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FREE-RADICAL MEDIATED PROCESSES AS MARKERS OF AIR POLLUTION STRESS IN TREES Curtis J. Richardson Richard T. Di Giulio Norman E. Tandy School of Forestry and Environmental Studies Duke University ABSTRACT Some of the air pollutants believed responsible for the decline of forests, particularly 03, NOx, H2O2, and SO2 can generate toxic oxygen-free radicals when introduced into biological systems. Free radicals can be defined simply as molecules containing an unpaired electron. The generation of radical species can occur through oxidations, reductions or ionizing radiation. Dioxygen reflection commonly occurs in both biotic and abiotic systems and the intermediates of this reduction (oxyradicals) are the superoxide radical (O2-), hydrogen peroxide (H2O2), and the very reactive hydroxyl radical (OH ). The extremely reactive hydroxyl radical oxidizes organic molecules, including biomolecules which results in enzyme breakdown, membrane damage (lipid peroxidation), and DNA alterations. The primary defense against the oxygen compounds superoxide and hydrogen peroxide involves the enzymes superoxide dismutase (SOD), peroxidase (Px) and catalase. In addition, non-enzymatic defenses include such compounds as vitamin E (a-tocopherol), B-carotenes, glutathione and ascorbic acid. We hypothesize that an increase in the production of these antioxidant enzymes by some genotypes in the population will result in increased tree resistance to air pollutants. Changes in the levels of production of these antioxidants and an increase of malondialdehyde (an index of lipid peroxidation) could also be used as biomarkers of oxidant stress in trees. Current air pollution research on 3 loblolly pine families (Pinus taeda) subjected to 5 ozone and 2 acid level treatments has shown increases in SOD, peroxidase, total glutathione and malondialdehyde at high O3 levels in conjunction with significant decreases in net photosynthesis. Our preliminary work and that of others suggest that we may be able to utilize antioxidants as early warning biomarkers of oxidative stress in trees. INTRODUCTION Photochemical oxidants such as SO2, NOx, and ozone are recognized as having phytotoxic effects on plants via the generation of free radicals (Mehlhorn et al. 1986, Castillo et al. 1987, Tandy et al. 1987, Wellburn 1987~. A free radical is defined as any atom, group of atoms or molecule in a particular state with one unpaired electron occupying an outer orbital (Del Maestro 1980~. The one electron oxidation or reduction 251
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252 of compounds results in the production of either cation or anion radicals, respectively (Thomas and Aust, 1986). The univalent pathway for reduction of molecular oxygen results in the generation of the superoxide anion radical (O2-), hydrogen peroxide, (H202) and hydroxyl radical (OH ) intermediates as shown in Figure 1. In addition to direct oxidative degradation of lipids, destruction of intercellular and extracellular proteins, including enzyme inactivation, or DNA damage, indirect toxic effects like mutagenicity and carcinogenicity have been related to oxygen radical formation (Kappus 1987). 0~ o- e ~ 2H ll2Oz: OH ~ ~0 .~0 Figure 1. The univalent pathway for reduction of molecular oxygen (from Del Maestro, 1980). Aerobic cells have evolved a variety of enzymatic mechanisms against the toxic effects of the intermediates of oxygen reduction (oxyradicals) such as superoxide dismutase (SOD), peroxidase (Px), and catalase shown in Figure 2. In addition non- enzymatic defenses include such compounds as glutathione, vitamin E (a-tocopherol), 13-carotenes, and ascorbic acid (Di Giulio and Richardson 1987). CYTOCHROME OXIDASE COMPLEX 1 '- 1 CATALASES | PEROXIDASES ~ l O2 1 ~ ~OH._H2O SUPEROXIDE DISMUTASES Figure 2. Enzymatic defense mechanisms to prevent accumulation of reactive free radicals (from Del Maestro 1980). In this paper we briefly review the literature concerning the use of antioxidants as biomarkers of photochemical oxidant air pollution stress in trees as well as present
