Resolving the role of plant glutamate dehydrogenase. I. In vivo real time nuclear magnetic resonance spectroscopy experiments.
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 (15)N-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 [(15)N]Glu or (15)NH(4)(+) 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.
Labboun
Soraya
S
Génie Enzymatique et Cellulaire, UMR CNRS 6022, UFR des Sciences, Université de Picardie Jules Verne, 33, Rue Saint-Leu, 80039 Amiens cedex, France.
Tercé-Laforgue
Thérèse
T
Roscher
Albrecht
A
Bedu
Magali
M
Restivo
Francesco M
FM
Velanis
Christos N
CN
Skopelitis
Damianos S
DS
Moschou
Panagiotis N
PN
Moshou
Panagiotis N
PN
Roubelakis-Angelakis
Kalliopi A
KA
Suzuki
Akira
A
Hirel
Bertrand
B
D005969:Glutamate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000096:biosynthesis; D009682:Magnetic Resonance Spectroscopy; D009584:Nitrogen / Q000378:metabolism; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics
D010940:Plant Proteins; D000644:Quaternary Ammonium Compounds; D018698:Glutamic Acid; D005969:Glutamate Dehydrogenase; D009584:Nitrogen
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