The effect of elevated glutathione reductase and superoxide dismutase activities in stressed transgenic tobacco.

Abstract

Life as we know it would be impossible in the absence of oxygen. However, too much oxygen can be toxic to the aerobic organisms which depend on it for their very existence. This apparent paradox arises as a result of oxygen's ability to accept electrons, forming highly reactive (reduced) oxygen species such as superoxide, hydrogen peroxide and the hydroxyl radical. The toxicity of oxygen is greatly enhanced in illuminated plants, due to the photosynthetic reactions which produce both oxygen and highly energetic electrons in close proximity to one another. These problems are further exacerbated when plants are exposed to a variety of stress conditions, since these conditions reduce the ability of plants to utilise excess electrons. As a result of the danger posed by· these reactive oxygen species, plants have· evolved a complex antioxidant system for their scavenging. Research has shown that plants with naturally elevated levels of the components of the antioxidant system are better equipped to deal with stress conditions which enhance the production of reactive oxygen species. A considerable amount of research has thus been dedicated to the elucidation of the antioxidant system. Almost as much research has been dedicated to enhancing the antioxidant system, with the aim of improving plant productivity under stress conditions. This study sought to evaluate plants carrying elevated levels of two of the enzymes of the antioxidant system. For these purposes, tobacco was transformed with the gene for E. coli glutathione reductase (GR), an enzyme believed to catalyse the rate limiting reaction in the scavenging of hydrogen peroxide. This gene was fused to the gene for the RUBISCO small subunit transit peptide - a peptide capable of targeting proteins to the chloroplast. Due to the presence of this peptide the transformed plants exhibited high chloroplastic levels of GR activity. These plants were crossed with a second tobacco transformant carrying high levels of chloroplastic tomato superoxide dismutase (SOD) - an enzyme responsible for the scavenging of superoxide. These hybrid plants were shown to exhibit high GR and SOD activities in the chloroplast .- the subcellular compartment most susceptible to damage caused by reactive oxygen species. The transgenic hybrids were evaluated for their ability to tolerate oxidative stress by treating them with paraquat - a herbicide whose mode of action involves the production of large quantities of activated oxygen. Under stress conditions, plants carrying just E. coli GR showed a slight improvement in their ability to deal with oxidative stress. In contrast to this, the SOD transformants showed more cellular damage than untransformed control plants. This was attributed to the inability of other enzymes in the antioxidant pathway to deal with the increased flow of metabolites through the pathway. The hybrid transformants showed enhanced stress tolerance in the initial stages of oxidative stress, but this declined with ongoing exposure to stress conditions. As with the SOD transformants, this decline in protection was . ascribed to the relatively low activities of the other enzymes in the antioxidant pathway. It was concluded that elevated levels of the two enzymes conferred greater stress tolerance than just one of the enzymes, but for true stress tolerance it will be necessary to evaluate the antioxidant system and enhance the activity of further enzymes in the pathway. It may also be necessary to improve the regulation of transgene expression, ensuring that none of the enzymes are overwhelmed by the increased flow of metabolites through the system.