Modulation of higher-plant NAD(H)-dependent glutamate dehydrogenase activity in transgenic tobacco via alteration of beta subunit levels

Planta. 2005 Sep;222(1):167-80. doi: 10.1007/s00425-005-1510-z. Epub 2005 Apr 1.

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.

MeSH terms

  • Amino Acids / metabolism
  • Cloning, Molecular
  • Gene Expression Regulation, Enzymologic
  • Gene Expression Regulation, Plant*
  • Genotype
  • Glutamate Dehydrogenase (NADP+) / chemistry*
  • Glutamate Dehydrogenase (NADP+) / genetics
  • Glutamate Dehydrogenase (NADP+) / metabolism*
  • Isoenzymes / chemistry
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Mitochondria / enzymology
  • Phylogeny
  • Plants, Genetically Modified
  • Protein Processing, Post-Translational
  • Protein Subunits / biosynthesis
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • Quaternary Ammonium Compounds / metabolism
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Tobacco / enzymology*
  • Tobacco / genetics*
  • Transformation, Genetic

Substances

  • Amino Acids
  • Isoenzymes
  • Protein Subunits
  • Quaternary Ammonium Compounds
  • RNA, Messenger
  • Glutamate Dehydrogenase (NADP+)