Flux profiling of photosynthetic carbon metabolism in intact plants

• Published in: Nature protocols Vol. 9; no. 8; pp. 1803 - 1824
• Main Authors: Heise, Robert, Arrivault, Stéphanie, Szecowka, Marek, Tohge, Takayuki, Nunes-Nesi, Adriano, Stitt, Mark, Nikoloski, Zoran, Fernie, Alisdair R
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01-08-2014; Nature Publishing Group
• Subjects: 631/1647/2196; 631/1647/334/2244/710; 631/449/1734; 631/92/1643; Analytical Chemistry; Arabidopsis - metabolism; Arabidopsis thaliana; Biological Techniques; Carbon; Carbon - metabolism; Carbon dioxide; Chromatography; Chromatography, Gas - methods; Chromatography, Liquid - methods; Computational Biology/Bioinformatics; Enzyme kinetics; Fluctuations; Gas chromatography; Innovations; Instrumentation; Labeling; Life Sciences; Liquid chromatography; Mass spectrometry; Mass Spectrometry - methods; Metabolic Networks and Pathways; Metabolism; Metabolites; Metabolomics - methods; Microarrays; Monte Carlo Method; Organic Chemistry; Photosynthesis; Physiological aspects; Protocol; Sucrose; Germany
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2014.115

Full characterization of the metabolic state of a biological system involves quantifying the flux of the biochemical reactions. In this protocol,
Arabidopsis
rosettes are treated with
13
CO
2
, and
13
C-labeled metabolites are analyzed by GC- and LC-MS.
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
CO
2
. 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.