Phytochemistry (v.68, #16-18)

Phytochemistry and the new technologies: Tackling the critical barriers to advancing systems biology by Richard Robins; G. Paul Bolwell; Norman G. Lewis (2134-2135).

Dynamic metabolic networks: Going with the flow by N.J. Kruger; R.G. Ratcliffe (2136-2138).

Recent computational strategies to evaluate the dynamic capabilities of metabolic networks are described and exemplified using paradigmatic models of metabolic pathways.Cellular metabolism is characterized by an intricate network of interactions between biochemical fluxes, metabolic compounds and regulatory interactions. To investigate and eventually understand the emergent global behavior arising from such networks of interaction is not possible by intuitive reasoning alone. This contribution seeks to describe recent computational approaches that aim to asses the topological and functional properties of metabolic networks. In particular, based on a recently proposed method, it is shown that it is possible to acquire a quantitative picture of the possible dynamics of metabolic systems, without assuming detailed knowledge of the underlying enzyme-kinetic rate equations and parameters. Rather, the method builds upon a statistical exploration of the comprehensive parameter space to evaluate the dynamic capabilities of a metabolic system, thus providing a first step towards the transition from topology to function of metabolic pathways. Utilizing this approach, the role of feedback mechanisms in the maintenance of stability is discussed using minimal models of cellular pathways.
Keywords: Kinetic modeling; Metabolomics; Systems biology; Complex networks; Nonlinear dynamics; Bifurcations;

Enzyme kinetics can be studied under in vivo conditions by modulating the activities of certain target enzymes. We extend this approach and apply it to the Calvin photosynthesis cycle with the aim to identify the set of modulations that allows maximum information gain at minimal experimental effort.To measure the kinetics of enzymes, the proteins are usually assayed in vitro after isolation from their parent organisms. We make an attempt to show how one might determine enzyme elasticities in an intact system by a multiple modulation approach. Certain target enzymes are modulated in their activities and the changes in metabolite concentrations and flux rates upon the modulations are used to calculate the enzyme elasticities. Central to this approach is that the modulations must be independent of each other, and an algorithm is developed for finding all independent modulations that allow determining the elasticities of a given enzyme. This approach is applied to a mass-action model of the Calvin cycle. The goal is to determine the elasticities of as many enzymes as possible by modulating the activities of as few of them as possible. It is shown that the elasticities of 20 (out of 22) Calvin cycle enzymes can be determined by modulating just five reactions. Moreover, visualization of independence of modulations may be used to decompose the Calvin cycle into several sections that are independent of each other regarding flow of matter and information.
Keywords: Calvin cycle; Enzyme kinetics; Elasticity; Metabolic control analysis; Multiple modulation;

On the processing of metabolic information through metabolite–gene communication networks: An approach for modelling causality by Jedrzej Szymanski; Monika Bielecka; Fernando Carrari; Alisdair R. Fernie; Rainer Hoefgen; Victoria J. Nikiforova (2163-2175).
The dynamic behaviour of biological systems is accomplished through informational exchange at different levels of the cellular hierarchy. Here, we present an approach integrating metabolic and transcript data into a causally directed network of inter-level informational flows. Using simple statistical tools we identify putative metabolic regulators of the adaptive response to environmental and developmental challenges.Gene-metabolite correlation networks of three independent biological systems were interrogated using an approach to define, and subsequently model, causality. The major goal of this work was to analyse how information from those metabolites, that displayed a rapid response to perturbation of the biological system, is processed through the response network to provide signal-specific adaptation of metabolism. For this purpose, comparison of network topologies was carried out on three different groups of system elements: transcription factors, other genes and metabolites, with special emphasis placed on those features which are possible sites of metabolic regulation or response propagation. The degree of connectivity in all three analysed gene-metabolite networks followed power-law and exponential functions, whilst a comparison of connectivities of the various cellular entities suggested, that metabolites are less involved in the regulation of the sulfur stress response than in the ripening of tomatoes (in which metabolites seem to have an even greater regulatory role than transcription factors). These findings reflect different degree of metabolic regulation for distinct biological processes. Implementing causality into the network allowed classification of metabolite-gene associations into those with causal directionality from gene to metabolite and from metabolite to gene. Several metabolites were positioned relatively early in the causal hierarchy and possessed many connections to the downstream elements. Such metabolites were considered to have higher regulatory potential. For the biological example of hypo-sulfur stress response in Arabidopsis, the highest regulatory potential scores were established for fructose and sucrose, isoleucine, methionine and sinapic acid. Further developments in profiling techniques will allow greater cross-systems comparisons, necessary for reliability and universality checks of inferred regulatory capacities of the particular metabolites.
Keywords: Solanum lycopersicum; Solanaceae; Tomato; Arabidopsis thaliana; Brassicaceae; Sulfur starvation; Fruit ripening; Correlation network; Causality; Metabolic regulation;

