Search

Guard cell metabolism is crucial for stomatal dynamics, but a full understanding of its role is hampered by experimental limitations and the flexible nature of the metabolic network. To tackle this challenge, we constructed a time-resolved stoichiometric model of guard cell metabolism that accounts for energy and osmolyte requirements and which is integrated with the mesophyll. The model resolved distinct roles for starch, sugars, and malate in guard cell metabolism and revealed several unexpected flux patterns in central metabolism. During blue light-mediated stomatal opening, starch breakdown was the most efficient way to generate osmolytes with downregulation of glycolysis allowing starch-derived glucose to accumulate as a cytosolic osmolyte. Maltose could also accumulate as a cytosolic osmoticum, although this made the metabolic system marginally less efficient. The metabolic energy for stomatal opening was predicted to be derived independently of starch, using nocturnally accumulated citrate which was metabolized in the tricarboxylic acid cycle to malate to provide mitochondrial reducing power for ATP synthesis. In white light-mediated stomatal opening, malate transferred reducing equivalents from guard cell photosynthesis to mitochondria for ATP production. Depending on the capacity for guard cell photosynthesis, glycolysis showed little flux during the day but was crucial for energy metabolism at night. In summary, our analyses have corroborated recent findings in Arabidopsis guard cell research, resolved conflicting observations by highlighting the flexibility of guard cell metabolism, and proposed new metabolic flux modes for further experimental testing., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

The root is a well-studied example of cell specialisation, yet little is known about the metabolism that supports the transport functions and growth of different root cell types. To address this, we used computational modelling to study metabolism in the elongation zone of a maize lateral root. A functional-structural model captured the cell-anatomical features of the root and modelled how they changed as the root elongated. From these data, we derived constraints for a flux balance analysis model that predicted metabolic fluxes of the 11 concentric rings of cells in the root. We discovered a distinct metabolic flux pattern in the cortical cell rings, endodermis and pericycle (but absent in the epidermis) that involved a high rate of glycolysis and production of the fermentation end-products lactate and ethanol. This aerobic fermentation was confirmed experimentally by metabolite analysis. The use of fermentation in the model was not obligatory but was the most efficient way to meet the specific demands for energy, reducing power and carbon skeletons of expanding cells. Cytosolic acidification was avoided in the fermentative mode due to the substantial consumption of protons by lipid synthesis. These results expand our understanding of fermentative metabolism beyond that of hypoxic niches and suggest that fermentation could play an important role in the metabolism of aerobic tissues., (© 2023 Society for Experimental Biology and John Wiley & Sons Ltd.)

Fluxes are the ultimate phenotype of metabolism and their accurate quantification is fundamental to any understanding of metabolic networks. Steady state metabolic flux analysis has been the method of choice for quantifying fluxes in heterotrophic cells, but it is unable to measure fluxes during short-lived metabolic states, such as a transient oxidative load. Isotopically non-stationary metabolic flux analysis (INST-MFA) can be performed over shorter timescales (minutes - hours) and might overcome this limitation. INST-MFA has recently been applied to photosynthesising leaves, but agriculturally important tissues such as roots and storage organs, or plants during the night are heterotrophic. Here we outline the application of INST-MFA to heterotrophic plant cells. Using INST-MFA we were able to identify changes in the fluxes supported by phosphoenolpyruvate carboxylase and malic enzyme under oxidative load, highlighting the potential of INST-MFA to measure fluxes during short-lived metabolic states. We discuss the challenges in applying INST-MFA, and highlight further development required before it can be routinely used to quantify fluxes in heterotrophic plant cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Smith, Ratcliffe and Kruger.)

