Reimport of carbon from cytosolic and vacuolar sugar pools into the Calvin–Benson cycle explains photosynthesis labeling anomalies

Significance

Photosynthesis metabolites are quickly labeled when ¹³CO ₂ is fed to leaves, but the time course of labeling reveals additional contributing processes involved in the metabolic dynamics of photosynthesis. The existence of three such processes is demonstrated, and a metabolic flux model is developed to explore and characterize them. The model is consistent with a slow return of carbon from cytosolic and vacuolar sugars into the Calvin–Benson cycle through the oxidative pentose phosphate pathway. Our results provide insight into how carbon assimilation is integrated into the metabolic network of photosynthetic cells with implications for global carbon fluxes.

Abstract

When isotopes of carbon are fed to photosynthesizing leaves, metabolites of the Calvin–Benson cycle (CBC) are rapidly labeled initially, but then the rate of labeling slows considerably, raising questions about the integration of the CBC within leaf metabolism. We have used 2-h time courses of labeling of Camelina sativa leaf metabolites to test models of ¹²C washout when the CO ₂ source is rapidly switched to ¹³CO ₂. Fitting exponential functions to the time course of CBC metabolites, we found evidence for three temporally distinct processes contributing to the labeling but none for metabolically inactive pools. We next modeled the data of all metabolites by ¹³C isotopically nonstationary metabolic flux analysis, testing a variety of flux networks. In the model that best explains measured data, three processes determine CBC metabolite labeling. First is fixation of incoming ¹³CO ₂; second is dilution by weakly labeled carbon in cytosolic glucose reentering the CBC following oxidative pentose phosphate pathway reactions, which forms a shunt bypassing much of the CBC. Third, very weakly labeled carbon from the vacuole further dilutes the labeling. This model predicts the shunt proceeds at about 5% of the rate of net CO ₂ fixation and explains the three phases of labeling. In showing the interconnection of three compartments, we have drawn a more complete picture of how carbon moves through photosynthetic metabolism in a way that integrates the CBC, cytosolic sugar pools, glucose-6-phosphate shunt, and vacuolar sugars into a single system.