Glutamate dehydrogenase (GDH) is an enzyme ubiquitous in nature that in vitro catalyses the reversible amination of α-ketoglutarate to glutamate. The in vivo direction of the GDH reaction in higher plants and hence the role of this enzyme is unclear, and at times has been a topic of controversy. This matter is confounded by the existence of several GDH isozymes, which result from the association of two separately encoded subunits (named α and β) into the GDH holoenzyme. The major aims of this thesis were to clarify the in vivo reaction direction of GDH in higher plants and to subsequently infer the role of this enzyme.
Transgenic tobacco plants were generated with between 0.5x and 34x normal GDH activity, via modulation of β subunit levels. Plant growth and amino acid and ammonium levels were unaffected under ideal growing conditions, suggesting that under these conditions GDH does not play a role in metabolism. The modulation of GDH activity in these lines also revealed possible cryptic translational control of GDH subunit levels.
The transgenic plants were used to clarify the in vivo direction of the GDH reaction. GDH over-expressing (OE) and under-expressing (UE) plants were supplied 15N-glutamate and a GS inhibitor via the roots. After 4 hours of labelling, ~27% more 15N-ammonium accumulated within the roots of two independent OEs compared with a control line, whereas 17% less 15N-ammonium accumulated within the roots of an UE (all P<0.01), Hence, the amount of accumulated 15N-ammonium within the roots of a particular genotype correlated with the amount of GDH activity within that genotype, indicating that GDH catabolises glutamate in vivo producing α-ketoglutarate and ammonium. In another experiment, an OE line was sprayed with a GS-inhibiting herbicide which causes photorespiratory ammonium accumulation and subsequent cell death. The same amount of ammonium accumulated in OE and control plants, suggesting that GDH does not assimilate ammonium in vivo.
Two cDNAs encoding either GDH α (ntgdhl) or β (ntgdh2) subunits were cloned from tobacco. Both genes were expressed at very low levels in leaves, and at higher levels in stems and roots, in correspondence with GDH protein and activity levels. In addition, ntgdhl, but not ntgdh2, was also expressed in flowers and senescing leaves. Neither gene was induced during the night, nor was GDH activity post-translationally up-regulated by prolonged dark-stress. Preliminary in situ hybridisation results with a tomato GDH β subunit cDNA, legdhl, suggested that this gene was expressed throughout tomato roots and flowers. In addition, legdhl was apparently expressed more strongly in lateral root primordia, as well as in axillary buds that were activated via plant decapitation. Exposure of tobacco leaf discs to various environmental stresses and phytohormones revealed that the tobacco GDH subunit genes were differentially induced: ntgdhl was induced by water stress, salt stress and wounding, whereas ntgdh2 was induced by cold stress and wounding. In addition, ntgdhl was induced slightly by 1-aminocyclopropane-l-carboxylic acid (ACC), strongly by indole acetic acid (lAA), and most strongly by salicylic acid (SA), ntgdh2 was induced by ACC, benzylaminopurine, epibrassinolide, gibberellic acid, LAA, and most strongly by SA. ntgdh2, but not ntgdhl, was also induced by exogenously supplied ammonium, but induction was delayed by several hours by co-supplying sucrose. These results indicated that GDH gene expression is regulated by a complex interaction between developmental and environmental signals which may be mediated through sugar/ATP and phytohormone levels. Moreover, although the GDH isozymes comprised totally of either α or β subunits apparently both catabolise glutamate, these isozymes seemingly have different physiological roles.
From these combined results the role of GDH in source leaves, sink tissues, and senescing leaves has been inferred. In source leaves it is proposed that GDH gene expression is induced rapidly by effects of environmental stresses such as decreases in ATP/sugar levels and/or increases in reactive oxygen species (ROS). Under these conditions GDH catabolises glutamate to fuel the TCA cycle with α-ketoglutarate to facilitate ATP production or ROS-reducing alternative oxidase activity. In sink tissues it is proposed that a permanent GDH pool remains inactive under ideal growth conditions, but in times of carbon limitation is activated to conduct a role similar to GDH in leaves. In addition to this role, GDH in roots may also help supply organic acids for heavy metal chelation in the rhizosphere. In senescing tissues it is proposed that a constantly active GDH pool not only helps produce ATP, but also supplies ammonium to GS to allow glutamine export.