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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.)

Genome-scale metabolic network models can be used for various analyses including the prediction of metabolic responses to changes in the environment. Legumes are well known for their rhizobial symbiosis that introduces nitrogen into the global nutrient cycle. Here, we describe a fully compartmentalised, mass and charge-balanced, genome-scale model of the clover Medicago truncatula, which has been adopted as a model organism for legumes. We employed flux balance analysis to demonstrate that the network is capable of producing biomass components in experimentally observed proportions, during day and night. By connecting the plant model to a model of its rhizobial symbiont, Sinorhizobium meliloti, we were able to investigate the effects of the symbiosis on metabolic fluxes and plant growth and could demonstrate how oxygen availability influences metabolic exchanges between plant and symbiont, thus elucidating potential benefits of inter organism amino acid cycling. We thus provide a modelling framework, in which the interlinked metabolism of plants and nodules can be studied from a theoretical perspective.

There is considerable interest in transferring crassulacean acid metabolism (CAM) to C 3 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 C 3 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 C 3 network. We conclude that CAM does not impose a significant productivity penalty and that engineering CAM into C 3 crops is likely to lead to a major increase in water-use efficiency without substantially affecting yield.

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.)

In this Update, we cover the basic principles of the estimation and prediction of the rates of the many interconnected biochemical reactions that constitute plant metabolic networks. This includes metabolic flux analysis approaches that utilize the rates or patterns of redistribution of stable isotopes of carbon and other atoms to estimate fluxes, as well as constraints-based optimization approaches such as flux balance analysis. Some of the major insights that have been gained from analysis of fluxes in plants are discussed, including the functioning of metabolic pathways in a network context, the robustness of the metabolic phenotype, the importance of cell maintenance costs, and the mechanisms that enable energy and redox balancing at steady state. We also discuss methodologies to exploit 'omic data sets for the construction of tissue-specific metabolic network models and to constrain the range of permissible fluxes in such models. Finally, we consider the future directions and challenges faced by the field of metabolic network flux phenotyping., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Identifying the correct subcellular locations for all enzymes and metabolites in plant metabolic networks is a major challenge, but is critically important for the success of the new generation of large-scale metabolic models that are driving a network-level appreciation of metabolic behavior. Even though the subcellular compartmentation of many central metabolic processes is thought to be well understood, recent gene-by-gene studies have revealed several unexpected enzyme localizations. Metabolite transport between subcellular compartments is crucial because it fundamentally affects the metabolic network structure. Although new metabolite transporters are being steadily identified, modeling work suggests that we have barely scratched the surface of the catalog of intracellular metabolite transporter proteins. In addition to compartmentation among organelles, it is increasingly apparent that microcompartment formation via the interactions of enzyme groups with intracellular membranes, the cytoskeleton, or other proteins is an important regulatory mechanism. In particular, this mechanism can promote metabolite channeling within the metabolic microcompartment, which can help control reaction specificity as well as dictate flux routes through the network. This has clear relevance for both synthetic biology in general and the engineering of plant metabolic networks in particular.

Research into plant metabolism has a long history, and analytical approaches of ever-increasing breadth and sophistication have been brought to bear. We now have access to vast repositories of data concerning enzymology and regulatory features of enzymes, as well as large-scale datasets containing profiling information of transcripts, protein and metabolite levels. Nevertheless, despite this wealth of data, we remain some way off from being able to rationally engineer plant metabolism or even to predict metabolic responses. Within the past 18 months, rapid progress has been made, with several highly informative plant network interrogations being discussed in the literature. In the present review we will appraise the current state of the art regarding plant metabolic network analysis and attempt to outline what the necessary steps are in order to further our understanding of network regulation.