Abstract
Following the discovery of glutamine synthetase/glutamate (Glu) synthase, the physiological roles of Glu dehydrogenase (GDH) in nitrogen metabolism in plants remain obscure and is the subject of considerable controversy. Recently, transgenics were used to overexpress the gene encoding for the beta-subunit polypeptide of GDH, resulting in the GDH-isoenzyme 1 deaminating in vivo Glu. In this work, we present transgenic tobacco (Nicotiana tabacum) plants overexpressing the plant gdh gene encoding for the alpha-subunit polypeptide of GDH. The levels of transcript correlated well with the levels of total GDH protein, the alpha-subunit polypeptide, and the abundance of GDH-anionic isoenzymes. Assays of transgenic plant extracts revealed high in vitro aminating and low deaminating activities. However, gas chromatography/mass spectrometry analysis of the metabolic fate of (15)NH(4) or [(15)N]Glu revealed that GDH-isoenzyme 7 mostly deaminates Glu and also exhibits low ammonium assimilating activity. These and previous results firmly establish the direction of the reactions catalyzed by the anionic and cationic isoenzymes of GDH in vivo under normal growth conditions and reveal a paradox between the in vitro and in vivo enzyme activities.
Highlights
Following the discovery of glutamine synthetase/glutamate (Glu) synthase, the physiological roles of Glu dehydrogenase (GDH) in nitrogen metabolism in plants remain obscure and is the subject of considerable controversy
GDH is a hexamer of a- and b-subunit polypeptides associated in an ordered ratio to form two homohexamers and five hybrids [b6, b5,a1..b1, a5, a6; Loulakakis and RoubelakisAngelakis, 1991; Turano et al, 1996]. gdh cDNAs encoding for the two polypeptides have been isolated from several higher plant species (Purnell et al, 2005)
The two subunit polypeptides of GDH a and b are encoded by two genes, gdh-NAD;A and gdh-NAD;B, respectively
Summary
Following the discovery of glutamine synthetase/glutamate (Glu) synthase, the physiological roles of Glu dehydrogenase (GDH) in nitrogen metabolism in plants remain obscure and is the subject of considerable controversy. Gas chromatography/mass spectrometry analysis of the metabolic fate of 15NH4 or [15N]Glu revealed that GDH-isoenzyme 7 mostly deaminates Glu and exhibits low ammonium assimilating activity These and previous results firmly establish the direction of the reactions catalyzed by the anionic and cationic isoenzymes of GDH in vivo under normal growth conditions and reveal a paradox between the in vitro and in vivo enzyme activities. High intracellular ammonium, provided either externally (Cammaerts and Jacobs, 1985; Srivastava and Singh Rana, 1987; Lea and Ireland, 1999) or as a result of protein hydrolysis (Masclaux et al, 2000; Limami et al, 2002), generally leads to de novo synthesis of the a-subunit, assembly of the anionic isoenzymes, and increased in vitro aminating activity (Loulakakis and Roubelakis-Angelakis, 1992). Purnell and Botella (2007), using transgenic tobacco (Nicotiana tabacum) plants overexpressing the tomato (Solanum lycopersicum) gene encoding for the b-subunit polypeptide of GDH (Purnell et al, 2005), found that GDH-isoenzyme 1 deaminates in vivo Glu
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