Network flux analysis: Impact of 13C-substrates on metabolism in Arabidopsis thaliana cell suspension cultures by Nicholas J. Kruger; Joanna E. Huddleston; Pascaline Le Lay; Nicholas D. Brown; R. George Ratcliffe (2176-2188).
Cell suspension cultures of Arabidopsis thaliana grown in media differing in 13C-enrichment are metabolically indistinguishable. There was no significant difference in the pattern of metabolism of either specific 14C-labelled or [U-14C]glucose between the cultures. Principal component analysis of 13C-decoupled 1H NMR metabolite fingerprints of cell extracts failed to discriminate between the different culture conditions. It is concluded that 13C-enrichment of the growth substrate has no effect on flux through the network of central carbon metabolism.The aim of this study was to test the assumption that 13C-enrichment of respiratory substrate does not perturb metabolism. Cell suspension cultures of Arabidopsis thaliana were grown in MS medium containing unlabelled glucose (with 13C at natural abundance), 100% [1-13C]glucose, 100% [U-13C6]glucose or 10% [U-13C6]glucose plus 90% unlabelled glucose. There was no significant difference in the metabolism of [U-14C]glucose between the cultures. Similarly, the pattern of 14CO2 release from specifically labelled [14C]-substrates was unaffected. Principal component analysis of 13C-decoupled 1H NMR metabolite fingerprints of cell extracts was unable to discriminate between the different culture conditions. It is concluded that 13C-enrichment of the growth substrate has no effect on flux through the central pathways of carbon metabolism in higher plants. This conclusion supports the implicit assumption in metabolic flux analysis that steady-state 13C-labelling does not perturb fluxes through the reactions of the metabolic network it seeks to quantify.
Keywords: Arabidopsis thaliana; Brassicaceae; [13C]glucose; Steady-state labelling; Metabolic flux analysis; Kinetic isotope effects; [14C]glucose metabolism; 1H NMR; Metabolite fingerprinting;

Failure to consider vacuolar compartmentation of glucose can have a marked influence on estimates of sucrose cycling from steady-state metabolic flux analysis. Mathematical modelling shows that measurements of the labelling of both cytosolic and vacuolar glucose are required to resolve the fluxes involving intracellular glucose in plants.Steady-state stable isotope labelling provides a method for generating flux maps of the compartmented network of central metabolism in heterotrophic plant tissues. Theoretical analysis of the contribution of the vacuole to the regeneration of glucose by endogenous processes shows that numerical fitting of isotopomeric data will only generate an accurate map of the fluxes involving intracellular glucose if information is available on the labelling of both the cytosolic and vacuolar glucose pools. In the absence of this information many of the calculated fluxes are at best unreliable or at worst indeterminate. This result suggests that the anomalously high rates of sucrose cycling and glucose resynthesis that have been reported in earlier steady-state analyses of tissues labelled with 13C-glucose precursors may be an artefact of assuming that the labelling pattern of extracted glucose reflected the labelling of the cytosolic pool. The analysis emphasises that although subcellular information can sometimes be deduced from a steady-state analysis without recourse to subcellular fractionation, the success of this procedure depends critically on the structure of the metabolic network. It is concluded that methods need to be implemented that will allow measurement of the subcellular labelling pattern of glucose and other metabolites, as part of the routine analysis of the redistribution of label in steady-state stable isotope labelling experiments, if the true potential of network flux analysis for generating metabolic phenotypes is to be realized.
Keywords: Metabolic modelling; Network flux analysis; Steady-state stable isotope labelling; Subcellular compartmentation; Sucrose cycling; Vacuole;

