A Diel Flux Balance Model Captures Interactions between Light and Dark Metabolism during Day-Night Cycles in C3 and Crassulacean Acid Metabolism Leaves

Abstract

Although leaves have to accommodate markedly different metabolic flux patterns in the light and the dark, models of leaf metabolism based on flux-balance analysis (FBA) have so far been confined to consideration of the network under continuous light. An FBA framework is presented that solves the two phases of the diel cycle as a single optimization problem and, thus, provides a more representative model of leaf metabolism. The requirement to support continued export of sugar and amino acids from the leaf during the night and to meet overnight cellular maintenance costs forces the model to set aside stores of both carbon and nitrogen during the day. With only minimal constraints, the model successfully captures many of the known features of C₃ leaf metabolism, including the recently discovered role of citrate synthesis and accumulation in the night as a precursor for the provision of carbon skeletons for amino acid synthesis during the day. The diel FBA model can be applied to other temporal separations, such as that which occurs in Crassulacean acid metabolism (CAM) photosynthesis, allowing a system-level analysis of the energetics of CAM. The diel model predicts that there is no overall energetic advantage to CAM, despite the potential for suppression of photorespiration through CO₂ concentration. Moreover, any savings in enzyme machinery costs through suppression of photorespiration are likely to be offset by the higher flux demand of the CAM cycle. It is concluded that energetic or nitrogen use considerations are unlikely to be evolutionary drivers for CAM photosynthesis.

Figure 1.

Flux balance framework for integrated metabolic modeling of the day and night phases of a mature leaf. The light and dark phases are represented by the white and gray backgrounds of the diagram, respectively. Metabolites shown in the dashed rectangles between the two phases represent potential storage compounds. Starch, Glc, Fru, malate, fumarate, citrate, and nitrate were allowed to accumulate in the light and the dark as denoted by the arrows pointing to the light and dark states. Twenty common amino acids were allowed to accumulate in the light but not the dark as denoted by the arrow pointing from the light phase to the dark phase. Export to the phloem was set to four Suc to one amino acid, with 18 different amino acids in the proportions shown in Supplemental Table S1. The export rate was set to be 3 times greater in the light than the dark. Nitrate was set as the sole nitrogen source, with the ratio of nitrate uptake from the phloem in the light:nitrate uptake from the phloem in the dark set to 3:2. Cellular maintenance costs in the dark were set to a fixed value, where the carbon exported during the night roughly equaled the carbon released as CO₂ in the dark phase. Maintenance costs were assumed to be the same in the light and the dark. The ratio of ATP maintenance cost:NADPH maintenance cost was set to 3:1.

Figure 2.

Metabolic routes for Glu biosynthesis from two modeling approaches. Left, A single steady-state model in constant light; right, the diel modeling framework. In constant light, the carbon skeletons for Gln synthesis were predicted to be supplied through a metabolic route, in which Thr is metabolized in the cytosol to acetate, transported to the peroxisome, and metabolized to citrate, which is exported to the cytosol and converted to 2-OG. Using the diel modeling framework, the model predicted the use of citrate stored in the dark to provide the carbon skeletons for Glu synthesis in the light. The thickness of the arrows is scaled to indicate relative flux magnitudes (in molar units). OAA, Oxaloacetate.

Figure 3.

Predicted flux map of metabolism for a mature C₃ leaf over a day-night cycle. The light and dark phases are represented by the white and gray backgrounds, respectively. Metabolites shown in the dashed rectangles between the two phases represent storage compounds. The thickness of the arrows is proportional to the metabolic flux through the reactions (in molar units). Metabolic processes listed within rounded rectangles carry fluxes too large to be represented on the flux map. TCA, Tricarboxylic acid.

Figure 4.

Flux predictions through the tricarboxylic acid cycle and pyruvate dehydrogenase in a C₃ leaf in the light and the dark. A noncyclic mode with two separate branches, citrate to 2-OG and oxaloacetate and fumarate to malate, was predicted to operate in the light. A cyclic mode of the tricarboxylic acid cycle was predicted to operate in the dark, mainly to produce ATP through oxidative phosphorylation. Fluxes illustrated are net conversion between metabolites over all subcellular compartments. The thickness of the arrows is proportional to the metabolic fluxes through the reactions (in molar units). Citrate stored in the dark is represented by an arrow pointing from the dark phase to the light phase. Malate storage is not illustrated in this diagram.

Figure 5.

Reductant shuttling between subcellular compartments and the production and consumption of NADH in mitochondria in the light. Left-pointing arrows represent reductant-consuming reactions; right-pointing arrows represent reductant-producing reactions. The thickness of the arrows is proportional to the metabolic flux through the reactions (in molar units), except for the conversion between 3PGA and GAP in the chloroplast, where the zigzag line across the arrow indicates that the flux is too large to be illustrated to scale in the diagram. Reductant shuttles among four subcellular compartments (cytosol, chloroplast, mitochondria, and peroxisome) are shown with dashed arrows representing transfer of metabolites between compartments. OAA, Oxaloacetate; 2OG, 2-OG. [See online article for color version of this figure.]

Figure 6.

Flux predictions through the tricarboxylic acid cycle and related reactions in a CAM leaf. A noncyclic mode with two distinct branches, citrate to 2-OG and succinate to oxaloacetate, was predicted to operate in the light. The two branches of the tricarboxylic acid cycle are connected by isocitrate lyase, which converts isocitrate into succinate and glyoxylate. A cyclic mode of the tricarboxylic acid cycle was predicted to operate in the dark, mainly to contribute to ATP production through oxidative phosphorylation. Fluxes illustrated are net conversion between metabolites over all subcellular compartments. The thickness of the arrows is proportional to the metabolic flux through the reactions (in molar units), except for the conversion between malate and oxaloacetate, where the zigzag line across the arrow indicates that the flux is too large to be illustrated to scale in the diagram. Citrate stored in the dark is represented by an arrow pointing from the dark phase to the light phase. Malate storage is not illustrated in this diagram.

Figure 7.

Comparison of model predictions between C₃ and the various subtypes of CAM defined in Table II. The model predictions for photon use, Rubisco carboxylase flux, Rubisco oxygenase flux, total flux through Rubisco, and total flux in the metabolic model are shown with values scaled as a percentage of the value in the C₃ leaf model.