While flux balance analysis (FBA) provides a framework for predicting steady-state leaf metabolic network fluxes, it does not readily capture the response to environmental variables without being coupled to other modelling formulations. To address this, we coupled an FBA model of 903 reactions of soybean (Glycine max) leaf metabolism with e-photosynthesis, a dynamic model that captures the kinetics of 126 reactions of photosynthesis and associated chloroplast carbon metabolism. Successful coupling was achieved in an iterative formulation in which fluxes from e-photosynthesis were used to constrain the FBA model and then, in turn, fluxes computed from the FBA model used to update parameters in e-photosynthesis. This process was repeated until common fluxes in the two models converged. Coupling did not hamper the ability of the kinetic module to accurately predict the carbon assimilation rate, photosystem II electron flux, and starch accumulation of field-grown soybean at two CO 2 concentrations. The coupled model also allowed accurate predictions of additional parameters such as nocturnal respiration, as well as analysis of the effect of light intensity and elevated CO 2 on leaf metabolism. Predictions included an unexpected decrease in the rate of export of sucrose from the leaf at high light, due to altered starch-sucrose partitioning, and altered daytime flux modes in the tricarboxylic acid cycle at elevated CO 2 . Mitochondrial fluxes were notably different between growing and mature leaves, with greater anaplerotic, tricarboxylic acid cycle and mitochondrial ATP synthase fluxes predicted in the former, primarily to provide carbon skeletons and energy for protein synthesis., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

The pyridine nucleotides nicotinamide adenine dinucleotide [NAD(H)] and nicotinamide adenine dinucleotide phosphate [NADP(H)] simultaneously act as energy transducers, signalling molecules, and redox couples. Recent research into photosynthetic optimisation, photorespiration, immunity, hypoxia/oxygen signalling, development, and post-harvest metabolism have all identified pyridine nucleotides as key metabolites. Further understanding will require accurate description of NAD(P)(H) metabolism, and genetically encoded fluorescent biosensors have recently become available for this purpose. Although these biosensors have begun to provide novel biological insights, their limitations must be considered and the information they provide appropriately interpreted. We provide a framework for understanding NAD(P)(H) metabolism and explore what fluorescent biosensors can, and cannot, tell us about plant biology, looking ahead to the pressing questions that could be answered with further development of these tools., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Rhizobia induce nodule formation on legume roots and differentiate into bacteroids, which catabolize plant-derived dicarboxylates to reduce atmospheric N 2 into ammonia. Despite the agricultural importance of this symbiosis, the mechanisms that govern carbon and nitrogen allocation in bacteroids and promote ammonia secretion to the plant are largely unknown. Using a metabolic model derived from genome-scale datasets, we show that carbon polymer synthesis and alanine secretion by bacteroids facilitate redox balance in microaerobic nodules. Catabolism of dicarboxylates induces not only a higher oxygen demand but also a higher NADH/NAD + ratio than sugars. Modeling and 13 C metabolic flux analysis indicate that oxygen limitation restricts the decarboxylating arm of the tricarboxylic acid cycle, which limits ammonia assimilation into glutamate. By tightly controlling oxygen supply and providing dicarboxylates as the energy and electron source donors for N 2 fixation, legumes promote ammonia secretion by bacteroids. This is a defining feature of rhizobium-legume symbioses., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

Crassulacean acid metabolism (CAM) evolved in arid environments as a water-saving alternative to C 3 photosynthesis. There is great interest in engineering more drought-resistant crops by introducing CAM into C 3 plants. However, it is unknown whether full CAM or alternative water-saving modes would be more productive in the environments typically experienced by C 3 crops. To study the effect of temperature and relative humidity on plant metabolism in the context of water saving, we coupled a time-resolved diel (based on a 24-h day-night cycle) model of leaf metabolism to an environment-dependent gas-exchange model. This combined model allowed us to study the emergence of CAM as a trade-off between leaf productivity and water saving. We show that vacuolar storage capacity in the leaf is a major determinant of the extent of CAM. Moreover, our model identified an alternative CAM cycle involving mitochondrial isocitrate dehydrogenase as a potential contributor to initial carbon fixation at night. Simulations across a range of environmental conditions show that the water-saving potential of CAM strongly depends on the daytime weather conditions and that the additional water-saving effect of carbon fixation by isocitrate dehydrogenase can reach 11% total water saving for the conditions tested., (© 2020 American Society of Plant Biologists. All rights reserved.)

