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.

The amount of inorganic carbon represents one of the main environmental factors determining productivity of photoautotrophic organisms. Using the model cyanobacterium Synechocystis sp. PCC 6803, we performed a first metabolome study with cyanobacterial cells shifted from high CO 2 (5% in air) into conditions of low CO 2 (LC; ambient air with 0.035% CO 2 ). Using gas chromatography-mass spectrometry, 74 metabolites were reproducibly identified under different growth conditions. Shifting wild-type cells into LC conditions resulted in a global metabolic reprogramming and involved increases of, for example, 2-oxoglutarate (2OG) and phosphoenolpyruvate, and reductions of, for example, sucrose and fructose-1,6-bisphosphate. A decrease in Calvin-Benson cycle activity and increased usage of associated carbon cycling routes, including photorespiratory metabolism, was indicated by synergistic accumulation of the fumarate, malate, and 2-phosphoglycolate pools and a transient increase of 3-phosphoglycerate. The unexpected accumulation of 2OG with a concomitant decrease of glutamine pointed toward reduced nitrogen availability when cells are confronted with LC. Despite the increase in 2OG and low amino acid pools, we found a complete dephosphorylation of the PII regulatory protein at LC characteristic for nitrogen-replete conditions. Moreover, mutants with defined blocks in the photorespiratory metabolism leading to the accumulation of glycolate and glycine, respectively, exhibited features of LC-treated wild-type cells such as the changed 2OG to glutamine ratio and PII phosphorylation state already under high CO 2 conditions. Thus, metabolome profiling demonstrated that acclimation to LC involves coordinated changes of carbon and interacting nitrogen metabolism. We hypothesize that Synechocystis has a temporal lag of acclimating carbon versus nitrogen metabolism with carbon leading.

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BackgroundRecent studies using transcript and metabolite profiles of wild-type and gene deletion mutants revealed that photorespiratory pathways are essential for the growth of Synechocystis sp. PCC 6803 under atmospheric conditions. Pool size changes of primary metabolites, such as glycine and glycolate, indicated a link to photorespiration.Methodology/Principal FindingsThe 13C labelling kinetics of primary metabolites were analysed in photoautotrophically grown cultures of Synechocystis sp. PCC 6803 by gas chromatography-mass spectrometry (GC-MS) to demonstrate the link with photorespiration. Cells pre-acclimated to high CO2 (5%, HC) or limited CO2 (0.035%, LC) conditions were pulse-labelled under very high (2% w/w) 13C-NaHCO3 (VHC) conditions followed by treatment with ambient 12C at HC and LC conditions, respectively. The 13C enrichment, relative changes in pool size, and 13C flux of selected metabolites were evaluated. We demonstrate two major paths of CO2 assimilation via Rubisco in Synechocystis, i.e., from 3PGA via PEP to aspartate, malate and citrate or, to a lesser extent, from 3PGA via glucose-6-phosphate to sucrose. The results reveal evidence of carbon channelling from 3PGA to the PEP pool. Furthermore, 13C labelling of glycolate was observed under conditions thought to suppress photorespiration. Using the glycolate-accumulating ΔglcD1 mutant, we demonstrate enhanced 13C partitioning into the glycolate pool under conditions favouring photorespiration and enhanced 13C partitioning into the glycine pool of the glycine-accumulating ΔgcvT mutant. Under LC conditions, the photorespiratory mutants ΔglcD1 and ΔgcvT showed enhanced activity of the additional carbon-fixing PEP carboxylase pathway.Conclusions/SignificanceWith our approach of non-steady-state 13C labelling and analysis of metabolite pool sizes with respective 13C enrichments, we identify the use and modulation of major pathways of carbon assimilation in Synechocystis in the presence of high and low inorganic carbon supplies.