Compartment-specific labeling information in 13C metabolic flux analysis of plants by Doug K. Allen; Yair Shachar-Hill; John B. Ohlrogge (2197-2210).
Plants and particularly their seeds have great potential for low cost production of chemical feedstocks and novel compounds, but rational metabolic engineering requires a better understanding of central carbon metabolism. To aid flux quantification we report new and improved measurements of 13C labeling information from specific compartments within the plant cell.Metabolic engineering of plants has great potential for the low cost production of chemical feedstocks and novel compounds, but to take full advantage of this potential a better understanding of plant central carbon metabolism is needed. Flux studies define the cellular phenotype of living systems and can facilitate rational metabolic engineering. However the measurements usually made in these analyses are often not sufficient to reliably determine many fluxes that are distributed between different subcellular compartments of eukaryotic cells. We have begun to address this shortcoming by increasing the number and quality of measurements that provide 13C labeling information from specific compartments within the plant cell. The analysis of fatty acid groups, cell wall components, protein glycans, and starch, using both gas chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy are presented here. Fatty acid labeling determinations are sometimes highly convoluted. Derivatization to butyl amides reduces the errors in isotopomer resolution and quantification, resulting in better determination of fluxes into seed lipid reserves, including both plastidic and cytosolic reactions. While cell walls can account for a third or more of biomass in many seeds, no quantitative cell wall labeling measurements have been reported for plant flux analysis. Hydrolyzing cell wall and derivatizing sugars to the alditol acetates, provides novel labeling information and thereby can improve identification of flux through upper glycolytic intermediates of the cytosol. These strategies improve the quantification of key carbon fluxes in the compartmentalized flux network of plant cells.
Keywords: Subcellular compartmentation; 13C MFA; Stable isotope tracer; Soybean; Cell wall; Protein glycans; Starch; Glycerolipids; Oilseed; GC–MS; NMR;

Rapeseed embryos are a model system for studying plant metabolism. In steady state 13C-feeding experiments, metabolic fluxes are determined using measurements of labeling in metabolic end-products. Using previously measured flux values, this criterion-based optimal design study determines substrate label combinations that provide the highest possible information content.Steady state metabolic flux analysis using 13C labeled substrates is of growing importance in plant physiology and metabolic engineering. The quality of the flux estimates in 13C metabolic flux analysis depend on the: (i) network structure; (ii) flux values; (iii) design of the labeling substrate; and (iv) label measurements performed. Whereas the first two parameters are facts of nature, the latter two are to some extent controlled by the experimenter, yet they have received little attention in most plant studies. Using the metabolic flux map of developing Brassica napus (Rapeseed) embryos, this study explores the value of optimal substrate label designs obtained with different statistical criteria and addresses the applicability of different optimal designs to biological questions. The results demonstrate the value of optimizing the choice of labeled substrates and show that substrate combinations commonly used in bacterial studies can be far from optimal for mapping fluxes in plant systems. The value of performing additional experiments and the inclusion of measurements is also evaluated.
Keywords: Metabolic flux analysis; Optimal design; Brassica napus; 13C labeling;

Substrate cycles in the central metabolism of maize root tips under hypoxia by Ana Paula Alonso; Philippe Raymond; Dominique Rolin; Martine Dieuaide-Noubhani (2222-2231).
This work describes a metabolic flux analysis (C13 and C14 labeling experiments for flux quantification) performed to study the response of maize root tips to hypoxia. ATP production was severely reduced, and the flux through the substrate cycles all decreased. However, substrate cycles remain important in terms of ATP consumption (50% of the produced ATP) and their importance was discussed.Substrate cycles, also called “futile” cycles, are ubiquitous and lead to a net consumption of ATP which, in the normoxic maize root, have been estimated at about 50% of the total ATP produced [Alonso, A.P., Vigeolas, H., Raymond, P., Rolin, D., Dieuaide-Noubhani, M., 2005. A new substrate cycle in plants. Evidence for a high glucose-phosphate-to-glucose turnover from in vivo steady-state and pulse-labeling experiments with [13C] glucose and [14C] glucose. Plant Physiol. 138, 2220–2232]. To evaluate their role we studied the substrate cycles of maize root tips under an oxygen limitation of respiration (3% O2). Short-time labeling experiments with [U-14C]-Glc were performed to quantify the fluxes through sucrose and starch cycles of synthesis and degradation. Steady-state labeling with [1-13C]-Glc followed by 1H NMR and 13C NMR analysis of sugars and free alanine was used to quantify fluxes in the central metabolic pathways, including the Glc-P/Glc cycle and the fructose-P/triose-P cycle of glycolysis. Comparison with results previously obtained in normoxia [Alonso et al., as mentioned above] showed that 3% O2 induced fermentation and reduced respiration, which led to a lesser amount of ATP produced. The rates of Glc consumption, glycolytic flux and all substrate cycles were lower, but the proportion of ATP consumed in the substrate cycles remained unchanged. These findings suggest that substrate cycles are not a luxury but an integral part of the organization of the plant central metabolism.
Keywords: Metabolic flux analysis; Maize root; Isotopic labeling; NMR; Hypoxia; Substrate cycle;