Cell expansion is a significant contributor to organ growth and is driven by the accumulation of osmolytes to increase cell turgor pressure. Metabolic modelling has the potential to provide insights into the processes that underpin osmolyte synthesis and transport, but the main computational approach for predicting metabolic network fluxes, flux balance analysis, often uses biomass composition as the main output constraint and ignores potential changes in cell volume. Here we present growth-by-osmotic-expansion flux balance analysis (GrOE-FBA), a framework that accounts for both the metabolic and ionic contributions to the osmotica that drive cell expansion, as well as the synthesis of protein, cell wall and cell membrane components required for cell enlargement. Using GrOE-FBA, the metabolic fluxes in dividing and expanding cells were analysed, and the energetic costs for metabolite biosynthesis and accumulation in the two scenarios were found to be surprisingly similar. The expansion phase of tomato fruit growth was also modelled using a multiphase single-optimization GrOE-FBA model and this approach gave accurate predictions of the major metabolite levels throughout fruit development, as well as revealing a role for transitory starch accumulation in ensuring optimal fruit development., (© 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Plant tissues, particularly roots, can be subjected to periods of hypoxia due to environmental circumstances. Plants have developed various adaptations in response to hypoxic stress and these have been described extensively. Less well-appreciated is the body of evidence demonstrating that scavenging of nitric oxide (NO) and the reduction of nitrate/nitrite regulate important mechanisms that contribute to tolerance to hypoxia. Although ethylene controls hyponasty and aerenchyma formation, NO production apparently regulates hypoxic ethylene biosynthesis. In the hypoxic mitochondrion, cytochrome c oxidase, which is a major source of NO, also is inhibited by NO, thereby reducing the respiratory rate and enhancing local oxygen concentrations. Nitrite can maintain ATP generation under hypoxia by coupling its reduction to the translocation of protons from the inner side of mitochondria and generating an electrochemical gradient. This reaction can be further coupled to a reaction whereby nonsymbiotic haemoglobin oxidizes NO to nitrate. In addition to these functions, nitrite has been reported to influence mitochondrial structure and supercomplex formation, as well as playing a role in oxygen sensing via the N-end rule pathway. These studies establish that nitrite and NO perform multiple functions during plant hypoxia and suggest that further research into the underlying mechanisms is warranted., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

NADPH is the primary source of cellular reductant for biosynthesis, and strategies for increasing productivity via metabolic engineering need to take account of the requirement for reducing power. In plants, while the oxidative pentose phosphate pathway is the most direct route for NADPH production in heterotrophic tissues, there is increasing evidence that other pathways make significant contributions to redox balance. Deuterium-based isotopic labelling strategies have recently been developed to quantify the relative production of NADPH from different pathways in mammalian cells, but the application of these methods to plants has not been critically evaluated. In this study, LC-MS was used to measure deuterium incorporation into metabolites extracted from heterotrophic Arabidopsis cell cultures grown on [1- 2 H]glucose or D 2 O. The results show that a high rate of flavin-enzyme-catalysed water exchange obscures labelling of NADPH from deuterated substrates and that this exchange cannot be accurately accounted for due to exchange between triose- and hexose-phosphates. In addition, the duplication of NADPH generating reactions between subcellular compartments can confound analysis based on whole cell extracts. Understanding how the structure of the metabolic network affects the applicability of deuterium labelling methods is a prerequisite for development of more effective flux determination strategies, ensuring data are both quantitative and representative of endogenous biological processes.

