2014
DOI: 10.1038/nprot.2014.115
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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 ma… Show more

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Cited by 62 publications
(50 citation statements)
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“…Our approach, which involves comparison of a single precursor pool and a single product, can be applied to nonsteady-state conditions. While there are more sophisticated computational methods that analyze dynamic labeling patterns in large metabolic networks, they can only be applied when the metabolism is in steady state (Antoniewicz et al, 2006;Yuan et al, 2006Yuan et al, , 2008Young et al, 2011) and, in higher plants, also require assumptions about the spatial compartmentation (Szecowka et al, 2013;Heise et al, 2014;Ma et al, 2014). Although recent theoretical developments allow the analysis of dynamic labeling in metabolic nonsteady state in microbes (Antoniewicz, 2013), it remains challenging to apply these methods in higher plants.…”
Section: Estimation Of Growth By Measuring Enrichment In Glc In the Cmentioning
confidence: 99%
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“…Our approach, which involves comparison of a single precursor pool and a single product, can be applied to nonsteady-state conditions. While there are more sophisticated computational methods that analyze dynamic labeling patterns in large metabolic networks, they can only be applied when the metabolism is in steady state (Antoniewicz et al, 2006;Yuan et al, 2006Yuan et al, , 2008Young et al, 2011) and, in higher plants, also require assumptions about the spatial compartmentation (Szecowka et al, 2013;Heise et al, 2014;Ma et al, 2014). Although recent theoretical developments allow the analysis of dynamic labeling in metabolic nonsteady state in microbes (Antoniewicz, 2013), it remains challenging to apply these methods in higher plants.…”
Section: Estimation Of Growth By Measuring Enrichment In Glc In the Cmentioning
confidence: 99%
“…Homogenized frozen plant material (30 mg) was extracted with methanol followed by phase separation using chloroform-water as described by Heise et al (2014). Ice-cold 100% (v/v) methanol and ribitol (1.31 mM) solution as an internal quantitative standard were added to the homogenized plant material and incubated for 10 min at 70°C.…”
Section: Metabolite and Protein Analysismentioning
confidence: 99%
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“…PPi as a substrate during Suc oxidation and as a product during Suc synthesis; Fig. 7; Heise et al, 2014;Ma et al, 2014).…”
Section: Discussionmentioning
confidence: 99%
“…the labeling of metabolites changes over time, but the concentrations of the metabolites remain constant). In this way, the reach of the kinetic flux profiling approach can be extended to estimate fluxes in a more complex set of reactions such as the Calvin-Benson cycle (Szecowka et al, 2013;Heise et al, 2014). The labeling information used in these analyses considered only the unlabeled fraction of each metabolite during a labeling pulse chase, but additional constraints on the fluxes can be gained by considering the labeling of fragments of metabolites (using mass spectrometry) or of specific carbon atoms (using NMR).…”
Section: General Principles Of Inference and The Prediction Of Metabomentioning
confidence: 99%