Quantitative metabolic profiling by 1-dimensional ¹H-NMR analyses: application to plant genetics and functional...

Metabolic profiling by 1-dimensional (1-D) ¹H-nuclear magnetic resonance (NMR) was tested for absolute quantification of soluble sugars, organic acids, amino acids and some secondary metabolites in fruit, roots and leaves. The metabolite responsible for each peak of the ¹H-NMR spectra was identified from spectra of pure compounds. Peak identity was confirmed by the addition of a small amount of commercially-available pure substance. ¹H-NMR spectra acquisition was automated. ¹H-NMR absolute quantification was performed with a synthesised electronic reference signal and validated by comparison with enzymatic or HPLC analyses; the correlation coefficients between ¹H-NMR data and enzymatic or HPLC data were highly significant. Depending on the species and tissues, 14-17 metabolites could be quantified with 15-25 min acquisition time. The detection limit was approximately 1-9 µg in the NMR tube, depending on the compound. Quantitative data were used for (1) a genetic study of strawberry fruit quality, (2) a functional study of tomato transformants overexpressing hexokinase and (3) a study of Arabidopsis phosphoenolpyruvate carboxylase transformants with several lines showing decreased activity of the enzyme. Biochemical phenotyping of the fruits of a strawberry offspring allowed the detection of quantitative trait loci (QTL) controlling fruit quality. Comparison of the roots of wild types and hexokinase tomato transformants using principal component analysis of metabolic profiles revealed that environmental factors, i.e. culture conditions, can significantly modify the metabolic status of plants and thus hide or emphasise the expression of a given genetic background. The decrease in phosphoenolpyruvate carboxylase activity (up to 75%) in Arabidopsis transformants impacted on the metabolic profiles without compromising plant growth, thus supporting the idea that the enzyme has a low influence on the carbon flux through the anaplerotic pathway.