In higher plants ammonium (NH 4 + ) assimilation occurs mainly through the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway. Nevertheless, when plants are exposed to stress conditions, such as excess of ammonium, the contribution of alternative routes of ammonium assimilation such as glutamate dehydrogenase (GDH) and asparagine synthetase (AS) activities might serve as detoxification mechanisms. In this work, the in vivo functions of these pathways were studied after supplying an excess of ammonium to tomato (Solanum lycopersicum L. cv. Agora Hybrid F1) roots previously adapted to grow under either nitrate or ammonium nutrition. The short-term incorporation of labelled ammonium ( 15 NH 4 + ) into the main amino acids was determined by GC-MS in the presence or absence of methionine sulphoximine (MSX) and azaserine (AZA), inhibitors of GS and GOGAT activities, respectively. Tomato roots were able to respond rapidly to excess ammonium by enhancing ammonium assimilation regardless of the previous nutritional regime to which the plant was adapted to grow. The assimilation of 15 NH 4 + could take place through pathways other than GS/GOGAT, since the inhibition of GS and GOGAT did not completely impede the incorporation of the labelled nitrogen into major amino acids. The in vivo formation of Asn by AS was shown to be exclusively Gln-dependent since the root was unable to incorporate 15 NH 4 + directly into Asn. On the other hand, an in vivo aminating capacity was revealed for GDH, since newly labelled Glu synthesis occurred even when GS and/or GOGAT activities were inhibited. The aminating GDH activity in tomato roots responded to an excess ammonium supply independently of the previous nutritional regime to which the plant had been subjected., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Key aspects of leaf mitochondrial metabolism in the light remain unresolved. For example, there is debate about the relative importance of exporting reducing equivalents from mitochondria for the peroxisomal steps of photorespiration versus oxidation of NADH to generate ATP by oxidative phosphorylation. Here, we address this and explore energetic coupling between organelles in the light using a diel flux balance analysis model. The model included more than 600 reactions of central metabolism with full stoichiometric accounting of energy production and consumption. Different scenarios of energy availability (light intensity) and demand (source leaf versus a growing leaf) were considered, and the model was constrained by the nonlinear relationship between light and CO 2 assimilation rate. The analysis demonstrated that the chloroplast can theoretically generate sufficient ATP to satisfy the energy requirements of the rest of the cell in addition to its own. However, this requires unrealistic high light use efficiency and, in practice, the availability of chloroplast-derived ATP is limited by chloroplast energy dissipation systems, such as nonphotochemical quenching, and the capacity of the chloroplast ATP export shuttles. Given these limitations, substantial mitochondrial ATP synthesis is required to fulfill cytosolic ATP requirements, with only minimal, or zero, export of mitochondrial reducing equivalents. The analysis also revealed the importance of exporting reducing equivalents from chloroplasts to sustain photorespiration. Hence, the chloroplast malate valve and triose phosphate-3-phosphoglycerate shuttle are predicted to have important metabolic roles, in addition to their more commonly discussed contribution to the avoidance of photooxidative stress., (© 2019 The author(s). All Rights Reserved.)

Euglenids are a group of algae of great interest for biotechnology, with a large and complex metabolic capability. To study the metabolic network, it is necessary to know where the component enzymes are in the cell, but despite a long history of research into Euglena , the subcellular locations of many major pathways are only poorly defined. Euglena is phylogenetically distant from other commonly studied algae, they have secondary plastids bounded by three membranes, and they can survive after destruction of their plastids. These unusual features make it difficult to assume that the subcellular organization of the metabolic network will be equivalent to that of other photosynthetic organisms. We analysed bioinformatic, biochemical, and proteomic information from a variety of sources to assess the subcellular location of the enzymes of the central metabolic pathways, and we use these assignments to propose a model of the metabolic network of Euglena . Other than photosynthesis, all major pathways present in the chloroplast are also present elsewhere in the cell. Our model demonstrates how Euglena can synthesise all the metabolites required for growth from simple carbon inputs, and can survive in the absence of chloroplasts.

Protein complexes of sequential metabolic enzymes, often termed metabolons, may permit direct channelling of metabolites between the enzymes, providing increased control over metabolic pathway fluxes. Experimental evidence supporting their existence in vivo remains fragmentary. In the present study, we test binary interactions of the proteins constituting the plant tricarboxylic acid (TCA) cycle. We integrate (semi-)quantitative results from affinity purification-mass spectrometry, split-luciferase and yeast-two-hybrid assays to generate a single reliability score for assessing protein-protein interactions. By this approach, we identify 158 interactions including those between catalytic subunits of sequential enzymes and between subunits of enzymes mediating non-adjacent reactions. We reveal channelling of citrate and fumarate in isolated potato mitochondria by isotope dilution experiments. These results provide evidence for a functional TCA cycle metabolon in plants, which we discuss in the context of contemporary understanding of this pathway in other kingdoms.

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 14 CO 2 from a range of positionally labeled forms of [ 14 C]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 utilization. Such information can be used to determine whether a 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 14 CO 2 release during metabolism of exogenously supplied [1- 14 C]-, [2- 14 C]-, [3,4- 14 C]-, and [6- 14 C]glucose. The method is exemplified by studies on Arabidopsis cell suspension cultures, but the protocol can be easily adapted for the investigation of other plant materials.

