Strategies for investigating the plant metabolic network with steady-state metabolic flux analysis: lessons from an Arabidopsis cell culture and other systems.

Steady-state (13)C metabolic flux analysis (MFA) is currently the experimental method of choice for generating flux maps of the compartmented network of primary metabolism in heterotrophic and mixotrophic plant tissues. While statistically robust protocols for the application of steady-state MFA to plant tissues have been developed by several research groups, the implementation of the method is still far from routine. The effort required to produce a flux map is more than justified by the information that it contains about the metabolic phenotype of the system, but it remains the case that steady-state MFA is both analytically and computationally demanding. This article provides an overview of principles that underpin the implementation of steady-state MFA, focusing on the definition of the metabolic network responsible for redistribution of the label, experimental considerations relating to data collection, the modelling process that allows a set of metabolic fluxes to be deduced from the labelling data, and the interpretation of flux maps. The article draws on published studies of Arabidopsis cell cultures and other systems, including developing oilseeds, with the aim of providing practical guidance and strategies for handling the issues that arise when applying steady-state MFA to the complex metabolic networks encountered in plants

The metabolic flux phenotype of heterotrophic Arabidopsis cells reveals a complex response to changes in nitrogen supply.

The extent to which individual plants utilise nitrate and ammonium, the two principal nitrogen sources in the rhizosphere, is variable and many species require a balance between the two forms for optimal growth. The effects of nitrate and ammonium on gene expression, enzyme activity and metabolite composition have been documented extensively with the aim of understanding the way in which plant cells respond to the different forms of nitrogen, but ultimately the impact of these changes on the organisation and operation of the central metabolic network can only be addressed by analysing the fluxes supported by the network. Accordingly steady-state metabolic flux analysis was used to define the metabolic phenotype of a heterotrophic Arabidopsis thaliana cell culture grown in Murashige and Skoog and ammonium-free media, treatments that influenced growth and biomass composition. Fluxes through the central metabolic network were deduced from the redistribution of label into metabolic intermediates and end products observed when cells were labelled with [1-(13) C]-, [2-(13) C]- or [(13) C6 ]glucose, in tandem with (14) C-measurements of the net accumulation of biomass. Analysis of the flux maps showed that: (i) flux through the oxidative pentose phosphate pathway varied independently of the reductant demand for biosynthesis, (ii) non-plastidic processes made a significant and variable contribution to the provision of reducing power for the plastid, and (iii) the inclusion of ammonium in the growth medium increased cell maintenance costs, in agreement with the futile cycling model of ammonium toxicity. These conclusions highlight the complexity of the metabolic response to a change in nitrogen nutrition

The metabolic flux phenotype of heterotrophic Arabidopsis cells reveals a flexible balance between the cytosolic and plastidic contributions to carbohydrate oxidation in response to phosphate limitation

Understanding the mechanisms that allow plants to respond to variable and reduced availability of inorganic phosphate is of increasing agricultural importance because of the continuing depletion of the rock phosphate reserves that are used to combat inadequate phosphate levels in the soil. Changes in gene expression, protein levels, enzyme activities and metabolite levels all point to a reconfiguration of the central metabolic network in response to reduced availability of inorganic phosphate, but the metabolic significance of these changes can only be assessed in terms of the fluxes supported by the network. Steady-state metabolic flux analysis was used to define the metabolic phenotype of a heterotrophic Arabidopsis thaliana cell culture grown on a Murashige and Skoog medium containing 0, 1.25 or 5 mm inorganic phosphate. Fluxes through the central metabolic network were deduced from the redistribution of (13) C into metabolic intermediates and end products when cells were labelled with [1-(13) C], [2-(13) C], or [(13) C6 ]glucose, in combination with (14) C measurements of the rates of biomass accumulation. Analysis of the flux maps showed that reduced levels of phosphate in the growth medium stimulated flux through phosphoenolpyruvate carboxylase and malic enzyme, altered the balance between cytosolic and plastidic carbohydrate oxidation in favour of the plastid, and increased cell maintenance costs. We argue that plant cells respond to phosphate deprivation by reconfiguring the flux distribution through the pathways of carbohydrate oxidation to take advantage of better phosphate homeostasis in the plastid

The metabolic flux phenotype of heterotrophic Arabidopsis cells reveals a flexible balance between the cytosolic and plastidic contributions to carbohydrate oxidation in response to phosphate limitation

