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Glutamate (Glu) dehydrogenase (GDH) catalyses the reversible amination of 2-oxoglutarate for the synthesis of Glu using ammonium as a substrate. This enzyme preferentially occurs in the mitochondria of companion cells of a number of plant species grown on nitrate as the sole nitrogen source. For a better understanding of the controversial role of GDH either in ammonium assimilation or in the supply of 2-oxoglutarate (F. Dubois, T. Tercé-Laforgue, M.B. Gonzalez-Moro, M.B. Estavillo, R. Sangwan, A. Gallais, B. Hirel [2003] Plant Physiol Biochem 41: 565-576), we studied the localization of GDH in untransformed tobacco (Nicotiana tabacum) plants grown either on low nitrate or on ammonium and in ferredoxin-dependent Glu synthase antisense plants. Production of GDH and its activity were strongly induced when plants were grown on ammonium as the sole nitrogen source. The induction mainly occurred in highly vascularized organs such as stems and midribs and was likely to be due to accumulation of phloem-translocated ammonium in the sap. GDH induction occurred when ammonia was applied externally to untransformed control plants or resulted from photorespiratory activity in transgenic plants down-regulated for ferredoxin-dependent Glu synthase. GDH was increased in the mitochondria and appeared in the cytosol of companion cells. Taken together, our results suggest that the enzyme plays a dual role in companion cells, either in the mitochondria when mineral nitrogen availability is low or in the cytosol when ammonium concentration increases above a certain threshold.Ammonium is the ultimate form of inorganic nitrogen available to the plant. It can originate from a wide variety of metabolic processes such as nitrate reduction, photorespiration, phenylpropanoid metabolism, utilization of nitrogen transport compounds, amino acid catabolism, symbiotic nitrogen fixation (Hirel and Lea, 2001), and insect digestion in carnivorous plants (Schulze et al., 1997). It is then incorporated into an organic molecule, 2-oxoglutarate, by the combined action of the enzymes Gln synthetase (GS) and Glu synthase (Gln-2-oxoglutarate aminotransferase; GOGAT) to allow the synthesis of Gln and Glu. Both amino acids are further used as amino group donors for the synthesis of virtually all the nitrogencontaining molecules including the other amino acids needed for protein synthesis and nucleotides used as basic molecules for RNA and DNA synthesis (Hirel and Lea, 2001). The GS/GOGAT cycle is the major mechanism of ammonium assimilation in higher plants regardless of the various sources of ammonium listed above. However, it has often been argued that other enzymes have the capacity to assimilate ammonium, leading to the hypothesis that alternative pathways might operate under particular physiological conditions when the GS/GOGAT pathway may not be able to fulfill its function (Harrison et al., 2003).One of these alternative pathways is the reaction catalyzed by the mitochondrial NAD(H)-dependent Glu dehydrogenase (GDH; EC 1.4.1.2), which possesses the ...