Oxygen deprivation leads to changes in mitochondrial morphology and impaired flow of reducing equivalents through the electron transport chain. The extent of these changes depends on the duration and severity of the treatment as well as on the species and cell type. Nitrate is known to ameliorate these effects in some instances, but it is possible that it is nitrite, rather than nitrate, that is the key to the mechanism. To test this, mitochondria were isolated from 21-day-old pea (Pisum sativum) roots and incubated for 90 min under normoxia or hypoxia in the presence or absence of 0.5 mM nitrite. The supply of nitrite under hypoxia led to nitric oxide production, improved mitochondrial integrity, improved energization of the inner mitochondrial membrane, increased ATP synthesis, decreased production of reactive oxygen species and decreased lipid peroxidation. It also resulted in higher levels and activities of complex I and the supercomplex I + III2. It is concluded that nitrite has an important role in maintaining mitochondrial function under hypoxia, and that it achieves this through the reduction of nitrite to nitric oxide by the mitochondrial electron transport chain., (© The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The nonprotein amino acid γ-aminobutyric acid (GABA) is the most abundant amino acid in the tomato (Solanum lycopersicum) leaf apoplast and is synthesized by Arabidopsis thaliana in response to infection by the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (hereafter called DC3000). High levels of exogenous GABA have previously been shown to repress the expression of the type III secretion system (T3SS) in DC3000, resulting in reduced elicitation of the hypersensitive response (HR) in the nonhost plant tobacco (Nicotiana tabacum). This study demonstrates that the GABA permease GabP provides the primary mechanism for GABA uptake by DC3000 and that the gabP deletion mutant ΔgabP is insensitive to GABA-mediated repression of T3SS expression. ΔgabP displayed an enhanced ability to elicit the HR in young tobacco leaves and in tobacco plants engineered to produce increased levels of GABA, which supports the hypothesis that GABA uptake via GabP acts to regulate T3SS expression in planta. The observation that P. syringae can be rendered insensitive to GABA through loss of gabP but that gabP is retained by this bacterium suggests that GabP is important for DC3000 in a natural setting, either for nutrition or as a mechanism for regulating gene expression. [Formula: see text] Copyright © 2016 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Unlabelled: Within legume root nodules, rhizobia differentiate into bacteroids that oxidize host-derived dicarboxylic acids, which is assumed to occur via the tricarboxylic acid (TCA) cycle to generate NAD(P)H for reduction of N2 Metabolic flux analysis of laboratory-grown Rhizobium leguminosarum showed that the flux from [(13)C]succinate was consistent with respiration of an obligate aerobe growing on a TCA cycle intermediate as the sole carbon source. However, the instability of fragile pea bacteroids prevented their steady-state labeling under N2-fixing conditions. Therefore, comparative metabolomic profiling was used to compare free-living R. leguminosarum with pea bacteroids. While the TCA cycle was shown to be essential for maximal rates of N2 fixation, levels of pyruvate (5.5-fold reduced), acetyl coenzyme A (acetyl-CoA; 50-fold reduced), free coenzyme A (33-fold reduced), and citrate (4.5-fold reduced) were much lower in bacteroids. Instead of completely oxidizing acetyl-CoA, pea bacteroids channel it into both lipid and the lipid-like polymer poly-β-hydroxybutyrate (PHB), the latter via a type III PHB synthase that is active only in bacteroids. Lipogenesis may be a fundamental requirement of the redox poise of electron donation to N2 in all legume nodules. Direct reduction by NAD(P)H of the likely electron donors for nitrogenase, such as ferredoxin, is inconsistent with their redox potentials. Instead, bacteroids must balance the production of NAD(P)H from oxidation of acetyl-CoA in the TCA cycle with its storage in PHB and lipids., Importance: Biological nitrogen fixation by symbiotic bacteria (rhizobia) in legume root nodules is an energy-expensive process. Within legume root nodules, rhizobia differentiate into bacteroids that oxidize host-derived dicarboxylic acids, which is assumed to occur via the TCA cycle to generate NAD(P)H for reduction of N2 However, direct reduction of the likely electron donors for nitrogenase, such as ferredoxin, is inconsistent with their redox potentials. Instead, bacteroids must balance oxidation of plant-derived dicarboxylates in the TCA cycle with lipid synthesis. Pea bacteroids channel acetyl-CoA into both lipid and the lipid-like polymer poly-β-hydroxybutyrate, the latter via a type II PHB synthase. Lipogenesis is likely to be a fundamental requirement of the redox poise of electron donation to N2 in all legume nodules., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Flux balance analysis of plant metabolism is an established method for predicting metabolic flux phenotypes and for exploring the way in which the plant metabolic network delivers specific outcomes in different cell types, tissues, and temporal phases. A recurring theme is the need to explore the flexibility of the network in meeting its objectives and, in particular, to establish the extent to which alternative pathways can contribute to achieving specific outcomes. Unfortunately, predictions from conventional flux balance analysis minimize the simultaneous operation of alternative pathways, but by introducing flux-weighting factors to allow for the variable intrinsic cost of supporting each flux, it is possible to activate different pathways in individual simulations and, thus, to explore alternative pathways by averaging thousands of simulations. This new method has been applied to a diel genome-scale model of Arabidopsis (Arabidopsis thaliana) leaf metabolism to explore the flexibility of the network in meeting the metabolic requirements of the leaf in the light. This identified alternative flux modes in the Calvin-Benson cycle revealed the potential for alternative transitory carbon stores in leaves and led to predictions about the light-dependent contribution of alternative electron flow pathways and futile cycles in energy rebalancing. Notable features of the analysis include the light-dependent tradeoff between the use of carbohydrates and four-carbon organic acids as transitory storage forms and the way in which multiple pathways for the consumption of ATP and NADPH can contribute to the balancing of the requirements of photosynthetic metabolism with the energy available from photon capture., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Phosphate starvation compromises electron flow through the cytochrome pathway of the mitochondrial electron transport chain, and plants commonly respond to phosphate deprivation by increasing flow through the alternative oxidase (AOX). To test whether this response is linked to the increase in nitric oxide (NO) production that also increases under phosphate starvation, Arabidopsis thaliana seedlings were grown for 15 d on media containing either 0 or 1mM inorganic phosphate. The effects of the phosphate supply on growth, the production of NO, respiration, the AOX level and the production of superoxide were compared for wild-type (WT) seedlings and the nitrate reductase double mutant nia. Phosphate deprivation increased NO production in WT roots, and the AOX level and the capacity of the alternative pathway to consume electrons in WT seedlings; whereas the same treatment failed to stimulate NO production and AOX expression in the nia mutant, and the plants had an altered growth phenotype. The NO donor S-nitrosoglutathione rescued the growth phenotype of the nia mutants under phosphate deprivation to some extent, and it also increased the respiratory capacity of AOX. It is concluded that NO is required for the induction of the AOX pathway when seedlings are grown under phosphate-limiting conditions., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Although the flows of material through metabolic networks are central to cell function, they are not easy to measure other than at the level of inputs and outputs. This is particularly true in plant cells, where the network spans multiple subcellular compartments and where the network may function either heterotrophically or photoautotrophically. For many years, kinetic modelling of pathways provided the only method for describing the operation of fragments of the network. However, more recently, it has become possible to map the fluxes in central carbon metabolism using the stable isotope labelling techniques of metabolic flux analysis (MFA), and to predict intracellular fluxes using constraints-based modelling procedures such as flux balance analysis (FBA). These approaches were originally developed for the analysis of microbial metabolism, but over the last decade, they have been adapted for the more demanding analysis of plant metabolic networks. Here, the principal features of MFA and FBA as applied to plants are outlined, followed by a discussion of the insights that have been gained into plant metabolic networks through the application of these time-consuming and non-trivial methods. The discussion focuses on how a system-wide view of plant metabolism has increased our understanding of network structure, metabolic perturbations and the provision of reducing power and energy for cell function. Current methodological challenges that limit the scope of plant MFA are discussed and particular emphasis is placed on the importance of developing methods for cell-specific MFA.