Parallel determination of enzyme activities and in vivo fluxes in Brassica napus embryos grown on organic or inorganic nitrogen source by Björn H. Junker; Joachim Lonien; Lindsey E. Heady; Alistair Rogers; Jörg Schwender (2232-2242).
Brassica napus embryos were grown in culture with either glutamine and alanine or ammonium nitrate as the sole nitrogen source. Dependent on the nitrogen source, in vivo metabolic fluxes around the TCA cycle changed distinctly. The changes observed in enzyme activity were not consistent with the changes in metabolic flux. It is suggested that the observed flux adjustments are driven by mass balances rather than transcriptional regulation.After the completion of the genomic sequencing of model organisms, numerous post-genomic studies, integrating transcriptome and metabolome data, are aimed at developing a more complete understanding of cell physiology. Here, we measure in vivo metabolic fluxes by steady state labeling, and in parallel, the activity of enzymes in central metabolism in cultured developing embryos of Brassica napus. Embryos were grown on either the amino acids glutamine and alanine as an organic nitrogen source, or on ammonium nitrate as an inorganic nitrogen source. The type of nitrogen made available to developing embryos caused substantial differences in fluxes associated with the tricarboxylic acid cycle, including flux reversion. The changes observed in enzyme activity were not consistent with our estimates of metabolic flux. Furthermore, most extractable enzyme activities are in large surplus relative to the requirements for the observed in vivo fluxes. The results demonstrate that in this model system the metabolic response of central metabolism to changes in environmental conditions can be achieved largely without regulatory reprogramming of the enzyme machinery.
Keywords: Brassica napus; Central metabolism; Enzyme activity profiling; Metabolic flux analysis;

Metabolic flux quantification is a powerful profiling tool in plant metabolic engineering and systems biology. We introduce the application of “bondomers”, a computationally efficient and intuitively appealing alternative to the commonly used “isotopomers”, toward systemic evaluation of fluxes in central carbon metabolism of Catharanthus roseus hairy roots.Methods for accurate and efficient quantification of metabolic fluxes are desirable in plant metabolic engineering and systems biology. Toward this objective, we introduce the application of “bondomers”, a computationally efficient and intuitively appealing alternative to the commonly used isotopomer concept, to flux evaluation in plants, by using Catharanthus roseus hairy roots as a model system. We cultured the hairy roots on (5% w/w U–13C, 95% w/w naturally abundant) sucrose, and acquired two-dimensional [13C, 1H] and [1H, 1H] NMR spectra of hydrolyzed aqueous extract from the hairy roots. Analysis of these spectra yielded a data set of 116 bondomers of β-glucans and proteinogenic amino acids from the hairy roots. Fluxes were evaluated from the bondomer data by using comprehensive bondomer balancing. We identified most fluxes in a three-compartmental model of central carbon metabolism with good precision. We observed parallel pentose phosphate pathways in the cytosol and the plastid with significantly different fluxes. The anaplerotic fluxes between phosphoenolpyruvate and oxaloacetate in the cytosol and between malate and pyruvate in the mitochondrion were relatively high (60.1 ± 2.5 mol per 100 mol sucrose uptake, or 22.5 ± 0.5 mol per 100 mol mitochondrial pyruvate dehydrogenase flux). The development of a comprehensive flux analysis tool for this plant hairy root system is expected to be valuable in assessing the metabolic impact of genetic or environmental changes, and this methodology can be extended to other plant systems.
Keywords: Plant metabolic flux; Catharanthus roseus; Hairy roots; Bondomer; Compartmented metabolism; Anaplerotic flux; Plant metabolic engineering;