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253 some of our preliminary findings on free radicals and antioxidants in loblolly pine. Our research was designed to test critically the hypothesis that a key mechanism of action underlying "air emission" stress involves the generation of toxic free radical intermediates. We propose that the key gaseous pollutants associated with acid precipitation (i.e., NO2, SO2, and ozone) are the principal sources of this free radical-mediated toxicity in trees and that acidity per se exacerbates this toxicity. The specific goal of this phase of our research was to establish dose-response relationships between ozone, acid rain and selected free radical-related biochemical responses, photosynthesis, and growth in Pinus taeda (loblolly pine). MATERIALS AND METHODS Plant Material And Treatments: Detailed information outlining our experimental design, treatment levels and methods are found in Richardson and Di Giulio ( 1987~. Three half-sib families of loblolly pine seedlings were exposed to 5 ozone treatments in conjunction with 2 acid rain treatments in open-top chambers (Richardson and Di Giulio 1987~. Results were statistically analyzed as a split plot design with 2 replicates. Harvests and Physiological Measurements: Monthly harvests were conducted at approximately mid-month during the growing season. At each harvest at least six seedlings per chamber were examined for in situ physiological responses and subsequently harvested for biochemical assays. Immediately prior to harvest, rates of photosynthesis, and stomata! resistance were measured in situ with a LI-COR 6200 portable photosynthesis system. Biochemical Measurements: Immediately following the harvest of each seedling. needles were clipped into vials and immediately immersed in liquid nitrogen. These samples were shipped to the Ecotoxicology Laboratory at Duke and stored at - 70° C until analysis. Activities of SOD were determined by the methods of McCord and Fridovich (1969), Beauchamp and Fridovich (1971), and Asada et al. (1974~. Peroxidase (Px) was analyzed following the methods of Putter and Becker (1983~. Reduced and oxidized glutathione (GSH and GSSG) were measured by a modification of the spectrophotometric assay of Smith (1985~. Lipid peroxidation was determined by the malondialdehyde (MDA) method of Uchiyama and Mihara (1978~. REVIEW, RESULTS AND DISCUSSION Rabinowitch and Fridovich ( 1983) in an excellent review of superoxide radicals, superoxide dismutases (SOD), and oxygen toxicity in plants argued that superoxide dismutases found in plants resembles those found in other organisms and that varying forms of SOD ~ Cu/Zn and Mn) might provide a defense against SO2, sunscald, herbicides, and photooxidative injury. Our lab has also found varying types of SOD's in red spruce (Tandy et al. 1987~. It is interesting to speculate that a nutrient deficiency of Cu. Mn or Zn, as a result of acid rain leaching, might lead to a decrease in the ability of the plant to produce various forms of SOD. A model of how SO2 effects plant chloroplasts and cytoplasm was proposed by Schulz in 1986 (Fig. 3~. Once SO2 enters the leaf it is converted to bisulfite and sulfite. Bisulfite is photooxidized to less toxic SO4 and the superoxide radical (O2-~. The superoxide radical can be dismutated by SOD, to H2O2 and O2. H2O2 is converted to H2O and O2 by Px and catalase (Figs. 2 and 3~.
