Abstract
The presence of cytosolic and plastidic pathways of carbohydrate oxidation is a characteristic feature of plant cell metabolism. Ideally, steady-state metabolic flux analysis, an emerging tool for creating flux maps of heterotrophic plant metabolism, would capture this feature of the metabolic phenotype, but the extent to which this can be achieved is uncertain. To address this question, fluxes through the pathways of central metabolism in a heterotrophic Arabidopsis (Arabidopsis thaliana) cell suspension culture were deduced from the redistribution of label in steady-state (13)C-labeling experiments using [1-(13)C]-, [2-(13)C]-, and [U-(13)C(6)]glucose. Focusing on the pentose phosphate pathway (PPP), multiple data sets were fitted simultaneously to models in which the subcellular compartmentation of the PPP was altered. The observed redistribution of the label could be explained by any one of three models of the subcellular compartmentation of the oxidative PPP, but other biochemical evidence favored the model in which the oxidative steps of the PPP were duplicated in the cytosol and plastids, with flux through these reactions occurring largely in the cytosol. The analysis emphasizes the inherent difficulty of analyzing the PPP without predefining the extent of its compartmentation and the importance of obtaining high-quality data that report directly on specific subcellular processes. The Arabidopsis flux map also shows that the potential ATP yield of respiration in heterotrophic plant cells can greatly exceed the direct metabolic requirements for biosynthesis, highlighting the need for caution when predicting flux through metabolic networks using assumptions based on the energetics of resource utilization.
Highlights
The presence of cytosolic and plastidic pathways of carbohydrate oxidation is a characteristic feature of plant cell metabolism
The extent to which the steps of the phosphate pathway (PPP) are duplicated in the cytosol and plastids has not been fully established, most of the biochemical evidence suggests that the enzymes for the oxidative steps at least are present in both compartments (Kruger and von Schaewen, 2003)
The Arabidopsis cell culture was maintained in the dark with Glc as the sole respiratory substrate, and the principal activities of the metabolic network, together with the associated biosynthetic outputs, were assessed by determining the redistribution of radiolabel following metabolism of [U-14C]Glc (Table I)
Summary
The presence of cytosolic and plastidic pathways of carbohydrate oxidation is a characteristic feature of plant cell metabolism. Steady-state metabolic flux analysis, an emerging tool for creating flux maps of heterotrophic plant metabolism, would capture this feature of the metabolic phenotype, but the extent to which this can be achieved is uncertain. The limited compartmentation of the pathways of carbohydrate oxidation in many flux maps can be traced to an analysis of developing oilseed rape embryos (Schwender et al, 2003), which indicated that the labeling data could be satisfactorily explained without duplication of glycolysis and the PPP. Building on a steady-state MFA of the same culture (Williams et al, 2008), similar experiments were conducted with multiple substrates and the labeling data sets were simultaneously fitted to models in which the subcellular compartmentation of the PPP was varied.
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