Deuterium in marine organic biomarkers: toward a new tool for quantifying aquatic mixotrophy.

The traditional separation between primary producers (autotrophs) and consumers (heterotrophs) at the base of the marine food web is being increasingly replaced by the paradigm that mixoplankton, planktonic protists with the nutritional ability to use both phago(hetero)trophy and photo(auto)trophy to access energy are widespread globally. Thus, many 'phytoplankton' eat, while 50% of 'protozooplankton' also perform photosynthesis. Mixotrophy may enhance primary production, biomass transfer to higher trophic levels and the efficiency of the biological pump to sequester atmospheric CO2 into the deep ocean. Although this view is gaining traction, science lacks a tool to quantify the relative contributions of autotrophy and heterotrophy in planktonic protists. This hinders our understanding of their impacts on carbon cycling within marine pelagic ecosystems. It has been shown that the hydrogen (H) isotopic signature of lipids is uniquely sensitive to heterotrophy relative to autotrophy in plants and bacteria. Here, we explored whether it is also sensitive to the trophic status in protists. The new understanding of H isotope signature of lipid biomarkers suggests it offers great potential as a novel tool for quantifying the prevalence of mixotrophy in diverse marine microorganisms and thus for investigating the implications of the 'mixoplankton' paradigm.

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.

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.

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.

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.

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.

Stoichiometric analysis of the energetics and metabolic impact of photorespiration in C3 plants.

Analysis of the impact of photorespiration on plant metabolism is usually based on manual inspection of small network diagrams. Here we create a structural metabolic model that contains the reactions that participate in photorespiration in the plastid, peroxisome, mitochondrion and cytosol, and the metabolite exchanges between them. This model was subjected to elementary flux modes analysis, a technique that enumerates all the component, minimal pathways of a network. Any feasible photorespiratory metabolism in the plant will be some combination of the elementary flux modes (EFMs) that contain the Rubisco oxygenase reaction. Amongst the EFMs we obtained was the classic photorespiratory cycle, but there were also modes that involve photorespiration coupled with mitochondrial metabolism and ATP production, the glutathione-ascorbate cycle and nitrate reduction to ammonia. The modes analysis demonstrated the underlying basis of the metabolic linkages with photorespiration that have been inferred experimentally. The set of reactions common to all the elementary modes showed good agreement with the gene products of mutants that have been reported to have a defective phenotype in photorespiratory conditions. Finally, the set of modes provided a formal demonstration that photorespiration itself does not impact on the CO2 :O2 ratio (assimilation quotient), except in those modes associated with concomitant nitrate reduction.

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.

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.

Variability in the carbon isotope composition of individual amino acids in plant proteins from different sources: 1 Leaves.

The natural carbon isotope composition of individual amino acids from plant leaf proteins has been measured to establish potential sources of variability. The plant leaves studied, taken from a range of plant groups (forbs, trees, grasses, and freshwater aquatic plants), showed no significant influence of either season or environment (water and light availability) on their Δδ(13)C values. Plant groups did, however, differ in carbon isotope composition, although no consistent differences were identified at the species level. A discriminant analysis model was constructed which allowed leaves from (1) nettles, (2) Pooideae, (3) other Poales, (4) trees and (5) freshwater higher plants to be distinguished from each other on the basis of their natural abundance (13)C/(12)C ratios of individual amino acids. Differences in carbon isotope composition are known to be retained, to some extent, in the tissues of their consumers, and hence an understanding of compound-specific variation in (13)C/(12)C fractional abundance in plants has the potential to provide dietary insights of value in archaeological and ecological studies.

Lack of fructose 2,6-bisphosphate compromises photosynthesis and growth in Arabidopsis in fluctuating environments.

The balance between carbon assimilation, storage and utilisation during photosynthesis is dependent on partitioning of photoassimilate between starch and sucrose, and varies in response to changes in the environment. However, the extent to which the capacity to modulate carbon partitioning rapidly through short-term allosteric regulation may contribute to plant performance is unknown. Here we examine the physiological role of fructose 2,6-bisphosphate (Fru-2,6-P2 ) during photosynthesis, growth and reproduction in Arabidopsis thaliana (L.). In leaves this signal metabolite contributes to coordination of carbon assimilation and partitioning during photosynthesis by allosterically modulating the activity of cytosolic fructose-1,6-bisphosphatase. Three independent T-DNA insertional mutant lines deficient in 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (F2KP), the bifunctional enzyme responsible for both the synthesis and degradation of Fru-2,6-P2 , lack Fru-2,6-P2 . These plants have normal steady-state rates of photosynthesis, but exhibit increased partitioning of photoassimilate into sucrose and have delayed photosynthetic induction kinetics. The F2KP-deficient plants grow normally in constant environments, but show reduced growth and seed yields relative to wildtype plants in fluctuating light and/or temperature. We conclude that Fru-2,6-P2 is required for optimum regulation of photosynthetic carbon metabolism under variable growth conditions. These analyses suggest that the capacity of Fru-2,6-P2 to modulate partitioning of photoassimilate is an important determinant of growth and fitness in natural environments.

Fluxes through plant metabolic networks: measurements, predictions, insights and challenges.

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.

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.

Optimization of steady-state ¹³C-labeling experiments for metabolic flux analysis.

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.

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.

Metabolic engineering of tomato fruit organic acid content guided by biochemical analysis of an introgression line.

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.