Results from plants grown in inoculated carbon-free medium indicated that this assumption was valid. This natural ¹³C method was demonstrated to be advantageous for studying rhizosphere respiration and the effects of living roots on original soil carbon decomposition.     

Synthesis of methyl 4'-O-methyl-beta-D-cellobioside-13C12 from D-glucose-13C6. Part 2: solid-state NMR studies

Double Quantum (DQ) NMR, which utilizes the magnetic dipole interaction between the (13)C atoms, was used for the complete assignment of the (13)C NMR resonances to the corresponding carbon ring positions for the monoclinic and triclinic allomorphs of methyl 4'-O-methyl-beta-D-cellobioside-(13)C(12)(1-(13)C(12)), a cellodextrin model compound of cellulose (13)C-perlabeled at the cellobiose core. The through-space interactions were used to identify the direct chemical bonds between adjacent carbon atoms in the rings. More importantly, the (13)C NMR signals of the carbon sites C1' and C4 involved in the glycosidic bond were identified. This allowed for the complete (13)C chemical shift assignment, that when combined with the X-ray crystallography data provides a complete characterization.     

Measurement of amino acid metabolism derived from [1-13C]glucose in the rat brain using13C magnetic resonance spectroscopy

To clarify the unique characteristics of amino acid metabolism derived from glucose in the central nervous system (CNS), we injected [1-¹³C]glucose intraperitoneally to the rat, and extracted the free amino acids from several kinds of tissues and measured the amount of incorporation of¹³C derived from [1-¹³C]glucose into each amino acid using¹³C-magnetic resonance spectroscopy (NMR). In the adult rat brain, the intensities of resonances from¹³C-amino acids were observed in the following order: glutamate, glutamine, aspartate, -aminobutyrate (GABA) and alanine. There seemed no regional difference on this labeling pattern in the brain. However, only in the striatum and thalamus, the intensities of resonances from [2-¹³C]GABA were larger than that from [2,3-¹³C]aspartate. In the other tissues, such as heart, kidney, liver, spleen, muscle, lung and small intestine, the resonances from GABA were not detected and every intensity of resonances from¹³C-amino acids, except¹³C-alanine, was much smaller than those in the brain and spinal cord. In the serum,¹³C-amino acid was not detected at all. When the rats were decapitated, in the brain, the resonances from [1-¹³C]glucose greatly reduced and the intensities of resonances from [3-¹³C]lactate, [3-¹³C]alanine, [2, 3, 4-¹³C]GABA and [2-¹³C]glutamine became larger as compared with those in the case that the rats were sacrificed with microwave. In other tissues, the resonances from [1-¹³C]glucose were clearly detected even after the decapitation. In the glioma induced by nitrosoethylurea in the spinal cord, the large resonances from glutamine and alanine were observed; however, the intensities of resonances from glutamate were considerably reduced and the resonances from GABA and aspartate were not detected. These results show that the pattern of¹³C label incorporation into amino acids is unique in the central nervous tissues and also suggest that the metabolic compartmentalization could exist in the CNS through the metabolic trafficking between neurons and astroglia.Abbreviations NMR nuclear magnetic resonance - GABA -aminobutyrate - GFAP glial fibrillary acidic protein Special issue dedicated to Dr. Bernard W. Agranoff.     

Metabolism of [1-13C]glucose in a synaptosomally enriched fraction of rat cerebrum studied by1H/13C magnetic resonance spectroscopy

This study explored the utility of¹H and¹³C magnetic resonance spectroscopy to study a standard synaptosomally enriched fraction (P2 pellet) made from rat cerebrum. The preparations contained high concentrations of N-acetylaspartate and -aminobutyric acid and low concentrations of glutamine, indicating that they were in fact rich in neuronal cytosol. The metabolic competence of the preparation was assessed by quantitative measurements of its ability to convert [1-¹³C]glucose into lactate, glutamate, aspartate, and other metabolites under well oxygenated conditions in 30 minutes. The minimum mean glycolytic rate was 0.8 mM glucose/min and the flow through the tricarboxylic acid cycle was equivalent to 0.2 mM glucose/min.Abbreviations ppm parts per million (chemical shift scale) - NMR nuclear magnetic resonance - GABA -aminobutyric acid - PBS phosphate-buffered normal saline solution - TSP 3-trimethylsilylpropionate During the performance of these studies Dr. A.P. Burlina was on leave from Instituto di Clinica delle Malattie Nervose e Mentali, University of Padua, Padua, Italy.     

