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JXB Advance Access published online on April 8, 2004
Journal of Experimental Botany, doi:10.1093/jxb/erh119
© 2004 by Oxford University Press
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Received October 3, 2003; accepted January 29, 2004
© 2004 Society for Experimental Biology

RESEARCH PAPER

Altered nitrogen metabolism associated with de-differentiated suspension cultures derived from root cultures of Datura stramonium studied by heteronuclear multiple bond coherence (HMBC) NMR spectroscopy

Ophélie Fliniaux 1, François Mesnard 2*, Sophie Raynaud-Le Grandic 1, Sylvie Baltora-Rosset 3, Christophe Bienaimé 4, Richard J. Robins 5*, and Marc-André Fliniaux 1

1 Laboratoire de Phytotechnologie, EA 2085, Faculté de Pharmacie, 1, rue des Louvels, F-80037 Amiens cedex 1, France
2 Laboratoire de Phytotechnologie, EA 2085, Faculté de Pharmacie, 1, rue des Louvels, F-80037 Amiens cedex 1, France; Laboratoire des Biomolécules et Pathologies Dégénératives, EA 2629, Faculté de Pharmacie, 1, rue des Louvels, F-80037 Amiens cedex 1, France
3 Laboratoire des Biomolécules et Pathologies Dégénératives, EA 2629, Faculté de Pharmacie, 1, rue des Louvels, F-80037 Amiens cedex 1, France
4 Agro Industrie Recherches et Développements, route de Bazancourt, F-51110 Pomacle, France
5 Laboratoire d’Analyse Isotopique et Electrochimique de Métabolismes, CNRS UMR 6006, Faculté des Sciences, 2, rue de la Houssinière, F-44322 Nantes cedex 3, France

* To whom correspondence should be addressed. E-mail: richard.robins{at}chimbio.univ-nantes.fr.


  Abstract

De-differentiation of transformed root cultures of Datura stramonium has previously been shown to cause a loss of tropane alkaloid synthetic capacity. This indicates a marked shift in physiological status, notably in the flux of primary metabolites into tropane alkaloids. Nitrogen metabolism in transformed root cultures of D. stramonium (an alkaloid-producing system) and de-differentiated suspension cultures derived therefrom (a non-producing system) has been compared using Nuclear Magnetic Resonance (NMR) spectroscopy. 15N-Labelled precursors [(15NH4)2SO4 and K15NO3] were fed and their incorporation into nitrogenous metabolites studied using Heteronuclear Multiple Bond Coherence (HMBC) NMR spectroscopy. In both cultures, the same amino acids were resolved in the HMBC spectra. However, marked differences were found in the intensity of labelling of a range of nitrogenous compounds. In differentiated root cultures, cross-peaks corresponding to secondary metabolites, such as tropine, were observed, whereas these were absent in the de-differentiated cultures. By contrast, N- acetylputrescine and {gamma}-aminobutyric acid (GABA) accumulated in the de-differentiated cultures to a much larger extent than in the root cultures. It can therefore be suggested that the loss of alkaloid biosynthesis was compensated by the diversion of putrescine metabolism away from the tropane pathway and toward the synthesis of GABA via N-acetylputrescine.

Key words: Datura stramonium, HMBC NMR, nitrogen metabolism, NMR, transformed cultures (root), transformed cultures (de-differentiated), tropane alkaloids.


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