Environmental stress (abiotic and biotic) result in oxidative stress. This occurs most directly as a result of ozone pollution or ionizing radiation. However, oxidative stress is a secondary effect of many type of stress, from pathogen attack to water-deficit stress. The oxidative stress arises from the production of free radicals and the subsequent ensuring cascade of reactions. Much of the oxidative stress-induced damage to cells occurs as a result of the formation of reactive oxygen species (ROS) .Successive reactions that form superoxide, hydrogen peroxide and hydroxyl-radical species. The ROS cause damage as result of their reaction with cellular macromolecules. Damage to cellular membranes may result from a low concentration of hydroxyl radical triggering a chain reaction of lipid peroxidation. Oxidative damage to proteins may involve specific amino acid modifications, fragmentation of the peptide chain, aggregation cross-linked reaction products. Altered electrical charged increased susceptibility to proteolysis. In DNA, both the sugar and base degradation, single-strand breakage, and cross-linking to protein. These lesions in DNA can result in deletions, mutations and other damaging genetic effects.

Plants contain a number of enzymes that catalyse the cascade of reactive oxygen species and convert them to less reactive products. One key point to notice is the key role of certain enzymes in this cascade, notably superoxide dismutase. Catalase and peroxidases in neutralizing these reactive species. A number of antioxidant compounds which includes B-carotene (provitamin A), ascorbic acid (vitamin C) and -tocopherol (vitamin E). Other important antioxidant system is the ascorbate and glutathione and zeaxanthin. Major antioxidant system is the ascorbate-glutathione cycle. The coupling of ascorbate and glutathione redox-cycling has been most extensively characterized in the chloroplast. Chloroplasts produce superoxide and hydrogen peroxide in the light, particularly from photosystem I. The superoxide generated itself converted into hydrogen peroxide by either spontaneous dismutation or by the activity of superoxide dismutase. The hydrogen peroxide produced is scavenged by the ascorbate and the enzyme ascorbate peroxidase. The monodehydroascorbate produced from this reaction may be regenerated into two ways. One is by reduction of NAD (P) H catalysed by monodehydroascorbate reductase. The other occurs via the dismutation of two monodehydroascorbate molecules to form ascorbate and dehydroascorbate, followed by the reduction of the dehydroascorbate by glutathione,catalysed by dehydroascorbate reductase.

From the outline of plant antioxidants and oxygen-scavenging activities given above, two eneral stragegies for engineering tolerance to oxidactive stress are apparent-either to increase the level of enzymes that remove ROS,or to increase the level of antioxidant compound that react with ROS.

EXPRESSION OF ENZYMES INVOLVED IN SCAVENGING ROS

Superoxide dismutases (SODs) catalyse the reaction in which the superoxide radical with two hydrogen ions to form oxygen and hydrogen peroxide. The activity of this enzyme therefore determine the concentration of the two substrates (superoxide and hydrogen perioxide) that react to from the hydroxyl radical. There is a large family of SOD enzymes, requiring different metal ions for activity, and predominating in different cellular compartments. Thus, Mn-, Fe- and Cu-/Zn- forms are found, specific to different organelles.

Transgenic tobacco plants containing each class of enzymes have been produced and tested for their ability to withstand oxidative stress. In each case, there has been some measureable increase in the ability to withstand oxidative stress such as exposure to ozone.

PRODUCTION OF ANTIOXIDANTS

ascorbate peroxidase, glutathione peroxidase and glutathione reductase--have been transformed into Arabidopsis and tobacco plants and shown to have some effect on tolerance to various abiotic stress such as heat, cold and salinity. Glutathione reductase also provided resistance to the oxidative stress resulting from paraquat treatment. On the other hand,simply increasing the cellular concentration of glutathione by expressing glutathione synthase had no effect on stress tolerance.

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