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254 Toxicologically, O2- and H2O2 are considered particularly important as precursors for the highly reactive and destructive OH radical. Cell injury can be severe if even a small portion of the superoxide radical is converted to OH . Cytoplasma - ChioropZast H2SO3 1 ~ PS ~ SOD POD/Catalase HSO3 · HSO3 OH ~ F. ~ CP-complex SO3 SO4 Necrosis Figure 3. A model of how SO2 affects plant chloroplasts and cytoplasm as proposed by Schulz (modified form Schulz 1986~/ Activity of Px, SOD, and catalase in needles of Pinus sylvestris (scotch pine) after 240 hours of exposure to 0.3 ppm of SO2 was shown to depend on needle age and reached a maximum in November (Fig. 4, from Schulz 1986~. In addition he reported no visible needle injury or necrosis but a significant increase of Px and SOD over controls (Fig. 4~. Catalase did not show an increase over controls. The intercellular fluid of Picea abies (Norway spruce) also displayed a significant increase in SOD after 30 days of 7-hour exposures with 300 ug.m~3 of ozone (Fig. 5, data from Castillo et al. (1987). The 30-day fumigation doubled the SOD activity in cell material as compared to the 2 day treatment. They also noted a decrease in ascorbic acid content and attributed this to the scavenging of toxic-free radicals by ascorbate-specific peroxidase. Glutathione and vitamin E were reported to increase significantly in both Abies alba (white fir) and Norway spruce after exposure to O~, SO2 and SO2 + O~ treatments (Mehlhorn et al. 1986~. Treatment of these trees with SO2 gave a higher increase of antioxidants in needles than those exposed to O3 or charcoal filtered air. In addition, a combination of both gases resulted in the maximum increase in both vitamin E and total glutathione, significantly above the ozone and filtered treatment alone.
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255 as min/mg Unit/mg 0.20 _ O . 1 5 0.10 O.05 2.0 PEROXIDASE / Control \% ~~D I I ~ I ~ I ~ I I Sep Nov Jan Mar May 1.5 1.0 0.5 _ _ SUPEROXIDE DISMUTASE Unit/mg 2 0.8 0.6 / ~ \ ~nntr^' \ O .4 I I ~ I I I I I I \ ~00.2 CATALASE s O2' C~ , I ~ I ~ I ~ I Sep Nov Jan Mar May Sap Nov Jan Mar May \ Figure 4. Activity of peroxidase (POD, note: Px in our paper) superoxide dimustase (SOD) and catalase in needles of Pinus sylvestris after 240 hours of exposure to 0.8 ppm of SO2 (modified from Schulz, 1986~. SOD Activity in Intercellular Fluid of Ozone-Exposed NonNay Spruce Seedlings 600 1 200 800 400 o Filtered Ambient Treatment i~ 2-day 30-day Outside Ozone Figure 5. Superoxide dismustase activity in intercellular fluid of picea abies after 30 days of 7-hour exposures with 300 ug.m-3 of ozone (after Castillo et al., 1987).
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256 In our loblolly pine study first-flush 1987 needles did not show a significant response after 90 days of exposure at the 1.5 level of 24 hour/day ozone treatments (from March to June); photosynthesis was reduced only 1% ,8%, and 20% at 1.5 x ambient ozone (66 ppb) for families S-103, S-80, and S-130, respectively (Fig. 6~. Ambient ozone was 48 ppb on a seasonal average. The seasonal peak for photosynthesis was found in August for all 3 families in the control chambers (CF). In August, after 150 days of treatment, photosynthesis was reduced by an average of 29°h in the 3 families treated with 66 ppb of ozone as compared to charcoal filtered controls with 20 ppb ozone. Photosynthesis was significantly affected by 24-hour ozone exposure at the 1.5 treatment above ambient levels only in family S- 130 in August, and for all 3 families at the 3.0 level; there were no statistically significant effects of acid rain. By 210 days of exposure (October) all the seedlings had a reduced photosynthetic rate from the August high values. The 3.0 treatment could not be measured since the seedlings displayed premature senescence of the 1987 first flush fascicles, which normally function for 2 complete growing seasons. 6 _ - ~n c`i 3 4 - cn CJ) 3 I z U) 0 2 o 1 12 1 8-80 · 8-1 03 8-1 30 . . , . . . ,. ... . . , . . . ,........ .... ......... ..... ......... ..... .... , .. . . ,. . . ... .. .... ..... , ., 0 ~ JUNE CF 1.5 3.0 AUG CF 1.5 3.0 DATE AND LEVEL OF OZONE TREATMENTS (note: * = significantly different from control at 0.05 level) OCT CF 1 .5 Figure 6. Seasonal trends of photosynthesis in the 1987 first-flush needles for loblolly pine families, 8-130, 8-80, and 8-103 exposed to charcoal-filtered (control), 1.5 and 3.0 times ambient ozone for 24 hours per day. Each bar represents the mean of 18 plants. Premature senexcence of needles removed the 3.0 treatment from analysis in October.