The monitoring of isotope dilution after 13CO2 labelling was optimized using Arabidopsis thaliana Col-0 or Oryza sativa IR57111 plants, which were maximally labelled with 13C. Carbon isotope dilution was evaluated for short (2 h) and long-term (3 days) kinetic measurements of metabolite pools in roots and shoots. Both approaches were shown to enable the characterization of metabolite specific partitioning processes and kinetics. A current experimental design for the kinetic metabolic phenotyping of higher plants using GC-EI-TOF-MS profiling analysis is proposed.The established GC-EI-TOF-MS method for the profiling of soluble polar metabolites from plant tissue was employed for the kinetic metabolic phenotyping of higher plants. Approximately 100 typical GC-EI-MS mass fragments of trimethylsilylated and methoxyaminated metabolite derivatives were structurally interpreted for mass isotopomer analysis, thus enabling the kinetic study of identified metabolites as well as the so-called functional group monitoring of yet non-identified metabolites. The monitoring of isotope dilution after 13CO2 labelling was optimized using Arabidopsis thaliana Col-0 or Oryza sativa IR57111 plants, which were maximally labelled with 13C. Carbon isotope dilution was evaluated for short (2 h) and long-term (3 days) kinetic measurements of metabolite pools in root and shoots. Both approaches were shown to enable the characterization of metabolite specific partitioning processes and kinetics. Simplifying data reduction schemes comprising calculation of 13C-enrichment from mass isotopomer distributions and of initial 13C-dilution rates were employed. Metabolites exhibited a highly diverse range of metabolite and organ specific half-life of 13C-label in their respective pools (13C-half-life). This observation implied the setting of metabolite specific periods for optimal kinetic monitoring. A current experimental design for the kinetic metabolic phenotyping of higher plants is proposed.
Keywords: Arabidopsis thaliana Col-0; Oryza sativa IR57111; 13C-carbon; 13CO2-carbondioxide; Dynamic flux analysis; Electron impact ionization (EI); Gas chromatography (GC); Metabolite profiling; Stable isotope dilution; Time-of-flight mass spectrometry (TOF-MS);

13CO2 as a universal metabolic tracer in isotopologue perturbation experiments by Werner Römisch-Margl; Nicholas Schramek; Tanja Radykewicz; Christian Ettenhuber; Eva Eylert; Claudia Huber; Lilla Römisch-Margl; Christine Schwarz; Maria Dobner; Norbert Demmel; Bernhard Winzenhörlein; Adelbert Bacher; Wolfgang Eisenreich (2273-2289).
The pilot study shows that pulse/chase labeling with 13CO2 as precursor is a powerful tool for the study of quantitative aspects of plant metabolism in completely unperturbed whole plants.A tobacco plant was illuminated for 5 h in an atmosphere containing 13CO2 and then maintained for 10 days under standard greenhouse conditions. Nicotine, glucose, and amino acids from proteins were isolated chromatographically. Isotopologue abundances of isolated metabolites were determined quantitatively by NMR spectroscopy and mass spectrometry. The observed non-stochastic isotopologue patterns indicate (i) formation of multiply labeled photosynthetic carbohydrates during the 13CO2 pulse phase followed by (ii) partial catabolism of the primary photosynthetic products, and (iii) recombination of the 13C-labeled fragments with unlabeled intermediary metabolites during the chase period. The detected and simulated isotopologue profiles of glucose and amino acids reflect carbon partitioning that is dominated by the Calvin cycle and glycolysis/glucogenesis. Retrobiosynthetic analysis of the nicotine pattern is in line with its known formation from nicotinic acid and putrescine via aspartate, glyceraldehyde phosphate and α-ketoglutarate as basic building blocks. The study demonstrates that pulse/chase labeling with 13CO2 as precursor is a powerful tool for the analysis of quantitative aspects of plant metabolism in completely unperturbed whole plants.

The concept and methodology of dynamic labeling for metabolic flux analysis (MFA) of plant metabolic pathways are discussed by describing a MFA study of tryptophan biosynthetic pathway in cultured rice cells.The concept and methodology of using dynamic labeling for the MFA of plant metabolic pathways are described, based on a case study to develop a method for the MFA of the tryptophan biosynthetic pathway in cultured rice cells. Dynamic labeling traces the change in the labeling level of a metabolite in a metabolic pathway after the application of a stable isotope-labeled compound. In this study, [1-13C] l-serine was fed as a labeling precursor and the labeling level of Trp was determined by using the LC–MS/MS. The value of metabolic flux is determined by fitting a model describing the labeling dynamics of the pathway to the observed labeling data. The biosynthetic flux of Trp in rice suspension cultured cell was determined to be 6.0 ± 1.1 nmol (g FW h)−1. It is also demonstrated that an approximately sixfold increase in the biosynthetic flux of Trp in transgenic rice cells expressing the feedback-insensitive version of anthranilate synthase alpha-subunit gene (OASA1D) resulted in a 45-fold increase in the level of Trp. In this article, the basic workflow for the experiment is introduced and the details of the actual experimental procedures are explained. Future perspectives are also discussed by referring recent advances in the dynamic labeling approach.
Keywords: Anthranilate synthase; Dynamic labeling experiment; Metabolic flux analysis; Oryza sativa; Tryptophan over-production;