Plant mitochondria generate nitric oxide (NO) under anoxia through the action of cytochrome c oxidase and other electron transport chain components on nitrite. This reductive mechanism operates under aerobic conditions at high electron transport rates. Indirect evidence also indicates that the oxidative pathway of NO production may be associated with mitochondria. We review the consequences of mitochondrial NO production, including the inhibition of oxygen uptake by cytochrome c oxidase, the inhibition of aconitase and succinate dehydrogenase, the induction of alternative oxidase, and the nitrosylation of several proteins, including glycine decarboxylase. The importance of these events in adaptation to abiotic and biotic stresses is discussed., (Copyright © 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Although leaves have to accommodate markedly different metabolic flux patterns in the light and the dark, models of leaf metabolism based on flux-balance analysis (FBA) have so far been confined to consideration of the network under continuous light. An FBA framework is presented that solves the two phases of the diel cycle as a single optimization problem and, thus, provides a more representative model of leaf metabolism. The requirement to support continued export of sugar and amino acids from the leaf during the night and to meet overnight cellular maintenance costs forces the model to set aside stores of both carbon and nitrogen during the day. With only minimal constraints, the model successfully captures many of the known features of C 3 leaf metabolism, including the recently discovered role of citrate synthesis and accumulation in the night as a precursor for the provision of carbon skeletons for amino acid synthesis during the day. The diel FBA model can be applied to other temporal separations, such as that which occurs in Crassulacean acid metabolism (CAM) photosynthesis, allowing a system-level analysis of the energetics of CAM. The diel model predicts that there is no overall energetic advantage to CAM, despite the potential for suppression of photorespiration through CO 2 concentration. Moreover, any savings in enzyme machinery costs through suppression of photorespiration are likely to be offset by the higher flux demand of the CAM cycle. It is concluded that energetic or nitrogen use considerations are unlikely to be evolutionary drivers for CAM photosynthesis., (© 2014 American Society of Plant Biologists. All Rights Reserved.)