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257 A preliminary biochemical analysis (data from all 3 families combined) of August needle tissue exposed to 1.5 X ambient ozone and collected concurrently with gas 20°h above controls (Fig. 7~. while photosynthesis decreased 290/0 on average in the 3 families (Fig. S). Malonaldehyde levels were increased by 100% at 3.0 X ambient ozone compared to charcoal-filtered (CF) treated clams. exchange measurements showed that MDA increased about ~ . ~ . . . indicating a significant increase in lipid peroxidation, a common result of oxidant damage (Fig. 7~. Photosynthesis dropped an average of 67% at the 3.0 treatment level in the 3 families. The responses of Px, a key enzymatic component of the anti-oxidant defense, showed almost the same pattern of increases as MDA under increased exposure from 1.5 to 3.0 X ambient ozone compared to CF (Figs. 7 and 8~. Reduced glutathione (GSH) increased only at the highest ozone treatment. 120 100 In o - o C' IL o ce - o o— 80 60 40 20 o -20 - 1~/////~ 1.5X · 3.0X PX GSH MDA Responses in Loblolly Pines Figure 7. Biochemical analysis of malonaldehyde (MDA), peroxidase (PX) and glutathione (GSH) as compared to controls (percent increase above charcoal filter treatment) in lablolly pine exposed to 24 hours of ozone at 1.5 and 3.0 times ambient in August 1987.
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258 * 200 In - ° 100 _, o C' o t CO C' no on o -100 ; . . . * l * 03 PHOTOSYNTHESIS · SOD ~ Px JUNE (1.5) JUNE (3.0) AUG (1.5) AUG (3.0) OCT (1.5) Month and Treatment Level (note: * = significantly different from control at 0.05 level) C7 Figure 8. A seasonal comparison of the change from controls in photosynthetic rates and antioxidants SOD and Px in loblolly pine in response to 24 hours of exposure to 1.5 and 3.0 times ambient ozone levels found in Durham, North Carolina. A seasonal comparison of photosynthetic rates, SOD and Px response averaged over all 3 families reveals that highest levels of these antioxidants are found under the 3.0 treatments in June and August when photosynthesis was reduced by 33% and 67%, respectively (Fig. 8~. SOD increased nearly 200% over controls in June and 165% in August, while Px averaged nearly a 100% increase over controls in both months. At the 1.5 X treatment level in June, SOD and Px increased by 26% and 46% over controls
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259 while photosynthesis decreased by 10%. These data suggest that antioxidants may be used as an indicator of oxidative stress in trees early in the growing season. In August, photosynthesis decreased by 29% and SOD and Px increased by 19% and 17%, respectively. By fall SOD and Px both had increased over early summer values and treated plants had higher values than controls by nearly 50%; photosynthesis was decreased by 20% in the exposed plants (Fig. S). These data show that antioxidants and MDA increased significantly over controls while photosynthesis simultaneously decreased. Our data are too preliminary to establish direct relationships, dose response curves or even family differences, but they do indicate that antioxidants may be useful biomarkers for indexing oxidant air pollutant stress in trees. CONCLUSIONS The possibility of tracking oxidant stress from these biochemical markers is encouraging. However, much more experimental work is needed to establish mechanism of action, molecular basis of antioxidant increase, seasonal effects, effects of multiple gases, as well as dose response relationships for individual species and ecotypes. Further work is also needed to test adequately the hypothesis