A transient 13C metabolic flux analysis methodology to measure central carbon fluxes in purely photoautotrophic systems under metabolic steady-state is formulated. A mathematical framework of 13C isotopomer balances is used to assess various experimental requirements of the problem, including intracellular metabolite concentration measurements and photobioreactor operation.Metabolic flux analysis is increasingly recognized as an integral component of systems biology. However, techniques for experimental measurement of system-wide metabolic fluxes in purely photoautotrophic systems (growing on CO2 as the sole carbon source) have not yet been developed due to the unique problems posed by such systems. In this paper, we demonstrate that an approach that balances positional isotopic distributions transiently is the only route to obtaining system-wide metabolic flux maps for purely autotrophic metabolism. The outlined transient 13C-MFA methodology enables measurement of fluxes at a metabolic steady-state, while following changes in 13C-labeling patterns of metabolic intermediates as a function of time, in response to a step-change in 13C-label input. We use mathematical modeling of the transient isotopic labeling patterns of central intermediates to assess various experimental requirements for photoautotrophic MFA. This includes the need for intracellular metabolite concentration measurements and isotopic labeling measurements as a function of time. We also discuss photobioreactor design and operation in order to measure fluxes under precise environmental conditions. The transient MFA technique can be used to measure and compare fluxes under different conditions of light intensity, nitrogen sources or compare strains with various mutations or gene deletions and additions.
Keywords: Synechocystis sp. PCC 6803; Transient 13C-MFA; Photosynthesis; Modeling; Calvin cycle; Instationary 13C-MFA;

Determination of metabolic fluxes in a non-steady-state system by C.J. Baxter; J.L. Liu; A.R. Fernie; L.J. Sweetlove (2313-2319).
At non-steady-state, the labelling of a metabolite pool can be described by equations based on mass-balance of labelled molecules. Non-steady-state fluxes can be estimated from such equations given data on change in labelling and metabolite pool size.Estimation of fluxes through metabolic networks from redistribution patterns of 13C has become a well developed technique in recent years. However, the approach is currently limited to systems at metabolic steady-state; dynamic changes in metabolic fluxes cannot be assessed. This is a major impediment to understanding the behaviour of metabolic networks, because steady-state is not always experimentally achievable and a great deal of information about the control hierarchy of the network can be derived from the analysis of flux dynamics. To address this issue, we have developed a method for estimating non-steady-state fluxes based on the mass-balance of mass isotopomers. This approach allows multiple mass-balance equations to be written for the change in labelling of a given metabolite pool and thereby permits over-determination of fluxes. We demonstrate how linear regression methods can be used to estimate non-steady-state fluxes from these mass balance equations. The approach can be used to calculate fluxes from both mass isotopomer and positional isotopomer labelling information and thus has general applicability to data generated from common spectrometry- or NMR-based analytical platforms. The approach is applied to a GC–MS time-series dataset of 13C-labelling of metabolites in a heterotrophic Arabidopsis cell suspension culture. Threonine biosynthesis is used to demonstrate that non-steady-state fluxes can be successfully estimated from such data while organic acid metabolism is used to highlight some common issues that can complicate flux estimation. These include multiple pools of the same metabolite that label at different rates and carbon skeleton rearrangements.
Keywords: Non-steady-state; Flux; Arabidopsis;

We propose an approach for measuring dynamic metabolic networks that combines different extraction solvents and NMR pulse sequences. In addition, we present examples that involve monitoring the time-dependent changes in the 13C-bondomer composition of Arabidopsis thaliana labeled with [13C6]glucose.Novel technologies for measuring biological systems and methods for visualizing data have led to a revolution in the life sciences. Nuclear magnetic resonance (NMR) techniques can provide information on metabolite structure and metabolic dynamics at the atomic level. We have been developing a new method for measuring the dynamic metabolic network of crude extracts that combines [13C6]glucose stable isotope labeling of Arabidopsis thaliana and multi-dimensional heteronuclear NMR analysis, whereas most conventional metabolic flux analyses examine proteinogenic amino acids that are specifically labeled with partially labeled substrates such as [2-13C1]glucose or 10% [13C6]glucose. To show the validity of our method, we investigated how to obtain information about biochemical reactions, C–C bond formation, and the cleavage of the main metabolites, such as free amino acids, in crude extracts based on the analysis of the 13C–13C coupling pattern in 2D-NMR spectra. For example, the combination of different extraction solvents allows one to distinguish complicated 13C–13C fine couplings at the C2 position of amino acids. As another approach, f1–f3 projection of the HCACO spectrum also helps in the analysis of 13C–13C connectivities. Using these new methods, we present an example that involves monitoring the incorporation profile of [13C6]glucose into A. thaliana and its metabolic dynamics, which change in a time-dependent manner with atmospheric 12CO2 assimilation.
Keywords: Metabolic network; Stable isotope labeling; Heteronuclear multi-dimensional NMR; 13C–13C fine coupling; Crude extract; Extraction solvents;