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., (© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.)

The goal of this study was to characterise the metabolic flux phenotype of transgenic tobacco (Nicotiana tabacum) hairy roots engineered for increased biosynthesis of geraniol, an intermediate of the terpenoid indole alkaloid pathway. Steady state, stable isotope labelling was used to determine flux maps of central carbon metabolism for transgenic lines over-expressing (i) plastid-targeted geraniol synthase (pGES) from Valeriana officinalis, and (ii) pGES in combination with plastid-targeted geranyl pyrophosphate synthase from Arabidopsis thaliana (pGES+pGPPS), as well as for wild type and control-vector-transformed roots. Fluxes were constrained by the redistribution of label from [1-¹³C]-, [2-¹³C]- or [¹³C6]glucose into amino acids, sugars and organic acids at isotopic steady state, and by biomass output fluxes determined from the fractionation of [U-¹⁴C]glucose into insoluble polymers. No significant differences in growth and biomass composition were observed between the lines. The pGES line accumulated significant amounts of geraniol/geraniol glycosides (151±24 ng/mg dry weight) and the de novo synthesis of geraniol in pGES was confirmed by ¹³C labelling analysis. The pGES+pGPPS also accumulated geraniol and geraniol glycosides, but to lower levels than the pGES line. Although there was a distinct impact of the transgenes at the level of geraniol synthesis, other network fluxes were unaffected, reflecting the capacity of central metabolism to meet the relatively modest demand for increased precursors in the transgenic lines. It is concluded that re-engineering of the terpenoid indole alkaloid pathway will only require simultaneous manipulation of the steps producing the pathway precursors that originate in central metabolism in tissues engineered to produce at least an order of magnitude more geraniol than has been achieved so far., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The analysis of stable isotope incorporation following feeding of (13)C-labeled precursors to plant tissues provides the constraints necessary for metabolic flux analysis. This protocol describes the use of one-dimensional (1)H and (13)C nuclear magnetic resonance spectroscopy for the quantification of (13)C enrichments and isotopomer abundances in mixtures of metabolites or hydrolyzed biomass components.

While steady-state (13)C metabolic flux analysis is a powerful method for deducing multiple fluxes in the central metabolic network of heterotrophic and mixotrophic plant tissues, it is also time-consuming and technically challenging. Key steps in the design and interpretation of steady-state (13)C labeling experiments are illustrated with a generic protocol based on applications to plant cell suspension cultures.