that air pollutant oxidants significantly increase antioxidants in viva. Future research should employ combinations of ozone with other free radical generating pollutants, such as sulfur dioxide, nitrogen dioxide, and hydrogen peroxide to test the effect of complex gas mixtures on plant biochemical response. ACKNOWLEDGMENTS The loblolly pine research was funded by a USDA Forest Service grant through the Southern Commerical Forest Research Cooperative (29-260). The funding for the development of assays for antioxidants in spruce and pine came from a grant through the USDA Forest Service Spruce-Fir research Cooperative (23-020). We kindly thank Dr. Tom Sasek, Dave Keen, and Ed Fendick for assistance with photosynthesis and statistical analysis. REFERENCES Asada, K., M. Takahashi, and M. Nagate. 1974. superoxide dismutase. Agric. Biol. Chem. 38:471-473. Beauchamp, C., and I. Fridovich 1971. Superoxide dismutase: assay applicable to acrylamide gels. Anal. Biochem. 44:276-287. Assay and inhibitors of spinach improved assays and an Castillo, F. J., P. R. Miller, and H. Greppin. 1987. Extracellular biochemical markers of photochemical oxidant air pollution damage to Norway spruce. Experimentia 43: 111-115. Del Maestro, R. F. 1980. An approach to free radicals in medicine and biology. Acta Physiol. Scand. 492: 153-168.
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260 Di Giulio, R. T., and C. J. Richardson. 1987. Effects of atmospheric deposition on red spruce: A free radical-based approach. Annual report to Spruce/fir Coop. Broomall, Penn. Kappus, H. 1987. Oxidative stress in chemical toxicity. Arch. Toxicol. 60: 144-149. McCord, I.M., and I. Fridovich 1969. Superoxide Dismutase: an enzymic function of erythrocuprein. J. Biol. Chem. 244: 6049-6055. Mehlhorn, H., G. Seaufart, A. Schmidt, and K.J. Kurnert. 1986. Effect of SO2 and 0~ on production of antioxidants in conifers. Plant Physiol. 82: 336-338. Putter, J., and R. Becker. 1983. Peroxidases. In Bergmeyer, H.U. (ed.), Methods of Enzymatic Analysis. Verlag Chamie, Deerfield Beach, Florida. Rabinowitch, H. D., and I Fridovich. 1983. Superoxide radicals, superoxide dismutases and oxygen toxicity in plants. Photochem. and photobiol. 37:679-690. Richardson, C. J., and R. T. Di Giulio. 1987. Effects of gaseous pollutants and acid precipitation on open-top chambered loblolly seedlings in Duke Forest: physiology and biochemistry. Annual report (SC07) to SCFRC, Raleigh North Carolina. Schulz, H. 1986. Biochemical and factor analytical examinations for interpretations of SO2-indications in needles of Pinus sylvestris. Biochem. Physiol. Pflanzen 81: 241 -256. Smith, I.K. 1985. Stimulation of glutathione synthesis in photorespiring plants by catalase inhibitors. Plant Physiol. 79: 1044- 1047. Tandy, N. E., R. T. Di Giulio, and C. J. Richardson 1987. Isozymes of superoxide dismutase in red spruce and their importance in protecting against oxidative stress. (In Press, International Symposium on the Effects of Atmospheric pollutants on Spruce/Fir forests of Germany and the U. S.~. Thomas, C. E., and S.D. Aust. 1986. Free radicals and environmental toxins. In Anals. of Emergency Medicine. 15:1075- 1083. UchIyama, M., and M. Mihara. 1978. tissues by thiobarbituric acid test. Anal. Biochem. 86:271-278. Determination of malonaldehyde precursor in Wellburn, A. R. 1987. Biochemical mechanisms of combined action of atmospheric pollutants upon plants. Pp. ~ 13- 829 in V. B. Vouk, G. C. Butler, A. C. Upton, D. V. Parke and S. C. Asher (eds). Methods For Assessing the Effects of Mixtures of Chemicals. SCOPE 1987.
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