NMR-based fluxomics: Quantitative 2D NMR methods for isotopomers analysis by Stéphane Massou; Cécile Nicolas; Fabien Letisse; Jean-Charles Portais (2330-2340).
Combined with 13C-labelling experiments, NMR provides two different types of data valuable for the analysis of metabolic fluxes: specific enrichments and positional isotopomers. Both data types can be measured for various metabolites in complex mixtures by using relevant quantitative 2D-NMR methods.We have investigated the reliability of 2D-COSY and 2D-TOCSY experiments to provide accurate measurements of 13C-enrichments in complex mixtures of 13C-labelled metabolites. This was done from both theoretical considerations and experimental investigations. The results showed that 2D-TOCSY but not 2D-COSY could provide accurate measurements of 13C-enrichments, provided efficient zero-quantum filters were applied during the mixing period. This approach extends the range of NMR methods applicable in 13C-labelling experiments and is suitable to investigating the dynamic behaviour of metabolic systems.
Keywords: 13C-labelling experiments; Quantitative 2D NMR; Specific 13C-enrichments; 2D-COSY; 2D ZQF-TOCSY;

In vivo 13C NMR determines metabolic fluxes and steady state in linseed embryos by Stéphanie Troufflard; Albrecht Roscher; Brigitte Thomasset; Jean-Noël Barbotin; Stephen Rawsthorne; Jean-Charles Portais (2341-2350).
The analysis of time-course data from 13C-labelling experiments detected by in vivo NMR of developing linseed embryos is shown to be complementary to steady state metabolic flux analysis as it gives information on the isotopic and metabolic dynamics while reaching steady state and allows determination of complementary fluxes.The dynamics of developing linseed embryo metabolism was investigated using 13C-labelling experiments where the real-time kinetics of label incorporation into metabolites was monitored in situ using in vivo NMR. The approach took advantage of the occurrence in this plant tissue of large metabolite pools – such as sucrose or lipids – to provide direct and quantitative measurement of the evolution of the labelling state within central metabolism. As a pre-requisite for the use of steady state flux measurements it was shown that isotopic steady state was reached within 3 h at the level of central intermediates whereas it took a further 6 h for the sucrose pool. Complete isotopic and metabolic steady state took 18 h to be reached. The data collected during the transient state where label was equilibrated but the metabolic steady state was incomplete, enabled the rates of lipid and sucrose synthesis to be measured in situ on the same sample. This approach is suitable to get a direct assessment of metabolic time-scales within living plant tissues and provides a valuable complement to steady state flux determinations.
Keywords: Linum usitatissimum; Oilseed embryo; Carbon-13 labelling experiments; Nuclear magnetic resonance; Metabolic fluxes; Metabolic steady state; Isotopic steady state;

Experimental and mathematical approaches to modeling plant metabolic networks by Rigoberto Rios-Estepa; Bernd Markus Lange (2351-2374).
This article provides an overview of approaches for evaluating the control of metabolic flux in plants, with an emphasis on using simplified case studies and examples from the plant metabolism literature, to demonstrate the utility of combining mathematical modeling with experimental testing. The mathematical concepts are explained and discussed with a target readership of phytochemists, biochemists, biophysicists and geneticists in mind.To support their sessile and autotrophic lifestyle higher plants have evolved elaborate networks of metabolic pathways. Dynamic changes in these metabolic networks are among the developmental forces underlying the functional differentiation of organs, tissues and specialized cell types. They are also important in the various interactions of a plant with its environment. Further complexity is added by the extensive compartmentation of the various interconnected metabolic pathways in plants. Thus, although being used widely for assessing the control of metabolic flux in microbes, mathematical modeling approaches that require steady-state approximations are of limited utility for understanding complex plant metabolic networks. However, considerable progress has been made when manageable metabolic subsystems were studied. In this article, we will explain in general terms and using simple examples the concepts underlying stoichiometric modeling (metabolic flux analysis and metabolic pathway analysis) and kinetic approaches to modeling (including metabolic control analysis as a special case). Selected studies demonstrating the prospects of these approaches, or combinations of them, for understanding the control of flux through particular plant pathways are discussed. We argue that iterative cycles of (dry) mathematical modeling and (wet) laboratory testing will become increasingly important for simulating the distribution of flux in plant metabolic networks and deriving rational experimental designs for metabolic engineering efforts.
Keywords: BioPathAt; Bondomer; Cumomer; Elementary modes; Flux; Isotopomer; Metabolic control analysis;

