The thermal dependencies of the apparent Km of the glutathione reductases from spinach (Spinacia oleracea L.) corn (Zea mays L.), and cucumber (Cucumis sativus L.) were determined. The apparent Km of the enzymes were found to vary up to 9-fold between 12.5 and 450C. Values of the apparent Km in excess of 200% of the observed minimum are suggested to be detrimental to the normal function of the enzyme. We propose the term "thermal kinetic window" to describe to the range of temperatures over which the apparent Km of the glutathione reductase is within 200% of its minimum and suggest that it may be a useful indicator of the limits of thermal stress for a given species. The thermal kinetic windows determined in this study are: <160C for spinach, 23 to 320C for corn, and 35 to 410C for cucumber.The effects of thermal stress on plants are substantial and have been extensively investigated and reviewed (6,14). Thermal stress has been defined as a temperature-induced aberration in the metabolism of the plant that, under certain conditions, is expressed as a reduction in growth, yield, or value of the plant (7). Temperature-induced aberrations in metabolism are often related to changes in the activity of enzymes and thus the thermal dependence of enzyme function may prove to be a useful indicator of the onset of thermal stress.GR' is thought to play an important role in the protection of the plant from both high and low temperature stresses by preventing the oxidation of enzymes and membranes (5,8,13,16). In the light ofthis role, a temperature-induced decline in GR activity could adversely affect the metabolism of the plant and thereby increase its susceptibility to thermal stresses. The effect of temperature on reaction rate and molecular integrity of GR has been addressed by Burke and Hatfield (2) who predicted changes in maximal velocity associated with changes in plant temperature. However, maximal velocity is not generally indicative ofthe in vivo rates ofenzyme reactions and thus may not be predictive of the effects of temperature on metabolism (15).The in vivo rate of an enzyme reaction is dependent upon interactions between the enzyme and specific ligands. The value of the apparent Km has been used as an indicator of 'Abbreviations; GR, glutathione reductase; TKW, thermal kinetic window.enzyme:ligand interactions. These interactions are often temperature dependent and it has been suggested that the thermal dependence of the Km of various plant enzymes may play a role in the thermal dependence of whole plant responses (6, 15).The objective of this study was to determine if the differential sensitivity of some plant species to thermal stresses could be related to differences in the thermal dependence of the Km of their GRs. We have chosen spinach, corn, and cucumber as representatives of cool, moderate, and warm environment species, respectively. The thermal dependence of apparent Km of the GRs from these species for NADPH was determined over a 12.5 to 45°C thermal gradient.
MATERIALS AND METHODS
Plant Ma...
The plasmid pAMβ1 has been successfully transferred from Streptococcus faecalis to Bacillus sphaericus by the method of filter mating. Subsequently, transfer of this conjugative plasmid has also been demonstrated between derivatives of B. sphaericus 1593, though at reduced frequencies. The transfer process was determined to be unaffected by either endogenous restriction enzyme activity of the recipient or the presence of exogenous DNase.
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