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Flux profiling of photosynthetic carbon metabolism in intact plants

Abstract

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 13CO2. 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.

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Figure 1: A schematic representation of the concept of the NAF method for the determination of subcellular metabolite levels.
Figure 2: Schematic representation of the pathway model.
Figure 3: Preparation of the plants.
Figure 4: Labeling system used for Arabidopsis.

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Acknowledgements

We thank J. Lunn, R. Feil and J. Huege for useful discussions and H. Ishihara and C. Abel for help with the video. We also thank B.O. Hansen for help in formatting.

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R.H., M. Szecowka, S.A., T.T., A.N.-N., M. Stitt, Z.N. and A.R.F. contributed sections to the manuscript. M. Stitt, Z.N. and A.R.F. collated and standardized the text. All authors read and approved the final version.

Corresponding author

Correspondence to Alisdair R Fernie.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Histograms of Monte Carlo simulation.

Frequencies of selected parameters φ of the Monte-Carlo simulation including 500 repetitions. The parameters φ describe to steady-state net flux distribution according to Equation (7) (also see Supplementary Note 1). The selected parameters correspond to (A) φ(1) amount of photorespiration, (B) φ(2) amount of starch synthesis, (C) φ(3) amount of sucrose synthesis and (D) φ(4) amount of trehalse synthesis. The corresponding modes M1 - M4 are listed in Supplementary Data 2. The arrow indicates the optimal value and the dashed lines border the 95% confindence intervals.

Supplementary information

Supplementary Figure 1

Histograms of Monte Carlo simulation. (PDF 66 kb)

Supplementary Table 1

Metabolic contents used for flux estimation. (PDF 68 kb)

Supplementary Table 2

Parameters of the input models. (PDF 67 kb)

Supplementary Table 3

Estimates of fluxes. (PDF 68 kb)

Supplementary Table 4

Estimates of inactive fractions. (PDF 52 kb)

Supplementary Methods (PDF 79 kb)

Supplementary Data 1

Amounts for phosphorylated metabolites measured by LC-MS/MS (in pmol gFW-1). (XLSX 18 kb)

Supplementary Data 2

The pathway model and its modes. (PDF 107 kb)

Supplementary Equations

System of ordinary differential Equations. (PDF 74 kb)

Supplementary Note 1

Implementation of the model. (PDF 119 kb)

Supplementary Note 2

Decomposition. (PDF 66 kb)

Supplementary Video 1

Quenching of Arabidopsis. (MP4 18131 kb)

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Heise, R., Arrivault, S., Szecowka, M. et al. Flux profiling of photosynthetic carbon metabolism in intact plants. Nat Protoc 9, 1803–1824 (2014). https://doi.org/10.1038/nprot.2014.115

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