Antioxidative responses of duckweed (Lemna minor L.) to short-term copper exposure

Abstract

Elevated concentrations of copper in the environment result in accumulation of the metal in plants and cause an increase in reactive oxidative species (ROS). The first response to elevated amounts of ROS is increased levels of enzymatic and non-enzymatic antioxidants that reduce oxidative stress. The aim of our study was to evaluate the early stages of antioxidative responses to the low copper concentrations usually present in moderately polluted environments. In addition, some other parameters were examined to evaluate the effect of copper on plants. Duckweed (Lemna minor L.) was exposed to different concentrations of copper sulphate for up to 24 hours. Glutathione concentration and enzymatic activities of catalase, guaiacol peroxidase and glutathione reductase were measured spectrophotometrically. Additionally, delayed and prompt chlorophyll fluorescence was measured by luminometry and fluorometry, respectively. The accumulation of copper in plants exposed for 24 hours to various concentrations of copper sulphate was measured by flame atomic absorption spectrophotometry. The treatment of plants with copper sulphate resulted in an immediate decrease of the glutathione pool, which was replenished after 24 hours at CuSO4 concentrations lower than 2 microM. Higher CuSO4 concentrations caused a decrease of reduced glutathione. The responses of the antioxidant enzymes glutathione reductase, guaiacol peroxidase and catalase to CuSO4 differed during the first six hours of exposure, but their enzyme activities all increased after 24 hours of exposure. All these enzymes displayed biphasic activity curves with maximum values between 0.5 microM and 1 microM CuSO4. The response of guaiacol peroxidase was the most pronounced and statistically significantly specific and that of catalase the least. Delayed chlorophyll fluorescence decreased after exposure to 1 microM CuSO4, but no significant effect on maximum quantum yield of photosystem II (Fv/Fm) was observed. L. minor accumulated relatively high concentrations of copper. The accumulation rate was higher at lower concentrations of copper in the test medium (up to 2 microM CuSO4) than at concentrations above 2 microM CuSO4. One of the most pronounced antioxidative responses to copper exposure was modified levels of oxidized and reduced forms of glutathione. The decrease of the glutathione pool is most probably coupled with induced production of phytochelatins. Antioxidative enzymes showed the biphasic enzyme activity characteristic of stress response. Guaiacol peroxidase exhibited the greatest significant increase of activity, even at higher CuSO4 concentrations at which the activity of catalase and glutathione reductase dropped. The intensity of delayed chlorophyll fluorescence decreased, indicating reduced photosynthesis of plants under stress. All the measured parameters showed that plants respond to even low copper concentrations very soon after exposure. The accumulation rate of copper in duckweed tissues indicates that L. minor is an accumulator species. The synchronized and prompt inducibility of antioxidants indicates their involvement in a general plant defence strategy for coping with metal-induced oxidative stress. Glutathione concentration and guaiacol peroxidase activity were found to be the most sensitive of the early indicators of exposure to copper concentrations present in polluted water bodies. The experimental design of the present study allowed us to compare the sensitivity of various methods and parameters for detecting plant responses to heavy metal-induced oxidative stress. The level of glutathione and the enzyme activities of guaiacol peroxidase and glutathione reductase could be used as a rapidly determined early warning system in toxicity studies.