Professor George Ratcliffe
Metabolic networks supply the precursors, energy and reducing power required for the synthesis and turnover of cellular components. The associated flows of material – the metabolic fluxes – are crucial in determining the performance and productivity of cells and organisms. For example, in an agricultural context, the production of harvestable end-products of plant metabolism is entirely dependent on the flux phenotype of the plant; while in biotechnology, the exploitation of micro-organisms and plants hinges on an ability to reconfigure the metabolic network to favour a flux distribution that leads to the preferential synthesis of particular products. Thus the fluxes supported by the plant metabolic network play a pivotal role in determining both phenotype and productivity. My main interest lies in understanding the organisation and regulation of the metabolic fluxes that occur in the plant metabolic network. A knowledge of the transcriptome, proteome or metabolome does not lead easily to the metabolic flux phenotype, and internal fluxes within the metabolic network have to be deduced from a suite of computational and experimental tools. My research group is strongly involved in the development and application of steady-state metabolic flux analysis (MFA), a technique that allows fluxes to be deduced from a stoichiometric model of the network using stable isotope (13C) labelling data and measurements of biosynthetic outputs. We complement this MFA work with an in silico approach using genome-scale models and constraints-based flux balance analysis. Together these methods allow us to assess the metabolic phenotypes of wild type, mutant and transgenic plants, and thus the metabolic impact of genetic and environmental perturbations.
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Alternative Crassulacean Acid Metabolism Modes Provide Environment-Specific Water-Saving Benefits in a Leaf Metabolic Model.
December 2020|Journal article|The Plant cellCrassulacean acid metabolism (CAM) evolved in arid environments as a water-saving alternative to C<sub>3</sub> photosynthesis. There is great interest in engineering more drought-resistant crops by introducing CAM into C<sub>3</sub> plants. However, it is unknown whether full CAM or alternative water-saving modes would be more productive in the environments typically experienced by C<sub>3</sub> 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.
July 2020|Journal article|The Plant journal : for cell and molecular biologyCell 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. -
Limitations of Deuterium-Labelled Substrates for Quantifying NADPH Metabolism in Heterotrophic Arabidopsis Cell Cultures.
September 2019|Journal article|MetabolitesNADPH 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.
August 2019|Journal article|Plant physiologyKey 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.Mitochondria, Chloroplasts, Plant Leaves, Malates, NADP, Adenosine Triphosphate, Electron Transport, Energy Metabolism, Light, Models, Biological -
Euglena Central Metabolic Pathways and Their Subcellular Locations.
June 2019|Journal article|MetabolitesEuglenids 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.Euglena, central metabolic pathway, subcellular location -
The role of nitrite and nitric oxide under low oxygen conditions in plants.
May 2019|Journal article|The New phytologistPlant 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 extensively described. 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. Whilst 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, is also 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 non-symbiotic 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. This article is protected by copyright. All rights reserved. -
Euglena Central Metabolic Pathways and Their Subcellular Locations
May 2019|Journal articleEuglenids 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 the subcellular locations of the component enzymes, 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. Moreover, we show here that the presence of the secondary chloroplast means that it is not possible to make reliable predictions of the subcellular locations of enzymes in Euglena using existing informatics tools. In order to generate a model of the central metabolic pathway operating in Euglena we analysed biochemical and proteomic information from a variety of sources to assess the subcellular location of relevant enzymes. We use these assignments to propose the compartmentation of the core metabolic pathways in Euglena, a prerequisite for the further study of the metabolic network of Euglena. This model of the metabolic network shows that, other than photosynthesis, all major pathways present in the chloroplast are duplicated elsewhere in the cell, and that several biosynthetic pathways confined to plastids in higher plants are localized elsewhere in Euglena. Our model demonstrates how this organism can synthesise all the metabolites required for growth from simple carbon inputs, and can survive in the absence of chloroplasts. -
Tomato roots exhibit in vivo glutamate dehydrogenase aminating capacity in response to excess ammonium supply
April 2019|Journal article|Journal of Plant Physiology
E: | george.ratcliffe@plants.ox.ac.uk |
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