Flux profiling of photosynthetic carbon metabolism in intact plants View Full Text


Ontology type: schema:ScholarlyArticle     


Article Info

DATE

2014-08

AUTHORS

Robert Heise, Stéphanie Arrivault, Marek Szecowka, Takayuki Tohge, Adriano Nunes-Nesi, Mark Stitt, Zoran Nikoloski, Alisdair R Fernie

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 (13)CO2. 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. More... »

PAGES

1803-1824

Identifiers

URI

http://scigraph.springernature.com/pub.10.1038/nprot.2014.115

DOI

http://dx.doi.org/10.1038/nprot.2014.115

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1034777675

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/24992096


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