Photosynthesis is the basis for life, and its optimization is a key biotechnological aim given the problems of population explosion and environmental deterioration. We describe a method to resolve intracellular fluxes in intact Arabidopsis thaliana rosettes based on time-dependent labeling patterns in the metabolome. Plants photosynthesizing under limiting irradiance and ambient CO 2 in a custom-built chamber were transferred into a 13 CO 2 -enriched environment. The isotope labeling patterns of 40 metabolites were obtained using liquid or gas chromatography coupled to mass spectrometry. Labeling kinetics revealed striking differences between metabolites. At a qualitative level, they matched expectations in terms of pathway topology and stoichiometry, but some unexpected features point to the complexity of subcellular and cellular compartmentation. To achieve quantitative insights, the data set was used for estimating fluxes in the framework of kinetic flux profiling. We benchmarked flux estimates to four classically determined flux signatures of photosynthesis and assessed the robustness of the estimates with respect to different features of the underlying metabolic model and the time-resolved data set.

Flux analysis has been carried out in plants for decades, but technical innovations are now enabling it to be carried out in photosynthetic tissues in a more precise fashion with respect to the number of metabolites measured. Here we describe a protocol, using gas chromatography (GC)- and liquid chromatography (LC)-mass spectrometry (MS), to resolve intracellular fluxes of the central carbon metabolism in illuminated intact Arabidopsis thaliana rosettes using the time course of the unlabeled fractions in 40 major constituents of the metabolome after switching to (13)CO2. We additionally simplify modeling assumptions, specifically to cope with the presence of multiple cellular compartments. We summarize all steps in this 8-10-week-long process, including setting up the chamber; harvesting; liquid extraction and subsequent handling of sample plant material to chemical derivatization procedures such as silylation and methoxymation (necessary for gas chromatography only); choosing instrumentation settings and evaluating the resultant chromatogram in terms of both unlabeled and labeled peaks. Furthermore, we describe how quantitative insights can be gained by estimating both benchmark and previously unknown fluxes from collected data sets.

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.

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