Thermal Dependence of the Apparent K_m of Glutathione Reductases from Three Plant Species

Abstract

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...

Optimal Thermal Environments for Plant Metabolic Processes (Cucumis sativus L.) (Light-Harvesting Chlorophyll a/b Pigment-Protein Complex of Photosystem II and Seedling Establishment in Cucumber)

“…Using the criterion of 200% of the minimum K , for determining the TKW (Burke et al, 1988) resulted in a TKW encompassing temperatures from 23.5 to 39OC with a minimum apparent K, value at 32.5OC. These results differ from those reported previously for cucumber NADPH-glutathione reductase in that the range of temperatures delineating the TKW is from 23.5 to 39OC rather than from 35 to 41OC (Mahan et al, 1990). The upper limits of the TKW in the two studies are similar; however, the lower limits differ by more than 10°C.…”

“…Because of the difference in TKW between that determined for NADH-hydroxypyruvate reductase in the present study and that reported previously for NADPHglutathione reductase (Mahan et al, 1990), we have used the fluorescence technique to further establish the bounds of the temperature optimum of cucumber.…”

“…Alternatively, because the minimum apparent K, values for NADPH-glutathione reductase are in the 1-to 2-PM range, even a small error in determination would have a significant impact on the TKW. If the reported 4OoC apparent K, of the NADPH-glutathione reductase (Mahan et al, 1990) Plant Physiol. Vol.…”

“…The TKW has been shown to be a reliable indicator of the temperature range outside of which plants experience temperature stress and reduced plant performance (Burke et al, 1988;Burke, 1990;Mahan et al, 1990;Ferguson et al, 1991). TKWs have been identified for severa1 crop species (Burke et al, 1988;Burke, 1990;Mahan et al, 1990;Ferguson et al, 1991), and have proven to be narrower than plant temperatures experienced on a seasonal or daily basis (Burke et al, 1988;Ferguson et al, 1991). Temperature extremes (for example, heat shock and chilling injury) are not required for a plant to experience thermal stress.…”

Influence of Water and Temperature Stress on the Temperature Dependence of the Reappearance of Variable Fluorescence following Illumination

“…These measures are used as indicators of metabolic efficiency. The TKWs have been identified for several crop species (1,2,4,5). These TKWs are narrower than plant temperatures experienced on a seasonal or daily basis (2).…”