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. 2013 Feb;25(2):694-714.
doi: 10.1105/tpc.112.106989. Epub 2013 Feb 26.

Metabolic fluxes in an illuminated Arabidopsis rosette

Marek Szecowka  1 , Robert Heise, Takayuki Tohge, Adriano Nunes-Nesi, Daniel Vosloh, Jan Huege, Regina Feil, John Lunn, Zoran Nikoloski, Mark Stitt, Alisdair R Fernie, Stéphanie Arrivault
Affiliations

Metabolic fluxes in an illuminated Arabidopsis rosette

Marek Szecowka et al. Plant Cell. 2013 Feb.

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 CO2 in a custom-built chamber were transferred into a (13)CO2-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.

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Figures

Figure 1.
Figure 1.
Overview of 13C Labeling Kinetics from Primary C Metabolism. (A) k-means clustering. Gray lines show the 13C enrichment (calculated as in Methods) of individual metabolites, and magenta lines show average 13C enrichment of all metabolites in the cluster. (B) Schematic representation of labeling kinetics. The scheme distinguishes the plastidic compartment but not the cytosol, peroxisomes, mitochondria, and vacuole. Metabolites are highlighted according to cluster following the color scheme in (A). Solid and double-headed arrows represent irreversible and reversible reactions, respectively. Dashed arrows represent conversions involving several steps.
Figure 2.
Figure 2.
13C Labeling Kinetics of Metabolites from CBC, Starch, Suc, and Trehalose Biosynthesis Pathways. (A) Time course of mass distribution. The relative abundance of each isotopomer (mn) for a given metabolite is represented; n is the number of 13C atoms incorporated. (B) and (C) 13C enrichment. The x axis corresponds to the labeling time on a log10 scale. In (C), the y axis corresponds to 13C enrichments on a log10 scale. Values (%) are average of three biological replicates ± sd, with the exception of Suc6P and Tre6P at time 180 s (n = 1). For clarity, sds are not shown in (B) and (C).
Figure 3.
Figure 3.
13C Enrichment of Sugars, Organic Acids, and Amino Acids. Sugars (A), photorespiration cycle intermediates (B), and organic acids and amino acids (C). Values (%) are average of three biological replicates ± sd.
Figure 4.
Figure 4.
Time Course of Mass Distribution of Photorespiration Cycle Intermediates. Gly (A), glycerate (B), and Ser (C). The relative abundance of each isotopomer (mn) for a given metabolite is represented, and n corresponds to the number of 13C atoms incorporated in the metabolite. Values (%) are average of three biological replicates ± sd.
Figure 5.
Figure 5.
Pathway Model, Elementary Flux Modes, and Flux Estimates. (A) Pathway model. The model includes the CBC, photorespiration, and Suc, starch synthesis, myo-inositol, and trehalose synthesis. There is explicit separation of cytosolic (blue, subscript c) and plastidic pools (yellow, subscript p) that are not equilibrated by transporters. Plastidic and cytosolic pools of 3PGA and DHAP are treated as a single pool (gray) that is in isotopic equilibrium (Stitt et al., 1983) due to rapid exchange via the triose-phosphate phosphate transporter. Metabolite pools involved in photorespiration are shown in pink. Reactions are set as irreversible (single-headed arrow) or reversible (double-headed arrow), based on experimental data (Bassham and Krause, 1969; Stitt et al., 1980). (B) to (F) Elementary flux modes of the model: synthesis of starch (B), synthesis of Suc (C), synthesis of myo-inositol (D), synthesis of trehalose (E), and photorespiration (F). (G) Simulation of best fit. Measured data for the proportion of the total pool present as the 12C isotopomer are shown as crosses (±sd) and the predicted decay dynamics of the 12C isotopomer modeled using the unadjusted data set (red line) and after excluding the inactive pool (blue line). Gray dotted lines indicate the inactive pool. For Tre6P and myo-inositol (asterisks), which have a small total pool size, the active pool assumption is not applied. The RuBP panel shows the input models for the influx to the pool of 3PGA (purple solid line) and Gly (purple dotted line). The corresponding crosses indicate the data used for parameter estimation. (H) Selected fluxes and rates for the two scenarios included that were used for benchmarking. The rates of Rubisco carboxylation and oxygenation are given as RuBP consumption (i.e., use of CO2 or O2, respectively). Fluxes were estimated for two scenarios (1) All, using unadjusted 12C isotopomer decay and metabolite content data sets. (2) Active, using the pool of each metabolite that is actively involved in photosynthetic fluxes. The inactive pool was nominally defined as the proportion that remains as 12C isotopomer at 60 min. It was subtracted from the 12C isotope kinetic (i.e., the 60-min value is set as zero). The absolute pool (Table 1) was also decreased in the same proportion. For each scenario, flux estimates are denoted by the optimal value obtained with the fit “optimum” and the “lower” and “upper” 95% confidence limits obtained from the Monte-Carlo simulation.
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