אסיף מאגר המחקר החקלאי

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

Year:

2004

Source of publication :

Authors :

Volume :

31

Co-Authors:

Moing, A., U. Rech. Sur Les Especes F., INRA, Université de Bordeaux 2, BP 81, F-33 883 Villenave d'Ornon, Cedex, France, UMR Physiol. et Biotech. Vég., Inst. Natl. de la Rech. Agronomique, Centre de Bordeaux, BP 81, F-33 883 Villenave d'Ornon, Cedex, France
Maucourt, M., UMR Physiol. et Biotech. Vég., INRA, Université de Bordeaux 2, BP 81, F-33 883 Villenave d'Ornon, Cedex, France
Renaud, C., U. Rech. Sur Les Especes F., INRA, Université de Bordeaux 2, BP 81, F-33 883 Villenave d'Ornon, Cedex, France
Gaudillère, M., UMR Physiol. et Biotech. Vég., INRA, Université de Bordeaux 2, BP 81, F-33 883 Villenave d'Ornon, Cedex, France
Brouquisse, R., UMR Physiol. et Biotech. Vég., INRA, Université de Bordeaux 2, BP 81, F-33 883 Villenave d'Ornon, Cedex, France
Lebouteiller, B., Inst. de Biotechnologie des Plantes, UMR CNRS 8618, Université Paris XI Sud, F-91 405 Orsay Cedex, France
Gousset-Dupont, A., Inst. de Biotechnologie des Plantes, UMR CNRS 8618, Université Paris XI Sud, F-91 405 Orsay Cedex, France
Vidal, J., Inst. de Biotechnologie des Plantes, UMR CNRS 8618, Université Paris XI Sud, F-91 405 Orsay Cedex, France
Granot, D., Institute of Field and Garden Crops, Agricultural Research Organisation, Volcani Center, Bet Dagan 50 250, Israel
Denoyes-Rothan, B., U. Rech. Sur Les Especes F., INRA, Université de Bordeaux 2, BP 81, F-33 883 Villenave d'Ornon, Cedex, France
Lerceteau-Köhler, E., CIREF, Lanxade, F-24 130 Prigonrieux, France
Rolin, D., UMR Physiol. et Biotech. Vég., INRA, Université de Bordeaux 2, BP 81, F-33 883 Villenave d'Ornon, Cedex, France

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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.

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