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.

There is clear potential for the genetic manipulation of key enzymes involved in stress metabolism in transgenic plants. However, the data emerging so far from such experiments are equivocal. The detailed analysis of stress responses in progeny of primary transgenics, coupled with comparisons with control transgenic plants that do not contain the GR transgene, allows us to take into account the possible variation in response to stress associated with regeneration of plants from tissue culture. The picture that is now beginning to emerge with respect to the role of GR in stress protection is that, although there are clearly benefits to be had from overexpression of the enzymes, there is no direct correlation between enzyme levels and stress tolerance. It may be that overexpression of the cytosolic isoform (gor2) will prove to be of greater benefit. Furthermore, the types of stresses to which transgenic plants have been exposed in order to assess the consequences of oxidative stress tolerance cannot reproduce those that will experienced in field conditions. Only when plants with higher GR levels and increased glutathione synthesis capacity are grown in field trials will it be possible to make a full assessment of the benefits of engineering plants with altered glutathione metabolism.

SynopsisThe activities of a number of enzymes of the ascorbate–glutathione pathway have been shown to rise under conditions of increased oxidative stress. The potential to alter the expression of specific enzymes of this pathway by genetic manipulation has provided the opportunity to attempt to develop transgenic plants with altered levels of oxidative stress defense enzymes which should have improved stress tolerances. We have cloned a cDNA for glutathione reductase from a higher plant (Pisum sativum L.) and have used this to construct chimeric genes for the expression of the pea enzyme in the chloroplast, mitochondrion or cytosol of transgenic tobacco plants. Some of the transformed lines with elevated levels of expression of glutathione reductase accumulate higher concentrations of glutathione and show increased tolerance to paraquat, however, no evidence was found for elevated tolerance to ozone fumigation.

The types of environmental stresses, both natural and anthropomorphic in origin, and physiological stresses that a plant is subjected to throughout its life are manifold. Examples are low temperatures combined with high light intensities, drought conditions, damage caused by pathogens, air-and waterborne pollutants and premature senescence [ 1,2].The physiological basis of the damage suffered by a plant during stress can often be explained as perturbations in oxygen metabolism leading to enhanced production of reactive oxygen intermediates (ROIs; hydrogen peroxide, superoxide anion and the hydroxyl radical). Most of the information available concerns the performance of photosynthesis in the leaf under stress [ 31. Disruption of the flow of energy from light-harvesting reactions, through electron-transport chains to CO, fixation, leads to 'leakage' of electrons to oxygen, causing the production of ROIs. If the protective mechanisms of the cell are compromised or overwhelmed, the effects on the plant are termed photooxidative damage. recognizing that during growth of the plant it is the chloroplast that is the primaryThe strategy of all aerobic organisms facing oxidative stress is to minimize production of KOls and to remove superoxide and hydrogen peroxide by reducing them to water by the concerted action of superoxide dismutases (S0I)s; EC 1.15.1 .l) and catalyses or peroxidases. In the chloroplast, the ~~ Abbreviations used: AI'X, ascorbate peroxidase; CaMV. cauliflower mosaic virus; GH, glutathione reductase; HOI. reactive oxygen intermediate: SO1 1, superoxide dis-$To whom correspondence should be addressed. 111utase.KOI-scavenging enzymes are Cu/%n-SOl)s (and sometimes Fe-SODS) [ 5 J and ascorbate peroxidase (APX; EC 1.11.1.7) [6J. The activities of these enzymes are supported by the antioxidants and enzymes of the glutathione-ascorbate cycle, which has been proposed to involve successive oxidations and reductions of ascorbate, glutathione and NADPI 1 by the enzymes APX. monodehydroascorbate reductase (EC 1.6.5.4), dehydroascorbate reductase (EC 1.8.5. I), glutathione reductase (GR; EC 1.6.4.2). Much of the information concerning this pathway has focused on its role in the prevention of photo-oxidative damage in the chloroplast, but the presence of enzymes of the glutathione-ascorbate cycle in non-photosynthetic tissues [7-9] demonstrates that this pathway may be functional in other cellular compartments. However, doubts about all, or part, of this cycle remain to be answered. In the chloroplast, an alternative route for the regeneration could be the direct reduction of monodehydroascorbate by electrons from photosystem I [ 101. The relative importance of glutathione-dependent versus glutathione-independent reactions for the regeneration of ascorbate and how the other roles of glutathione (see below) fit into this scheme have not been addressed.Our interest is to arrive at a more complete understanding of the roles of these enzymes in oxidative stress by applying molecular genetic techniques in conjunction wi...

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