Journal
NATURE PROTOCOLS
Volume 9, Issue 8, Pages 1803-1824Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2014.115
Keywords
-
Categories
Ask authors/readers for more resources
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 (CO2)-C-13. 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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available