Comparison between elementary flux modes analysis and 13C-metabolic fluxes measured in bacterial and plant cells

<p>Abstract</p> <p>Background</p> <p>¹³C metabolic flux analysis is one of the pertinent ways to compare two or more physiological states. From a more theoretical standpoint, the structural properties of metabolic networks can be analysed to explore feasible metabolic behaviours and to define the boundaries of steady state flux distributions. Elementary flux mode analysis is one of the most efficient methods for performing this analysis. In this context, recent approaches have tended to compare experimental flux measurements with topological network analysis.</p> <p>Results</p> <p>Metabolic networks describing the main pathways of central carbon metabolism were set up for a bacteria species (<it>Corynebacterium glutamicum</it>) and a plant species (<it>Brassica napus</it>) for which experimental flux maps were available. The structural properties of each network were then studied using the concept of elementary flux modes. To do this, coefficients of flux efficiency were calculated for each reaction within the networks by using selected sets of elementary flux modes. Then the relative differences - reflecting the change of substrate <it>i.e</it>. a sugar source for <it>C</it>. <it>glutamicum </it>and a nitrogen source for <it>B</it>. <it>napus </it>- of both flux efficiency and flux measured experimentally were compared. For both organisms, there is a clear relationship between these parameters, thus indicating that the network structure described by the elementary flux modes had captured a significant part of the metabolic activity in both biological systems. In <it>B</it>. <it>napus</it>, the extension of the elementary flux mode analysis to an enlarged metabolic network still resulted in a clear relationship between the change in the coefficients and that of the measured fluxes. Nevertheless, the limitations of the method to fit some particular fluxes are discussed.</p> <p>Conclusion</p> <p>This consistency between EFM analysis and experimental flux measurements, validated on two metabolic systems allows us to conclude that elementary flux mode analysis could be a useful tool to complement ¹³C metabolic flux analysis, by allowing the prediction of changes in internal fluxes before carbon labelling experiments.</p

Impaired cell growth under ammonium stress explained by modeling the energy cost of vacuole expansion in tomato leaves

Ammonium (NH4+)-based fertilization efficiently mitigates the adverse effects of nitrogen fertilization on the environment. However, high concentrations of soil NH4+ provoke growth inhibition, partly caused by the reduction of cell enlargement and associated with modifications of cell composition, such as an increase of sugars and a decrease in organic acids. Cell expansion depends largely on the osmotic-driven enlargement of the vacuole. However, the involvement of subcellular compartmentation in the adaptation of plants to ammonium nutrition has received little attention, until now. To investigate this, tomato (Solanum lycopersicum) plants were cultivated under nitrate and ammonium nutrition and the fourth leaf was harvested at seven developmental stages. The vacuolar expansion was monitored and metabolites and inorganic ion contents, together with intracellular pH, were determined. A data-constrained model was constructed to estimate subcellular concentrations of major metabolites and ions. It was first validated at the three latter developmental stages by comparison with subcellular concentrations obtained experimentally using non-aqueous fractionation. Then, the model was used to estimate the subcellular concentrations at the seven developmental stages and the net vacuolar uptake of solutes along the developmental series. Our results showed ammonium nutrition provokes an acidification of the vacuole and a reduction in the flux of solutes into the vacuoles. Overall, analysis of the subcellular compartmentation reveals a mechanism behind leaf growth inhibition under ammonium stress linked to the higher energy cost of vacuole expansion, as a result of alterations in pH, the inhibition of glycolysis routes and the depletion of organic acids.TP benefited from a cotutelle PhD (University of Bordeaux and University of the Basque Country) and thanks the University of the Basque Country (UPV/EHU, Spain) for his PhD grant during the execution of this work. This research was financially supported by the Basque Government (IT-932-16) and the Spanish Government (BIO2017-84035-R co-funded by Fondo Europeo para el Desarrollo Regional [FEDER]). Analytics were supported by MetaboHUB (ANR-11-INBS-0010) and PHENOME (ANR-11-INBS-0012) projects. Technical support was provided by Cedric Cassan, Ana Renovales and Mandy Bordas. The authors also thank SGIker (UPV/EHU, FEDER, EU) for the technical and human support provided

1H-NMR metabolomics: Profiling method for a rapid and efficient screening of transgenic plants

