A role for fermentation in aerobic conditions as revealed by computational analysis of maize root metabolism during growth by cell elongation.

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.

A hybrid kinetic and constraint-based model of leaf metabolism allows predictions of metabolic fluxes in different environments.

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 CO2 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 CO2 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 CO2 . 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.

Isotopically non-stationary metabolic flux analysis of heterotrophic Arabidopsis thaliana cell cultures.

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.

Shining a light on NAD- and NADP-based metabolism in plants.

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.

Metabolic control of nitrogen fixation in rhizobium-legume symbioses.

Rhizobia induce nodule formation on legume roots and differentiate into bacteroids, which catabolize plant-derived dicarboxylates to reduce atmospheric N2 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 13C 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 N2 fixation, legumes promote ammonia secretion by bacteroids. This is a defining feature of rhizobium-legume symbioses.

Alternative Crassulacean Acid Metabolism Modes Provide Environment-Specific Water-Saving Benefits in a Leaf Metabolic Model.

Crassulacean acid metabolism (CAM) evolved in arid environments as a water-saving alternative to C3 photosynthesis. There is great interest in engineering more drought-resistant crops by introducing CAM into C3 plants. However, it is unknown whether full CAM or alternative water-saving modes would be more productive in the environments typically experienced by C3 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.

Flux balance analysis of metabolism during growth by osmotic cell expansion and its application to tomato fruits.

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.

The role of nitrite and nitric oxide under low oxygen conditions in plants.

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.

Limitations of Deuterium-Labelled Substrates for Quantifying NADPH Metabolism in Heterotrophic Arabidopsis Cell Cultures.

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 [1-2H]glucose or D2O. 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.

Leaf Energy Balance Requires Mitochondrial Respiration and Export of Chloroplast NADPH in the Light.

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 CO2 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.

Euglena Central Metabolic Pathways and Their Subcellular Locations.

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.

Computational analysis of the productivity potential of CAM.

There is considerable interest in transferring crassulacean acid metabolism (CAM) to C3 crops to improve their water-use efficiency. However, because the CAM biochemical cycle is energetically costly, it is unclear what impact this would have on yield. Using diel flux balance analysis of the CAM and C3 leaf metabolic networks, we show that energy consumption is three-fold higher in CAM at night. However, this additional cost of CAM can be entirely offset by the carbon-concentrating effect of malate decarboxylation behind closed stomata during the day. Depending on the resultant rates of the carboxylase and oxygenase activities of rubisco, the productivity of the PEPCK-CAM subtype is 74-100% of the C3 network. We conclude that CAM does not impose a significant productivity penalty and that engineering CAM into C3 crops is likely to lead to a major increase in water-use efficiency without substantially affecting yield.

Cell-Type Specific Metabolic Flux Analysis: A Challenge for Metabolic Phenotyping and a Potential Solution in Plants.

Stable isotope labelling experiments are used routinely in metabolic flux analysis (MFA) to determine the metabolic phenotype of cells and tissues. A complication arises in multicellular systems because single cell measurements of transcriptomes, proteomes and metabolomes in multicellular organisms suggest that the metabolic phenotype will differ between cell types. In silico analysis of simulated metabolite isotopomer datasets shows that cellular heterogeneity confounds conventional MFA because labelling data averaged over multiple cell types does not necessarily yield averaged flux values. A potential solution to this problem-the use of cell-type specific reporter proteins as a source of cell-type specific labelling data-is proposed and the practicality of implementing this strategy in the roots of Arabidopsis thaliana seedlings is explored. A protocol for the immunopurification of ectopically expressed green fluorescent protein (GFP) from Arabidopsis thaliana seedlings using a GFP-binding nanobody is developed, and through GC-MS analysis of protein hydrolysates it is established that constitutively expressed GFP reports accurately on the labelling of total protein in root tissues. It is also demonstrated that the constitutive expression of GFP does not perturb metabolism. The principal obstacle to the implementation of the method in tissues with cell-type specific GFP expression is the sensitivity of the GC-MS system.

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 labeled 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 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 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 the investigation of other plant materials.

Protein-protein interactions and metabolite channelling in the plant tricarboxylic acid cycle.

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.

Nitrite Protects Mitochondrial Structure and Function under Hypoxia.

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.

Lipogenesis and Redox Balance in Nitrogen-Fixing Pea Bacteroids.

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-ß-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-ß-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.

A Method of Accounting for Enzyme Costs in Flux Balance Analysis Reveals Alternative Pathways and Metabolite Stores in an Illuminated Arabidopsis Leaf.

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.

Nitric oxide induces the alternative oxidase pathway in Arabidopsis seedlings deprived of inorganic phosphate.

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.