Construction of vectors for the overexpression and inhibition of iron superoxide dismutase in Nicotiana tabacum

Recent advances in plant biotechnology provide scientists with the opportunity to alter crops to meet the needs of society. Research reported in this thesis is the preliminary result of an attempt to genetically engineer plants to resist oxidative stress.

Both plants and animals use oxygen as an electron acceptor in oxidative phosphorylation, reducing oxygen to water. Plants also produce oxygen by oxidizing water to provide elecfrons for photosynthesis. The oxidation of water to oxygen during photosynthesis, which requires the transfer of four electrons per oxygen molecule produced, is completed in a single step. Therefore, no reactive oxygen intermediates are produced. High light raises the potential energy of the elecfrons in the photosynthetic pathway, providing reducing power for the elecfron transport chain (reviewed in Bowler et al., 1992). Reactive oxygen intermediates such as superoxide (-02'), hydrogen peroxide (H2O2), and the hydroxyl radical (-OH) are produced when the plant cannot utilize this energy by productive means (such as storing the energy of the elecfrons by using them to produce carbohydrates). The plant must drain the electrons nonproductively. Reactive oxygen intermediates cause cellular damage that is referred to as oxidative stress.

During photosynthesis, molecular oxygen competes with nicotinamide adenine dinucleotide phosphate (NADP) for elecfrons from the reduced photosystem. NADP is the primary electron acceptor and is reduced to NADPH, providing reducing power for carbohydrate synthesis. Under stressfiil conditions, including high light and cold temperatures, the Calvin Cycle cannot recycle NADP quickly enough, so that the NADP becomes scarce and the plant reduces O2. A plant under oxidative stress reduces oxygen by a single electron, instead of by four as in oxidative phosphorylation, resulting in a negatively charged oxygen molecule with an unpaired electron. This reactive molecule is the superoxide radical (•O2").

In addition to photosynthesis and respiration, the univalent reduction of oxygen to produce superoxide may result from a variety of biological reactions. These include the oxidation of flavins (Ballou et al, 1969), hydroquinones (Misra and Fridovich, 1972a), catecholamines (Cohen and Heikkila, 1974), thiols (Saez et al, 1982), hemoglobin (Misra and Fridovich, 1972b) and reduced ferredoxin (Misra and Fridovich, 1971). Several oxidative enzymes can also produce superoxide including xanthine oxidase, aldehyde oxidase, and several flavin dehydrogenases (reviewed by Fridovich, 1975). Also, neutrophils and macrophages produce toxic concentrations of superoxide during phagocytosis in response to infection (Bannister and Bannister, 1985).