Quantitative metabolic profiling by 1-dimensional 1H-NMR analyses: application to plant genetics and functional genomics
Annick Moing A F , Mickaël Maucourt B , Christel Renaud A , Monique Gaudillère B , Renaud Brouquisse B , Bénédicte Lebouteiller C , Aurélie Gousset-Dupont C , Jean Vidal C , David Granot D , Béatrice Denoyes-Rothan A , Estelle Lerceteau-Köhler E and Dominique Rolin BA Unité de Recherches sur les Espèces Fruitières et la Vigne, INRA, Université de Bordeaux 2, BP 81, F-33 883 Villenave d’Ornon, Cedex, France.
B UMR Physiologie et Biotechnologie Végétales, INRA, Université de Bordeaux 1, Université de Bordeaux 2, BP 81, F-33 883 Villenave d’Ornon, Cedex, France.
C Institut de Biotechnologie des Plantes, UMR CNRS 8618, Bat 630 Université Paris XI Sud, F-91 405 Orsay Cedex, France.
D Institute of Field and Garden Crops, Agricultural Research Organisation, Volcani Center, Bet Dagan 50 250, Israel.
E CIREF, Lanxade, F-24 130 Prigonrieux, France.
F Current address: UMR Physiologie et Biotechnologie Végétales, Institut National de la Recherche Agronomique, Centre de Bordeaux, BP 81, F-33 883 Villenave d’Ornon, Cedex, France. Corresponding author; email: moing@bordeaux.inra.fr
Functional Plant Biology 31(9) 889-902 https://doi.org/10.1071/FP04066
Submitted: 5 April 2004 Accepted: 2 June 2004 Published: 27 September 2004
Abstract
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.
Keywords: Arabidopsis, amino acids, hexokinase, metabolic profiling, metabolites, metabolomics, organic acids, phosphoenolpyruvate carboxylase, proton NMR, soluble sugars, strawberry, tomato, transformants, quantitative trait loci.
Acknowledgments
We thank CIREF for taking care of the strawberry offspring, D Camy for help with the preparation of strawberry samples, C Rolin for language corrections and C Deborde for critical reading of the manuscript. This work was partially funded by Région Aquitaine (contract 980305011).
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