Abstract
Pool size measurements are important for the estimation of absolute intracellular fluxes in particular scenarios based on data from heavy carbon isotope experiments. Recently, steady-state fluxes estimates were obtained for central carbon metabolism in an intact illuminated rosette of Arabidopsis thaliana grown photoautotrophically (Szecowka et al., 2013; Heise et al., 2014). Fluxes were estimated therein by integrating mass-spectrometric data of the dynamics of the unlabeled metabolic fraction, data on metabolic pool sizes, partitioning of metabolic pools between cellular compartments and estimates of photosynthetically inactive pools, with a simplified model of plant central carbon metabolism. However, the fluxes were determined by treating the pool sizes as fixed parameters. Here we investigated whether and, if so, to what extent the treatment of pool sizes as parameters to be optimized in three scenarios may affect the flux estimates. The results are discussed in terms of benchmark values for canonical pathways and reactions, including starch and sucrose synthesis as well as the ribulose-1,5-bisphosphate carboxylation and oxygenation reactions. In addition, we discuss pathways emerging from a divergent branch point for which pool sizes are required for flux estimation, irrespective of the computational approach used for the simulation of the observable labeling pattern. Therefore, our findings indicate the necessity for development of techniques for accurate pool size measurements to improve the quality of flux estimates from non-stationary flux estimates in intact plant cells in the absence of alternative flux measurements.
Highlights
Metabolism encompasses the entirety of largely enzyme-catalyzed reactions transforming the set of nutrients into molecules that support various functions
The findings from Scenarios B were the basis for conducting a sensitivity analysis which facilitates the investigation of the effect of pool size measurements
As demonstrated theoretically in the introduction, in the absence of alternative flux measurements, data on pool sizes are mandatory for the estimation of fluxes at divergent branch points from non-stat.-13C-labeling data
Summary
Metabolism encompasses the entirety of largely enzyme-catalyzed reactions transforming the set of nutrients into molecules that support various functions. Reaction fluxes mutually relate to the involved metabolic pools and influence growth and other cellular tasks (Fell, 2005). For this reason, understanding the determinants of fluxes and their change upon perturbation is key to studying and controlling cellular behavior (Yoon et al, 2013). Short-term radioactive labeling is used to estimate the rate of synthesis of intermediates (Lunn and Hatch, 1995) Both approaches require the accumulation of the end product or the tracer within the time frame of the experiment. Other more mathematically involved methods have been developed to facilitate the estimation of fluxes through selected components and at a network level, as detailed below
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