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- 13C 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.Extensive subcellular compartmentation, with unique locations for many steps and pathways, as well as the duplication of other steps and pathways in different compartments, adds greatly to the structural complexity of the plant metabolic network (Lunn, 2007;. The need to consider discrete pools of metabolites in specific compartments, and the transporters that link them, complicates the quest for a detailed, predictive understanding of the regulation of plant metabolism; as a result, it remains difficult to manipulate flows of material through the central metabolic network in a predictable way (Carrari et al., 2003;Sweetlove et al., 2008).The emergence of steady-state metabolic flux analysis (MFA) as a practicable systems biology tool for generating flux maps of the central metabolic pathways in plants offers new opportunities for analyzing plant metabolic phenotypes (Ratcliffe and ShacharHill, 2006;Libourel and Shachar-Hill, 2008;Schwender, 2008;Kruger and Ratcliffe, 2009). In this approach, substrates labeled with stable isotopes are introduced into the network, and fluxes are determined by measuring the labeling of the system after it has reached an isotopic and metabolic steady state. Subcellular compartmentation of steps and pathways can be incorporated into the model that describes the redistribution of t...

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