Ozone in Populus Leaves1

Atmospheric ozone causes formation of various highly reactive intermediates (e.g. peroxyl and superoxide radicals, H202, etc.) in plant tissues. A plant's productivity in environments with ozone may be related to its ability to scavenge the free radicals formed. The effects of ozone on photosynthesis and some free radical scavengers were measured in the fifth emergent leaf of poplars. Clonal poplars (Populus deltoides x Populus cv caudina) were fumigated with 180 parts per billion ozone for 3 hours. Photosynthesis was measured before, during, and after fumigation. During the first 90 minutes of ozone exposure, photosynthetic rates were unaffected but glutathione levels and superoxide dismutase activity increased. After 90 minutes of ozone exposure, photosynthetic rates began to decline while glutathione and superoxide dismutase continued to increase. Total glutathione (reduced plus oxidized) increased in fumigated leaves throughout the exposure period. The ratio of GSH/GSSG also decreased from 12.8 to 1.2 in ozone exposed trees. Superoxide dismutase levels increased twofold in fumigated plants. After 4 hours of ozone exposure, the photosynthetic rate was approximately half that of controls while glutathione levels and superoxide dismutase activity remained above that of the controls. The elevated antioxidant levels were maintained 21 hours after ozone exposure while photosynthetic rates recovered to about 75% of that of controls. Electron transport and NADPH levels remained unaffected by the treatment. Hence, elevated antioxidant metabolism may protect the photosynthetic apparatus during exposure to ozone. All organisms that have evolved in aerobic environments have a variety of enzymatic and nonenzymatic mechanisms to prevent oxidation of cellular components. It is quite probable that existing mechanisms for detoxifying oxyradicals are invoked in response to ozone since it causes the formation of some highly reactive oxyradicals in aqueous solutions, a major product being the superoxide anion (I1). The superoxide anion can be metabolized by several isozymes of superoxide dismutase found in plants (14). The hydrogen peroxide formed by this reaction is toxic. It can inactivate -SH containing enzymes (5, 13) or react with superoxide to form the hydroxyl radical, which can attack many macromolecular Supported by Environmental Protection Agency grant No. R814197-01-0 to Boyce Thompson Institute, Ithaca, NY, subcontract (BTI 88 No. 1) to R. G. A. , Virginia Polytechnic Institute and State University, Blacksburg, VA. species. It is to the cell's advantage, therefore, to remove the hydrogen peroxide quickly. This occurs through a series of oxidations and reductions of glutathione, ascorbic acid, and NADPH (9, 22). We have observed that ozone causes an increase in the activity of components of the superoxide dismutase-ascorbate-glutathione pathway in clonal poplars. In our experimental system, photosynthesis was inhibited even under conditions when levels of NADPH were not detectably affected. In another experimental system, reductant was found to accumulate as a result of ozone exposure, while the reductive pentose phosphate pathway was inhibited (17, 18). Since NADPH did not accumulate in our system on exposure to ozone, it may have been consumed in other metabolic processes; for example, the increased synthesis and rereduction of oxidized glutathione. In an ozone enriched environment, a successful plant would be one that effectively removes the increased free radicals formed by ozone. The formation of free radicals by ozone is very fast; thus, the antioxidant scavenging system should respond quickly before extensive ozone damage can occur. To our knowledge, there have been no published reports of the initial effects of ozone on the scavenging system of plants. Antioxidant increases have indeed been shown to occur in plants (21) after prolonged ozone exposure but no information is available on the pattern of antioxidant accumulation. In this report we document the initial effects of ozone on some aspects of the antioxidant scavenging system of plants. A preliminary report of our findings has appeared elsewhere (27). MATERIALS AND METHODS