Flux balance models of metabolism generally utilize synthesis of biomass as the main determinant of intracellular fluxes. However, the biomass constraint alone is not sufficient to predict realistic fluxes in central heterotrophic metabolism of plant cells because of the major demand on the energy budget due to transport costs and cell maintenance. This major limitation can be addressed by incorporating transport steps into the metabolic model and by implementing a procedure that uses Pareto optimality analysis to explore the trade-off between ATP and NADPH production for maintenance. This leads to a method for predicting cell maintenance costs on the basis of the measured flux ratio between the oxidative steps of the oxidative pentose phosphate pathway and glycolysis. We show that accounting for transport and maintenance costs substantially improves the accuracy of fluxes predicted from a flux balance model of heterotrophic Arabidopsis cells in culture, irrespective of the objective function used in the analysis. Moreover, when the new method was applied to cells under control, elevated temperature and hyper-osmotic conditions, only elevated temperature led to a substantial increase in cell maintenance costs. It is concluded that the hyper-osmotic conditions tested did not impose a metabolic stress, in as much as the metabolic network is not forced to devote more resources to cell maintenance., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)

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., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)

Organic acid content is regarded as one of the most important quality traits of fresh tomato (Solanum lycopersicum). However, the complexity of carboxylic acid metabolism and storage means that it is difficult to predict the best way to engineer altered carboxylic acid levels. Here, we used a biochemical analysis of a tomato introgression line with increased levels of fruit citrate and malate at breaker stage to identify a metabolic engineering target that was subsequently tested in transgenic plants. Increased carboxylic acid levels in introgression line 2-5 were not accompanied by changes in the pattern of carbohydrate oxidation by pericarp discs or the catalytic capacity of tricarboxylic acid cycle enzymes measured in isolated mitochondria. However, there was a significant decrease in the maximum catalytic activity of aconitase in total tissue extracts, suggesting that a cytosolic isoform of aconitase was affected. To test the role of cytosolic aconitase in controlling fruit citrate levels, we analyzed fruit of transgenic lines expressing an antisense construct against SlAco3b, one of the two tomato genes encoding aconitase. A green fluorescent protein fusion of SlAco3b was dual targeted to cytosol and mitochondria, while the other aconitase, SlAco3a, was exclusively mitochondrial when transiently expressed in tobacco (Nicotiana tabacum) leaves. Both aconitase transcripts were decreased in fruit from transgenic lines, and aconitase activity was reduced by about 30% in the transgenic lines. Other measured enzymes of carboxylic acid metabolism were not significantly altered. Both citrate and malate levels were increased in ripe fruit of the transgenic plants, and as a consequence, total carboxylic acid content was increased by 50% at maturity.

Cysteine synthesis is catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL) in the cytosol, plastids, and mitochondria of plants. Biochemical analyses of recombinant plant SAT and OAS-TL indicate that the reversible association of the proteins in the cysteine synthase complex (CSC) controls cellular sulfur homeostasis. However, the relevance of CSC formation in each compartment for flux control of cysteine synthesis remains controversial. Here, we demonstrate the interaction between mitochondrial SAT3 and OAS-TL C in planta by FRET and establish the role of the mitochondrial CSC in the regulation of cysteine synthesis. NMR spectroscopy of isolated mitochondria from WT, serat2;2, and oastl-C plants showed the SAT-dependent export of OAS. The presence of cysteine resulted in reduced OAS export in mitochondria of oastl-C mutants but not in WT mitochondria. This is in agreement with the stronger in vitro feedback inhibition of free SAT by cysteine compared with CSC-bound SAT and explains the high OAS export rate of WT mitochondria in the presence of cysteine. The predominant role of mitochondrial OAS synthesis was validated in planta by feeding [(3)H]serine to the WT and loss-of-function mutants for OAS-TLs in the cytosol, plastids, and mitochondria. On the basis of these results, we propose a new model in which the mitochondrial CSC acts as a sensor that regulates the level of SAT activity in response to sulfur supply and cysteine demand.

The transition from a pathway-centred view of plant metabolism to a network-wide perspective is still incomplete. Further progress in this direction requires tools to facilitate the structural description of the network on the basis of fully annotated genomes, techniques for modelling the properties of the network, and experimental methods for constraining the models and verifying their outputs. It also requires a focus on metabolic flux as the key to understanding the regulation of metabolic activity and the relationship between the inputs and outputs of the network. Progress is being made on several fronts and this Special Issue on 'Pathways and fluxes: exploring the plant metabolic network' describes current developments in the genomic reconstruction of metabolic networks, the application of flux-balance analysis to such networks, kinetic modelling, and both steady-state-and non-steady state isotope-based measurements of multiple fluxes in the network of central carbon metabolism. The papers also highlight insights that can be obtained from pathway analysis, particularly in relation to the thermodynamic and kinetic efficiency of the predicted and observed flux distributions.

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.