'" • ••••• AL.F. PI .... .. , © 1997 by Gustav Fischer Verlag, Jena Light Regulation of native and Escherichia coli Glutathione Reductase in Transgenic Tobacco PAUL w. BADENHoRSTI, ALAN M. AMORY*, and BARBARA I. HUCKETT 2 Biology Department, University of Natal, Durban 4041, South Africa Received May 8, 1997· Accepted July 15, 1997 Summary In plants, defence against oxygen free radicals relies on the Asada-Halliwell pathway, a series of enzyme- catalysed redox reactions that reduce toxic oxygen species to water at the expense of reductant. The rate limiting step in this cycle is believed to be the reduction of glutathione, by glutathione reductase (GR, EC 1.6.4.2), using NADPH. As a tool to understanding the plant antioxidant system, tobacco (Nicotiana tabacum cv. Samsun) containing the Escherichia coli GR gene (gor) has been engineered by Agrobacterium tumifaciem-mediated transformation. The response of these transformants to light has been examined and data indicates that both plant and E. coli glutathione reductases are subject to diurnal variation. Total GR activity (GRA) in control and transgenic plants increases following exposure to light, but the increases are greater in the transgenic plants. Separation of native and E. coli GR isoforrns indicates that this is due to increases in both tobacco and E. coli GRA. Rises in E. coli GRA can be ascribed primarily to a light- mediated post-translational activation. Although E. coli GR transcript and protein levels vary over the photoperiod, the nature and magnitude of the fluctuations do not account for the changes in E. coli GRA, suggesting that E. coli GR is subjected to a light-mediated post-translational activation. While tobacco GR levels could not be assessed, it is likely that tobacco GR is subject to the same post-translational activation. Key words: Nicotiana tabacum cv. Samsun, Glutathione reductase, Light, Oxidative stress. Introduction While the energetic advantages of life in the presence of oxygen are evident, such an existence has inherent claflgers. Unless controlled, the potential exists for the formation of oxygen free radicals and the resultant destruction of cell com- ponents. This is particularly valid in plants where, in the chloroplast, the juxtaposition of a potent electron generating pathway with oxygen production poses a hazard. Control of toxic oxygen species is mediated primarily by superoxide dis- mutase (SOD, EC 1.15.1.1) and the Asada-Halliwell pathway, a series of non-enzymatic and/or enzyme-catalysed reductions of ascorbate and glutathione, using NADPH as the source of reductant (Foyer and Halliwell, 1976; Asada and Takahashi, 1987). Glutathione reductase (GR, EC 1.6.4.2), the final step in the cycle, provides the link between the antioxidant system and photochemically produced reductant (NADPH) and has been proposed to be the rate limiting step Oablonski and Anderson, 1981). Under normal circumstances this system functions effi- ciently to limit oxygen damage. However, under adverse con- ditions leading to the release of large amounts of free radicals, it is likely that the system will be severely tested. Nearly all major environmental stresses on plants exert most, or at least a component, of their effects through the production of oxy- gen free radicals. Atmospheric pollutants such as ozone (Sharma and Davis, 1994) and sulphur dioxide generate free radicals (Asada and Takahashi, 1987). Drought and cold * Correspondence. 1 Present address: MRC Laboratory of Molecular Biology, Hills Rd, Cambridge CB2 2QH, England. 2 Present address: Biotechnology Department, SASA Experiment Station, Private Bag X02, Mount Edgecombe, 4300, South Africa. J Plant Physiol W,L 152. pp. 502-509 (1998)