Oxidative stress responses in transgenic tobacco containing altered levels of glutathione reductase activity

Abstract

SummaryA pea glutathione reductase cDNA was expressed in tobacco. Three classes of construct were used which gave a range of elevated levels of glutathione reductaee (GR) activity in the cytosol (GR32), chloroplasts (GR36), or in both chloroplasts and mitochondria (GR46}. In some transgenic progeny (T z) from self-fertilized GR32 and GR36 primary transformants, having approximately twofold elevation of GR activity as compared with recessive siblings, there was an amelioration of the effect on leaf discs of up to 15 pM paraquat. However, lines with similarly elevated levels of GR activity showed no decreased sensitivity to the herbicide. None of the GR32 and GR36 lines was less sensitive to ozone. Conversely, 1"2 progeny of GR46 lines, with greater than 4.5-fold elevations of GR activity, showed no reduced sensitivity to paraquat but two out of four of these lines were less sensitive to ozone fumigation. The differential response to stress cosegregated with the presence of the transgene but there was no relationship between the degree of stress response and the level of GR activity. There was an elevation in the total glutathione pool in all lines showing increased GR activity but there was no change in the ratio of oxidized to reduced glutathione. These results demonstrate that the mechanisms of protection against ozone and paraquat are different although both can be mediated by elevated GR activity.

Modification of reactive oxygen species scavenging capacity of chloroplasts through plastid transformation

“…We found that elevation of plastid GR activity over-rides feedback control of glutathione synthesis leading to elevated total glutathione levels. Broadbent et al (1995) and Foyer et al (1995) report a similar finding in nuclear transformants of tobacco and poplar respectively.…”

“…We contend that the increased MV sensitivity of the transplastomic lines over-expressing GR has a similar basis, although it is not reflected in a study in poplar (Foyer et al 1995) in which chloroplast targeted GR did result in improved oxidative stress tolerance. Another study with tobacco (Broadbent et al 1995) showed considerable interline variation, some GR over-expressing lines giving a modest increase in oxidative stress tolerance, while others did not. We found that elevation of plastid GR activity over-rides feedback control of glutathione synthesis leading to elevated total glutathione levels.…”

“…The variable results obtained amongst independent nuclear transformants (e.g. Broadbent et al 1995;McKersie et al 1999) should be minimised by the targeted gene insertion, via homologous recombination, into the plastome (Svab et al 1990), eliminating position effects and ensuring uniform expression levels. The lack of any known protein export mechanism from chloroplasts ensures the recombinant protein is localised to the target organelle, and obviates concerns over residual cytosolic expression due to inefficient chloroplast targeting.…”

Impact of Enhanced Capacity to Scavenge Reactive Oxygen Species on Cold Tolerance of Tobacco

“…Research on abiotic stress tolerance of transgenics overexpressing ROS-scavenging enzymes has yielded conflicting results: some studies have shown a beneficial impact of the overexpression on tolerance to a wide range of types of abiotic stress (Mohamed et al 2003;Badawi et al 2004;Eltayeb et al 2007), while others indicate a positive impact of overexpression in response to only some types of stress (Broadbent et al 1995;Kwon et al 2003;Le Martret et al 2011). Several studies on different enzymes have found that overexpression of only some of those enzymes enhanced performance under a particular stress (Foyer et al 1995;Payton et al 2001;Faize et al 2011;Le Martret et al 2011;Poage et al 2011).…”

“…For example, of three poplar lines into which g-glutamylcysteine synthetase (required for production of glutathione) had been introduced, one showed a reduction in F V /F M compared to NT, while another showed increased photosynthetic assimilation and F V /F M compared to NT, and there was also growth variation between the three (Herschbach et al 2010). Similarly, the impact on oxidative and ozone stress tolerance of inserting GR genes into tobacco has been found to vary between lines (Broadbent et al 1995). Plastid (in contrast to nuclear) transformation, however, proceeds through homologous recombination, that is, targeted integration, so less interline variation would be expected.…”