GC-EI-TOF-MS analysis of in vivo carbon-partitioning into soluble metabolite pools of higher plants by monitoring isotope dilution after 13CO2 labelling

• Published in: Phytochemistry (Oxford) 2007-08, Vol.68 (16-18), p.2258-2272
• Main Authors: Huege, J, Sulpice, R, Gibon, Y, Lisec, J, Koehl, K, Kopka, J
• Format: Article
• Language: English
• Subjects: Arabidopsis - metabolism; Arabidopsis thaliana; Carbon - chemistry; Carbon - metabolism; carbon dioxide; Carbon Dioxide - chemistry; Carbon Dioxide - metabolism; Carbon Isotopes; Chemical Fractionation; cultured cells; electrospray ionization mass spectrometry; gas chromatography; Gas Chromatography-Mass Spectrometry; Half-Life; Homeostasis; Isotope Labeling; Kinetics; Life Sciences; mass spectrometry; matrix-assisted laser desorption-ionization mass spectrometry; Oryza - metabolism; Oryza sativa; Reproducibility of Results; Solubility; stable isotopes; Vegetal Biology
• ISSN: 0031-9422; 1873-3700; 0031-9422
• DOI: 10.1016/j.phytochem.2007.03.026

The established GC-EI-TOF-MS method for the profiling of soluble polar metabolites from plant tissue was employed for the kinetic metabolic phenotyping of higher plants. Approximately 100 typical GC-EI-MS mass fragments of trimethylsilylated and methoxyaminated metabolite derivatives were structurally interpreted for mass isotopomer analysis, thus enabling the kinetic study of identified metabolites as well as the so-called functional group monitoring of yet non-identified metabolites. The monitoring of isotope dilution after 13CO2 labelling was optimized using Arabidopsis thaliana Col-0 or Oryza sativa IR57111 plants, which were maximally labelled with 13C. Carbon isotope dilution was evaluated for short (2 h) and long-term (3 days) kinetic measurements of metabolite pools in root and shoots. Both approaches were shown to enable the characterization of metabolite specific partitioning processes and kinetics. Simplifying data reduction schemes comprising calculation of 13C-enrichment from mass isotopomer distributions and of initial 13C-dilution rates were employed. Metabolites exhibited a highly diverse range of metabolite and organ specific half-life of 13C-label in their respective pools (13C-half-life). This observation implied the setting of metabolite specific periods for optimal kinetic monitoring. A current experimental design for the kinetic metabolic phenotyping of higher plants is proposed.