Understanding the mechanisms that allow plants to respond to variable and reduced availability of inorganic phosphate is of increasing agricultural importance because of the continuing depletion of the rock phosphate reserves that are used to combat inadequate phosphate levels in the soil. Changes in gene expression, protein levels, enzyme activities and metabolite levels all point to a reconfiguration of the central metabolic network in response to reduced availability of inorganic phosphate, but the metabolic significance of these changes can only be assessed in terms of the fluxes supported by the network. Steady-state metabolic flux analysis was used to define the metabolic phenotype of a heterotrophic Arabidopsis thaliana cell culture grown on a Murashige and Skoog medium containing 0, 1.25 or 5 mm inorganic phosphate. Fluxes through the central metabolic network were deduced from the redistribution of (13) C into metabolic intermediates and end products when cells were labelled with [1-(13) C], [2-(13) C], or [(13) C6 ]glucose, in combination with (14) C measurements of the rates of biomass accumulation. Analysis of the flux maps showed that reduced levels of phosphate in the growth medium stimulated flux through phosphoenolpyruvate carboxylase and malic enzyme, altered the balance between cytosolic and plastidic carbohydrate oxidation in favour of the plastid, and increased cell maintenance costs. We argue that plant cells respond to phosphate deprivation by reconfiguring the flux distribution through the pathways of carbohydrate oxidation to take advantage of better phosphate homeostasis in the plastid

Metabolic network fluxes in heterotrophic Arabidopsis cells: stability of the flux distribution under different oxygenation conditions.

Steady-state labeling experiments with [1-(13)C]Glc were used to measure multiple metabolic fluxes through the pathways of central metabolism in a heterotrophic cell suspension culture of Arabidopsis (Arabidopsis thaliana). The protocol was based on in silico modeling to establish the optimal labeled precursor, validation of the isotopic and metabolic steady state, extensive nuclear magnetic resonance analysis of the redistribution of label into soluble metabolites, starch, and protein, and a comprehensive set of biomass measurements. Following a simple modification of the cell culture procedure, cells were grown at two oxygen concentrations, and flux maps of central metabolism were constructed on the basis of replicated experiments and rigorous statistical analysis. Increased growth rate at the higher O(2) concentration was associated with an increase in fluxes throughout the network, and this was achieved without any significant change in relative fluxes despite differences in the metabolite profile of organic acids, amino acids, and carbohydrates. The balance between biosynthesis and respiration within the tricarboxylic acid cycle was unchanged, with 38% +/- 5% of carbon entering used for biosynthesis under standard O(2) conditions and 33% +/- 2% under elevated O(2). These results add to the emerging picture of the stability of the central metabolic network and its capacity to respond to physiological perturbations with the minimum of rearrangement. The lack of correlation between the change in metabolite profile, which implied significant disruption of the metabolic network following the alteration in the oxygen supply, and the unchanging flux distribution highlights a potential difficulty in the interpretation of metabolomic data

Subcellular flux analysis of central metabolism in a heterotrophic Arabidopsis cell suspension using steady-state stable isotope labeling.

The presence of cytosolic and plastidic pathways of carbohydrate oxidation is a characteristic feature of plant cell metabolism. Ideally, steady-state metabolic flux analysis, an emerging tool for creating flux maps of heterotrophic plant metabolism, would capture this feature of the metabolic phenotype, but the extent to which this can be achieved is uncertain. To address this question, fluxes through the pathways of central metabolism in a heterotrophic Arabidopsis (Arabidopsis thaliana) cell suspension culture were deduced from the redistribution of label in steady-state (13)C-labeling experiments using [1-(13)C]-, [2-(13)C]-, and [U-(13)C(6)]glucose. Focusing on the pentose phosphate pathway (PPP), multiple data sets were fitted simultaneously to models in which the subcellular compartmentation of the PPP was altered. The observed redistribution of the label could be explained by any one of three models of the subcellular compartmentation of the oxidative PPP, but other biochemical evidence favored the model in which the oxidative steps of the PPP were duplicated in the cytosol and plastids, with flux through these reactions occurring largely in the cytosol. The analysis emphasizes the inherent difficulty of analyzing the PPP without predefining the extent of its compartmentation and the importance of obtaining high-quality data that report directly on specific subcellular processes. The Arabidopsis flux map also shows that the potential ATP yield of respiration in heterotrophic plant cells can greatly exceed the direct metabolic requirements for biosynthesis, highlighting the need for caution when predicting flux through metabolic networks using assumptions based on the energetics of resource utilization

Assessing metabolic flux in plants with radiorespirometry

Carbohydrates are the dominant respiratory substrate in many plant cells. However, the route of carbohydrate oxidation varies depending on the relative cellular demands for energy, reductant and precursors for biosynthesis. During these processes individual substrate carbon atoms are differentially released as carbon dioxide by specific reactions in the network, and this can be measured by monitoring the release of 14CO2 from a range of positionally labelled forms of [14C]glucose. Although the relative amounts of carbon dioxide produced from different carbon positions do not allow precise determination of fluxes, they are indicative of the route of carbohydrate utilisation. Such information can be used to determine whether comprehensive metabolic flux analysis is merited, and also to facilitate independent verification of flux maps generated by other techniques. This chapter describes an approach to determine and interpret the pattern of oxidation of carbohydrates by monitoring 14CO2 release during metabolism of exogenously supplied [1-14C]-, [2-14C]-, [3,4-14C]- and [6-14C]glucose. The method is exemplified by studies on Arabidopsis cell suspension cultures, but the protocol can be easily adapted for investigation of other plant materials