An Enzymatic Preparation of UDP (U-13C) Glucose

Uniformly labeled uridine diphosphoglucose (UDP(U-¹³C)G) was prepared by a two-step enzymatic synthesis. (U-¹³C) G-6-P was prepared quantitatively by incubating (U-¹³C) glucose, ATP, MgS0₄, and hexokinase. UDP(U-¹³C) Glucose was prepared by incubation of (U-¹³C)G-6-P with UDPG pyrophosphorylase, phosphoglucomutase, inorganic pyrophosphatase, UTP, and glucose-1, 6-diphosphate in pH 7.5, 100 mM Tris-HCl buffer. After purification over Biogel P-2 and subsequent preparative HPLC, UDP (U-¹³C)G was obtained in 50% yield. UDP(U-¹³C)G was characterized by ¹³C NMR and FAB-MS.     

Carbon allocation and carbon transfer between t Betula papyrifera and t Pseudotsuga menziesii seedlings using a 13C pulse-labeling method

Here we describe a simple method for pulse-labeling tree seedlings with ¹³CO_2(gas), and then apply the method in two related experiments: t (i) comparison of carbon allocation patterns between t Betula papyrifera Marsh. and t Pseudotsuga menziesii (Mirb.) Franco, and t (ii) measurement of one-way belowground carbon transfer from t B. papyrifera to t P. menziesii. Intraspecific carbon allocation patterns and interspecific carbon transfer both influence resource allocation, and consequently development, in mixed communities of t B. papyrifera and t P. menziesii.In preparation for the two experiments, we first identified the appropriate ¹³CO_2(gas) pulse-chase regime for labeling seedlings: a range of pulse (100-mL and 200-mL 99 atom%¹³ CO_2(gas)) and chase (0, 3 and 6 d) treatments were applied to one year-old t B. papyrifera and t P. menziesii seedlings. The amount of ¹³CO₂ fixed immediately after 1.5 h exposure was greatest for both t B. papyrifera (40.8 mg excess ¹³C) and t P. menziesii (22.9 mg excess ¹³C) with the 200-mL pulse, but higher ¹³C loss and high sample variability resulted in little difference in excess¹³ C content between pulse treatments after 3 d for either species. The average excess ¹³C root/shoot ratio of t B. papyrifera and t P. menziesii changed from 0.00 immediately following the pulse to 0.61 and 0.87 three and six days later, which reflected translocation of 75% of fixed isotope out of foliage within 3 d following the pulse and continued enrichment in fine roots over 6 d. Based on these results, the 100-mL CO_2(gas) and 6-d chase were considered appropriate for the carbon allocation and belowground transfer experiments.In the carbon allocation experiment, we found after 6 d that t B. papyrifera allocated 49% (average 9.5 mg) and t P. menziesii 41% (average 5.8 mg) of fixed isotope to roots, of which over 55% occurred in fine roots in both species. Species differences in isotope allocation patterns paralleled differences in tissue biomass distribution. The greater pulse labeling efficiency of t B. papyrifera compared to t P. menziesii was associated with its two-fold and 13- fold greater leaf and whole seedling net photosynthetic rates, respectively, 53% greater biomass, and 35% greater root/shoot ratio.For the carbon transfer experiment, t B. papyrifera and t P. menziesii were grown together in laboratory rootboxes, with their roots intimately mingled. A pulse of 100 mL¹³ CO_2(gas) was applied to paper birch and one-way transfer to neighboring t P. menziesii was measured after 6 d. Of the excess ¹³C fixed by t B. papyrifera, 4.7% was transferred to neighboring t P. menziesii, which distributed the isotope evenly between roots and shoots. Of the isotope received by t P. menziesii, we estimated that 93% was taken up through belowground pathways, and the remaining 7% taken up by foliage as¹³ CO_2(gas) respired by t B. papyrifera shoots. These two experiments indicate that t B. papyrifera fixes more total carbon and allocates a greater proportion to its root system than does t P. menziesii, giving it a competitive edge in resource gathering; however, below-ground carbon sharing is of sufficient magnitude that it may help ensure co-existence of the two species in mixed communities.     

