Toward Arabidopsis thaliana hydrophilic metabolome: assessment of extraction methods and quantitative 1H NMR

Our goal was to establish the hydrophilic metabolome of heterotrophic Arabidopsis thaliana cells grown in suspension, a cellular model of plant sink tissues. Water-soluble metabolites were extracted using four protocols: perchloric acid, boiling ethanol, methanol and methanol/chloroform (M/Chl). They were detected and quantified using 1H nuclear magnetic resonance (NMR) spectroscopy at 400 MHz. Extraction yields and reproducibility of the extraction methods were investigated. The effects of cell harvest protocol, cell grinding and lyophilization and storage conditions on the measured metabolic profiles were also studied. These quantitative studies demonstrated for the first time that the four extraction protocols commonly used do lead to quite similar molecular compositions as analyzed by 1H NMR. The M/Chl method proved effective and reliable to prepare series of physiologically significant extracts from plant cells for 1H NMR analysis. Reproducibility of the detected metabolome was assessed over long periods of time by analyzing a large number of separate extracts prepared from independent cultures. Larger variations in the NMR metabolite profiles could be correlated to changes in physiological parameters of the culture medium. Quantitative resolved 1H NMR of cell extracts proved to be robust and reliable for routine metabolite profiling of plant cell cultures.