title: Modulation of higher-plant NAD(H)-dependent glutamate dehydrogenase activity in transgenic tobacco via alteration of beta subunit levels.
abstract: Glutamate dehydrogenase (GDH; EC 1.4.1.2-1.4.1.4) catalyses in vitro the reversible amination of 2-oxoglutarate to glutamate. In vascular plants the in vivo direction(s) of the GDH reaction and hence the physiological role(s) of this enzyme remain obscure. A phylogenetic analysis identified two clearly separated groups of higher-plant GDH genes encoding either the alpha- or beta-subunit of the GDH holoenzyme. To help clarify the physiological role(s) of GDH, tobacco (Nicotiana tabacum L.) was transformed with either an antisense or sense copy of a beta-subunit gene, and transgenic plants recovered with between 0.5- and 34-times normal leaf GDH activity. This large modulation of GDH activity (shown to be via alteration of beta-subunit levels) had little effect on leaf ammonium or the leaf free amino acid pool, except that a large increase in GDH activity was associated with a significant decrease in leaf Asp (~51%, P=0.0045). Similarly, plant growth and development were not affected, suggesting that a large modulation of GDH beta-subunit titre does not affect plant viability under the ideal growing conditions employed. Reduction of GDH activity and protein levels in an antisense line was associated with a large increase in transcripts of a beta-subunit gene, suggesting that the reduction in beta-subunit levels might have been due to translational inhibition. In another experiment designed to detect post-translational up-regulation of GDH activity, GDH over-expressing plants were subjected to prolonged dark-stress. GDH activity increased, but this was found to be due more likely to resistance of the GDH protein to stress-induced proteolysis, rather than to post-translational up-regulation.
authors.affiliation: Department of Botany, University of Queensland, QLD 4072, Australia.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Purnell
authors.forename: Damianos S
authors.initials: DS
authors.lastname: Skopelitis
authors.forename: Kalliopi A
authors.initials: KA
authors.lastname: Roubelakis-Angelakis
authors.forename: José R
authors.initials: JR
authors.lastname: Botella
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D003001:Cloning, Molecular; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D005838:Genotype; D018086:Glutamate Dehydrogenase (NADP+) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology; D010802:Phylogeny; D030821:Plants, Genetically Modified; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics*; D014170:Transformation, Genetic
chemical_list: D000596:Amino Acids; D007527:Isoenzymes; D021122:Protein Subunits; D000644:Quaternary Ammonium Compounds; D012333:RNA, Messenger; D018086:Glutamate Dehydrogenase (NADP+)