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

Abstract

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 fluxweighting 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 CalvinBenson cycle revealed the potential for alternative transitory carbon stores in leaves and led to predictions about the lightdependent 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.

In silico metabolic network analysis of Arabidopsis leaves

“…Furthermore, the metabolic fluxes that are most influenced by a decreased ATP: NADPH ratio, appear to handle an excess of redox power by channeling NADPH through the malate/oxaloacetate shuttle into the mitochondrion, where ATP is produced by oxidative phosphorylation (Additional file 1: Figure S2). This supports the hypothesis that mitochondria can act as a sink for reduced NADPH [96–98]. In addition, plastidic triose phosphate is increasingly exported to the cytosolic EMP pathway, thus effectively reducing the plastidic ATP requirement.

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“…In this way, also the output ratio of ATP: NADPH can be influenced significantly by the light environment, which has been investigated extensively [65–67, 96, 101]. How cellular metabolism copes with the changed ATP: NADPH supply on the level of intracellular fluxes is investigated here.…”

Inference and prediction of metabolic network fluxes

“…This can be misleading because multiple alternative pathways will often operate simultaneously in planta. A recent methodology that uses reaction weighting factors to account for the differing costs of the enzymatic machinery provides a potential solution (Cheung et al, 2015). Using this approach, the flux space was analyzed in thousands of simulations using different sets of weighting factors for the fluxes, and the averaged results show operation of alternative pathways.…”

“…The fluxes can be compared under different states, allowing, for example, the effect of genotype or environment on the metabolic system to be characterized. The resulting integrated view also allows access to important aspects of metabolism, such as energy and redox balancing, that are properties of the system as a whole (Kramer and Evans, 2011;Cheung et al, 2015). Finally, knowledge of system-wide metabolic fluxes is an invaluable tool for guiding efforts to engineer the plant metabolic system, allowing predictions of the ideal network configuration and fluxes for the overproduction of desirable end products (Farré et al, 2014).…”

“…The fluxes can be compared under different states, allowing, for example, the effect of genotype or environment on the metabolic system to be characterized. The resulting integrated view also allows access to important aspects of metabolism, such as energy and redox balancing, that are properties of the system as a whole (Kramer and Evans, 2011;Cheung et al, 2015). Finally, knowledge of system-wide * Address correspondence to lee.sweetlove@plants.ox.ac.uk.…”