Kinetic model of sucrose accumulation in maturing sugarcane culm tissue by Lafras Uys; Frederik C. Botha; Jan-Hendrik S. Hofmeyr; Johann M. Rohwer (2375-2392).
Kinetic modelling was used to investigate sucrose accumulation in the storage parenchyma of sugarcane (Saccharum officinarum). This approach yielded a profile of metabolic changes associated with the maturation of sugarcane internodes. The control over the sucrose accumulation process could also be quantified.Biochemically, it is not completely understood why or how commercial varieties of sugarcane (Saccharum officinarum) are able to accumulate sucrose in high concentrations. Such concentrations are obtained despite the presence of sucrose synthesis/breakdown cycles (futile cycling) in the culm of the storage parenchyma. Given the complexity of the process, kinetic modelling may help to elucidate the factors governing sucrose accumulation or direct the design of experimental optimisation strategies.This paper describes the extension of an existing model of sucrose accumulation (Rohwer, J.M., Botha, F.C., 2001. Analysis of sucrose accumulation in the sugar cane culm on the basis of in vitro kinetic data. Biochem. J. 358, 437–445) to account for isoforms of sucrose synthase and fructokinase, carbon partitioning towards fibre formation, and the glycolytic enzymes phosphofructokinase (PFK), pyrophosphate-dependent PFK and aldolase. Moreover, by including data on the maximal activity of the enzymes as measured in different internodes, a growth model was constructed that describes the metabolic behaviour as sugarcane parenchymal tissue matures from internodes 3–10.While there was some discrepancy between modelled and experimentally determined steady-state sucrose concentrations in the cytoplasm, steady-state fluxes showed a better fit. The model supports a hypothesis of vacuolar sucrose accumulation against a concentration gradient. A detailed metabolic control analysis of sucrose synthase showed that each isoform has a unique control profile. Fructose uptake by the cell and sucrose uptake by the vacuole had a negative control on the futile cycling of sucrose and a positive control on sucrose accumulation, while the control profile for neutral invertase was reversed. When the activities of these three enzymes were changed from their reference values, the effects on futile cycling and sucrose accumulation were amplified.
Keywords: Sugarcane; Kinetic modelling; Metabolic control analysis; Plant metabolism; Sucrose accumulation;

Dynamic flux cartography of hairy roots primary metabolism by M. Cloutier; M. Perrier; M. Jolicoeur (2393-2404).
A dynamic metabolic model is proposed to analyze and visualize hairy roots primary metabolism. The visualization allows for a better understanding of the interactions between nutrients, metabolites and pathways in plant metabolism.A dynamic model for plant cell and hairy root primary metabolism is presented. The model includes nutrient uptake (Pi, sugars, nitrogen sources), the glycolysis and pentose phosphate pathways, the TCA cycle, amino acid biosynthesis, respiratory chain, biosynthesis of cell building blocks (structural hexoses, organic acids, lipids, and organic phosphated molecules). The energy shuttles (ATP, ADP) and cofactors (NAD/H, NADP/H) are also included. The model describes the kinetics of 44 biochemical reactions (fluxes) of the primary metabolism of plant cells and includes 41 biochemical species (metabolites, nutrients, biomass components). Multiple Michaelis–Menten type kinetics are used to describe biochemical reaction rates. Known regulatory phenomena on metabolic pathways are included using sigmoid switch functions. A visualization framework showing fluxes and metabolite concentrations over time is presented. The visualization of fluxes and metabolites is used to analyze simulation results from Catharanthus roseus hairy root 50 d batch cultures. The visualization of the metabolic system allows analyzing split ratios between pathways and flux time-variations. For carbon metabolism, the cells were observed to have relatively high and stable fluxes for the central carbon metabolism and low and variable fluxes for anabolic pathways. For phosphate metabolism, a very high free intracellular Pi turnover rate was observed with higher flux variations than for the carbon metabolism. Nitrogen metabolism also exhibited large flux variations. The potential uses of the model are also discussed.
Keywords: Metabolic modelling; Hairy roots; Kinetic model; Metabolic regulation;