In vivo 13C NMR analysis of acetate metabolism in Thiobacillus versutus under denitrifying conditions

The disappearance of 2-¹³C-acetate and the subsequent incorporation of label into cellular metabolites were followed in denitrifying cells of Thiobacillus versutus by ¹³C NMR spectroscopy. In cells grown under acetate-limitation, the specific rate of consumption was idependent of the density of the cell suspension. An isotopic steady state was reached within 30 min if sufficient substrate was added to the cell suspension. In cells grown under nitrate-limitation, the consumption of 2-¹³C-acetate proceeded at a significantly lower rate. The decrease and final disappearance of 2-¹³C-acetate were accompanied by incorporation of ¹³C into glutamate, glutamine, and by the release of labeled HCO ₃ ^– and CO₂. The appearance of a broad resonance being the methyl endgroup of poly-3-hydroxybutyrate (PHB) was indicative for PHB mobilization during the incubation. The sequence of label incorporation and the distribution among the various carbon nuclei were consistent with the operation of the tricarboxylic acid cycle.     

Assimilate partitioning affects 13C fractionation of recently assimilated carbon in maize

Coupling ¹³C natural abundance and ¹⁴C pulse labelling enabled us to investigate the dependence of ¹³C fractionation on assimilate partitioning between shoots, roots, exudates, and CO₂ respired by maize roots. The amount of recently assimilated C in these four pools was controlled by three levels of nutrient supply: full nutrient supply (NS), 10 times diluted nutrient supply (DNS), and deionised water (DW). After pulse labelling of maize shoots in a ¹⁴CO₂ atmosphere, ¹⁴C was traced to determine the amounts of recently assimilated C in the four pools and the δ¹³C values of the four pools were measured. Increasing amounts of recently assimilated C in the roots (from 8% to 10% of recovered ¹⁴C in NS and DNS treatments) led to a 0.3‰ ¹³C enrichment from NS to DNS treatments. A further increase of C allocation in the roots (from 10% to 13% of recovered ¹⁴C in DNS and DW treatments) resulted in an additional enrichment of the roots from DNS to DW treatments by 0.3‰. These findings support the hypothesis that ¹³C enrichment in a pool increases with an increasing amount of C transferred into that pool. δ¹³C of CO₂ evolved by root respiration was similar to that of the roots in DNS and DW treatments. However, if the amount of recently assimilated C in root respiration was reduced (NS treatment), the respired CO₂ became 0.7‰ ¹³C depleted compared to roots. Increasing amounts of recently assimilated C in the CO₂ from NS via DNS to DW treatments resulted in a 1.6‰ δ¹³C increase of root respired CO₂ from NS to DW treatments. Thus, for both pools, i.e. roots and root respiration, increasing amounts of recently assimilated C in the pool led to a δ¹³C increase. In DW and DNS plants there was no ¹³C fractionation between roots and exudates. However, high nutrient supply decreased the amount of recently assimilated C in exudates compared to the other two treatments and led to a 5.3‰ ¹³C enrichment in exudates compared to roots. We conclude that ¹³C discrimination between plant pools and within processes such as exudation and root respiration is not constant but strongly depends on the amount of C in the respective pool and on partitioning of recently assimilated C between plant pools. Section Editor: H. Lambers