Metabolic Fluxes in an Illuminated Arabidopsis Rosette  

Abstract

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.

Purification and functional characterization of the vacuolar malate transporter tDT from Arabidopsis

“…Accordingly, we also conducted 13 CO 2 labelling of Wt-, tdt1-and tDT overexpressor rosette leaves under ambient steady state conditions, which allows quantification of the incorporation of 13 C into compounds of the primary carbon metabolism (25), here with a particular interest for malate malate and citrate. Due to technical limitation it was not possible to separate the isotopomers of citrate and isocitrate.…”

“…Labelling was performed on 5-week-old plants as described in Szecowka et al 2013. The experiment was started 2 h after the onset of light and the last labelled plant was harvested 1 h hour before the offset of lights.…”

“…All frozen plant material above the plastic foil was collected and stored at -80°C, then ground under liquid N 2 . For LC-MS/MS quantification, metabolites were extracted from 15mg (fresh weight) frozen plant powder (Szecowka et al 2013). Extracts were analyzed and data processed as described earlier (Szecowka et al 2013).…”

Elements Required for an Efficient NADP-Malic Enzyme Type C4 Photosynthesis

“…First, Fru-bisP plays a role not only in the Calvin-Benson cycle and Suc synthesis, but also in glycolysis and the pentose phosphate pathway, which are currently not included in this model. Slowly labeled FBP in a 13 CO 2 labeling experiment in Arabidopsis (Arabidopsis thaliana) indicated that a large part of the FBP pool is not directly involved in Calvin-Benson cycle (Szecowka et al, 2013). Second, the regulatory mechanisms controlling the activity of the enzymes of photosynthetic carbon metabolism were not described in the model.…”

“…More accurate measurements of kinetic parameters, metabolite concentrations (especially metabolites concentrations in different compartments of both BSCs and MCs), and metabolite fluxes under varied [CO 2 ] and PPFD will undoubtedly help improve the C4 systems model. Fortunately, various techniques in metabolomics (Fiehn, 2002;Sumner et al, 2003;Weckwerth, 2003;Kopka et al, 2005;Oksman-Caldentey and Saito, 2005) and fluxomics (Fernie et al, 2005;Wiechert et al, 2007;Szecowka et al, 2013) are rapidly emerging, which can help fill this data demand.…”