title: 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 (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.
authors.affiliation: 1] Max Planck Institut für Molekulare Pflanzenphysiologie, Potsdam-Golm, Germany. [2].
authors.forename: Robert
authors.initials: R
authors.lastname: Heise
authors.affiliation: 1] Max Planck Institut für Molekulare Pflanzenphysiologie, Potsdam-Golm, Germany. [2].
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Arrivault
authors.affiliation: 1] Max Planck Institut für Molekulare Pflanzenphysiologie, Potsdam-Golm, Germany. [2].
authors.forename: Marek
authors.initials: M
authors.lastname: Szecowka
authors.affiliation: Max Planck Institut für Molekulare Pflanzenphysiologie, Potsdam-Golm, Germany.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Tohge
authors.affiliation: 1] Max Planck Institut für Molekulare Pflanzenphysiologie, Potsdam-Golm, Germany. [2] Present address: Max Planck partner group, Departamento de Biologia Vegetal Universidade Federal de Viçosa MG, Brasil.
authors.forename: Adriano
authors.initials: A
authors.lastname: Nunes-Nesi
authors.affiliation: 1] Max Planck Institut für Molekulare Pflanzenphysiologie, Potsdam-Golm, Germany. [2].
authors.forename: Mark
authors.initials: M
authors.lastname: Stitt
authors.affiliation: 1] Max Planck Institut für Molekulare Pflanzenphysiologie, Potsdam-Golm, Germany. [2].
authors.forename: Zoran
authors.initials: Z
authors.lastname: Nikoloski
authors.affiliation: 1] Max Planck Institut für Molekulare Pflanzenphysiologie, Potsdam-Golm, Germany. [2].
authors.forename: Alisdair R
authors.initials: AR
authors.lastname: Fernie
mesh_terms: D017360:Arabidopsis / Q000378:metabolism*; D002244:Carbon / Q000378:metabolism*; D002849:Chromatography, Gas / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D053858:Metabolic Networks and Pathways; D055432:Metabolomics / Q000379:methods; D009010:Monte Carlo Method
chemical_list: D002244:Carbon