Metabolomics-based approaches are methods of choice for studying changes in fruit composition induced by  environmental or genetic modulation of biochemical pathways in the fruit. Owing to enzyme redundancy and  high plasticity of the metabolic network, transgenic alteration of the activity of the enzymes from the central metabolism very often results in only slight modifications of the fruit composition. In order to avoid costly and  time-consuming plant analysis, we used a fast and sensitive 1H-NMR-based metabolomic profiling technique  allowing discovery of slight metabolite variations in a large number of samples. Here, we describe the  screening of transgenic tomato plants in which two genes from the central metabolism, phosphoenolpyruvate  carboxylase (EC.3.4.1.1) and malate synthase (EC 2.3.3.9) were silenced by antisens and RNAi strategy.  1H-NMR metabolomic profiles of methanol-d4 D2O buffer extracts of tomato fruit flesh were acquired and  subjected to unsupervised multivariate statistical analysis. 1H-NMR spectra were binned into variable-size  spectral domains, making it possible to get an overall analysis of a large number of resonances, even in the  case of uncontrolled variation of the chemical shift. Principal component analysis was used to separate groups  of samples and to relate known and unknown metabolites to transgenic events. The screening of 100 samples,  from extraction to data mining, took 36 h. Thus, this procedure allows the rapid selection of metabolic  phenotypes of interest among about 30 transgenic lines.Key words: Metabolome, GMO, tomato, fruit, 1H-NMR profiling, screening

Mise en place d'une approche de fluxomique chez le fruit de tomate (étude de transformants surexprimant des hexokinases)

BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

Respiratory Metabolism in Heterotrophic Plant Cells as Revealed by Isotopic Labeling and Metabolic Flux Analysis

Mitochondrial respiration requires redox power that is mainly provided by the tricarboxylic acid (TCA) cycle in heterotrophic tissues. Glycolysis is commonly assumed to be the major pathway for carbon replenishment of the TCA cycle in most plant cells. However, the TCA cycle also provides precursors for amino acids and organic acids synthesis, a process that requires 4- or 5-C molecules supplied by anaplerotic pathways. The TCA cycle is thus involved in both anabolism and catabolism. Despite the good knowledge of enzymes and pathways involved in these processes, the regulation of carbon partitioning between catabolism and anabolism remains poorly understood. Metabolic flux analysis (MFA) aims at quantifying fluxes in metabolic networks and provides new insights for the study of the TCA cycle and associated pathways. This chapter presents briefly the principles of MFA, and describes how C-14-tracing evolved to C-13-MFA, and more recently to C-13-INST-MFA. Such analyses have provided insights about the origin of carbon atoms entering the TCA cycle and the partitioning between respiration and biosyntheses

Respiratory Metabolism in Heterotrophic Plant Cells as Revealed by Isotopic Labeling and Metabolic Flux Analysis

Mitochondrial respiration requires redox power that is mainly provided by the tricarboxylic acid (TCA) cycle in heterotrophic tissues. Glycolysis is commonly assumed to be the major pathway for carbon replenishment of the TCA cycle in most plant cells. However, the TCA cycle also provides precursors for amino acids and organic acids synthesis, a process that requires 4- or 5-C molecules supplied by anaplerotic pathways. The TCA cycle is thus involved in both anabolism and catabolism. Despite the good knowledge of enzymes and pathways involved in these processes, the regulation of carbon partitioning between catabolism and anabolism remains poorly understood. Metabolic flux analysis (MFA) aims at quantifying fluxes in metabolic networks and provides new insights for the study of the TCA cycle and associated pathways. This chapter presents briefly the principles of MFA, and describes how C-14-tracing evolved to C-13-MFA, and more recently to C-13-INST-MFA. Such analyses have provided insights about the origin of carbon atoms entering the TCA cycle and the partitioning between respiration and biosyntheses

Application of metabolic flux analysis to plants.

This volume compiles a series of chapters that cover the major aspects of plant metabolic flux analysis, such as but not limited to labeling of plant material, acquisition of labeling data, mathematical modeling of metabolic network at the cell, tissue, and plant level. A short revue, including methodological points and applications of flux analysis to plants, is presented in this introductory chapter

Methods for the qualitative and quantitative analysis of metabolic fluxes

International audienc

Quantification of compartmented metabolic fluxes in maize root tips using isotope distribution from 13C- or 14C-labeled glucose

International audienc