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

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 (12)C washout when the CO(2) source is rapidly switched to (13)CO(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 (13)C 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 (13)CO(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.