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Plant and Cell Physiology Advance Access originally published online on August 18, 2009
Plant and Cell Physiology 2009 50(10):1761-1773; doi:10.1093/pcp/pcp118
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© The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use distribution, and reproduction in any medium, provided the original work is properly cited.

Resolving the Role of Plant Glutamate Dehydrogenase. I. in vivo Real Time Nuclear Magnetic Resonance Spectroscopy Experiments

Soraya Labboun1,5, Thérèse Tercé-Laforgue2,5, Albrecht Roscher1, Magali Bedu2, Francesco M. Restivo3, Christos N. Velanis4, Damianos S. Skopelitis4, Panagiotis N. Moshou4, Kalliopi A. Roubelakis-Angelakis4, Akira Suzuki2 and Bertrand Hirel2,*

1Génie Enzymatique et Cellulaire, UMR CNRS 6022, UFR des Sciences, Université de Picardie Jules Verne, 33, Rue Saint-Leu, 80039 Amiens cedex, France
2Unité de Nutrition Azotée des Plantes, Unité de Recherche 511, Institut National de la Recherche Agronomique, Centre de Versailles-Grignon, Institut Jean-Pierre Bourgin, 78026 Versailles cedex, France
3Department of Genetics, Biology of Microorganisms, Anthropology and Evolution, University of Parma, Parco Area delle Scienze 11/A, 43100 Parma, Italy
4Department of Biology, University of Crete, 71409 Heraklion, Greece

*Corresponding author: E-mail, hirel{at}versailles.inra.fr; Fax, +33-1-30-83-30-96.


  Abstract

In higher plants the glutamate dehydrogenase (GDH) enzyme catalyzes the reversible amination of 2-oxoglutarate to form glutamate, using ammonium as a substrate. For a better understanding of the physiological function of GDH either in ammonium assimilation or in the supply of 2-oxoglutarate, we used transgenic tobacco (Nicotiana tabacum L.) plants overexpressing the two genes encoding the enzyme. An in vivo real time 15N-nuclear magnetic resonance (NMR) spectroscopy approach allowed the demonstration that, when the two GDH genes were overexpressed individually or simultaneously, the transgenic plant leaves did not synthesize glutamate in the presence of ammonium when glutamine synthetase (GS) was inhibited. In contrast we confirmed that the primary function of GDH is to deaminate Glu. When the two GDH unlabeled substrates ammonium and Glu were provided simultaneously with either [15N]Glu or 15NHFormula respectively, we found that the ammonium released from the deamination of Glu was reassimilated by the enzyme GS, suggesting the occurrence of a futile cycle recycling both ammonium and Glu. Taken together, these results strongly suggest that the GDH enzyme, in conjunction with NADH-GOGAT, contributes to the control of leaf Glu homeostasis, an amino acid that plays a central signaling and metabolic role at the interface of the carbon and nitrogen assimilatory pathways. Thus, in vivo NMR spectroscopy appears to be an attractive technique to follow the flux of metabolites in both normal and genetically modified plants.

Keywords: Ammonium - Glutamate - Glutamate dehydrogenase - Gene overexpression - Homoeostasis - Tobacco

Abbreviations: AlaAT, alanine aminotransferase; {alpha}-Glx, {alpha}-amino groups of Glu and Gln; AOA, aminooxyacetate; GABA, {gamma}-aminobutyrate; GDH, glutamate dehydrogenase; GS, glutamine synthetase; GOGAT, glutamate synthase; MSX, methionine sulfoximine; NMR, nuclear magnetic resonance; PAGE, polyacrylamide gel electrophoresis; WT, wild-type.

5These authors contributed equally to this work.

(Received April 7, 2009; Accepted August 11, 2009)
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