EMISSION OF ETHYLENE BY OAT PLANTS TREATED WITH OZONE AND SIMULATED ACID RAIN

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1 New Phytol. {\9Sb) 103, EMISSION OF ETHYLENE BY OAT PLANTS TREATED WITH OZONE AND SIMULATED ACID RAIN BY E. J. PELL AND M. PUENTE Department of Plant Pathology and Center for Air Environment Studies, 211 Buckhout Laboratory, University Park, PA 16802, USA {Accepted 4 April 1986) SUMMARY Experiments were conducted to determine whether acidic rain either exacerbated or predisposed Avena sativa L. cv. 'Ogle' to ozone injury as measured by foliar emission of ethylene. Three sets of experiments were conducted. Experiment I: Plants were exposed to 588 //.g m~' (0-30 ppm) ozone from 0830 to 1130 h. Plants were treated from 1300 to 1400 h with simulated acidic rain at ph 2-8, 3-8, 4-6 or 5-6. Experiment II: treatments were the same as in Experiment I except that only simulated acidic rain of ph 2-8 was utilized and larger numbers of plants per treatment were included. Experiment III; plants were treated with simulated acidic rain of ph 2-8 from 1500 to 1600 h followed by exposure to 588 fig m"-' ozone the next day (1200 to 1500 h). At the end of each experiment, ethylene emission was determined for a pooled sample of the first and second leaf of each plant. Seventy-two hours after the experiment foliar injury was assessed. Acidic rain of ph 2-8 and 3-8 induced small lesions on the foliage but never induced increases in ethylene emission. Ozone induced flecking on the foliage and initiated significant increases in ethylene production. Ethylene emission was not affected by interactions between ozone and acidic rain in any of the experiments. Key words; Ethylene, oat, ozone, acid rain, ph. INTRODUCTION The importance of ozone (Og) as a phytotoxic air pollutant is well documented. Physiological responses, foliar symptoms and growth and yield losses have all been associated with exposure of plants to O3. The effects of acidic rain on plants are less well defined. While there are reports of foliar injury and yield losses in response to simulated acidic rain (Evans, 1984), there is no consensus regarding the severity of the problem or its importance to plants in managed and natural ecosystems. The magnitude of the response of plants to O., is dependent upon the environmental conditions in which it is grown (US Environmental Protection Agency, 1978). Plants stressed by O3 may also be the recipients of acidic rain. Several researchers have conducted experiments designed to measure potential interactions between the two pollutants. Shriner (1978) found that Phaseolus vulgaris L. cv. 'Red Kidney', treated intermittently with simulated acidic rain and O3 over a five-week period, exhibited a significant reduction in dry weight of foliage, while treatments with acidic rain alone elicited no such adverse effect. In the latter study no effects on photosynthesis were detected, whether the pollutants were presented alone or in combination. Troiano et al. (1983) demonstrated that ozone-containing non-filtered air and simulated acidic rain interacted to reduce vigour but not viability, or length, of soybean seeds X/8f)/ $03.00/ The New Phytologist

2 7IO E. J. PELL AND M. PUENTE Prinz (1984) has proposed that the decline of red spruce trees may result from interactions between Og and acidic rain. In experiments in which O3 induced membrane leakage and ion efflux, subsequent acidic rain treatment leached ions from red spruce needles. Under simulated rain treatments with low ph, ion leakage was greater than at higher ph treatments. From the studies cited above it is apparent that the potential exists for O3 and acidic rain to elicit an interactive response from some plant species. It is also clear that not all plant responses will reflect the interaction of the two stresses. We have studied the potential of acidic rain to (1) exacerbate the response to O3 and (2) predispose plants to injury by O3. Ethylene emission was selected as the response of the plants by which effects of acidic rain and/or O3 would be measured. There have been numerous reports documenting the ability of O3 to initiate the emission of ethylene in many plant species (Tingey, Standley & Field, 1976; Adepipe & Tingey, 1978; Stan & Schicker, 1982). Fthylene emission is described as an early and sensitive response of plants to O3. The effects of acidic rain on ethylene production are less clear. It has been shown that Phaseolus vulgaris L. cv. 'Kinghorn' produced ethylene in response to simulated acidic rain ph of 3-0 (Chia, Mayfield & Thompson, 1984). Amy & Pell (1985) have shown that simulated acidic rain alone did not induce ethylene emission in leaves of Solanum tuberosum cv. ' Norland', Raphanus sativus cv. 'Cherry Belle' or Glycine max cv. 'Amsoy'. In the text which follows we will describe experiments which examined the effects of acidic rain and/or O3 on production of ethylene by Avena sativa L cv 'Ogle'. MATERIALS AND METHODS Plant culture Avena sativa L. cv. ' Ogle', three plants per pot, were planted in a commercial peat-vermiculite mix (1:1 by volume). Plants were maintained in a glasshouse with a 16-h photoperiod affected by supplement lighting of 227 jie m~^ s"^ Pots were watered from above until foliage emerged and, subsequently, were subirrigated to keep the foliage dry. When the plants were 7 to 12 d old and the first leaf was fully developed, they were fertilized with the nutrient solution described by Marshall & Kolb (1982). All experiments were conducted when plants were 13 to 16 d old and the second leaf was fully expanded. Plants were approximately 17 cm in height. s with pollutants Plants were exposed to 588 //g m"'' (0 30 ppm) O3 for 3 h treatments in environmentally controlled growth chambers (De Vos et al, 1983). Nonexposed plants were maintained in a companion chamber. Irradiance in both chambers was 160 ^E m^2 s~'. Plants were treated with simulated acidic rain for 1 h as described previously (Arny & Pell, 1985). Three sets of experiments were conducted. In Experiment I, 50 plants were exposed to O3 from 0830 to 1130 h. From 1300 to exposed and 10 nonexposed plants were treated with rain at ph 2-8, 3-8, 4 8, or 5-6. Ten plants exposed to O3 were not treated with acidic rain. Another 10 plants did not receive any treatment. The experiment was replicated three times. Experiment II was similar to Experiment I except that only one simulated rain treatment was

3 Pollutants and ethylene emissions by oat plants 711 included at ph 2-8. There were 25 pots per treatment. The experiment was repeated twice. Experiment III was designed to determine whether acid rain might predispose plants to O3. Fifty pots of oat plants were treated with simulated acidic rain ph 2-8 from 1500 to 1600 h. On the next day, half of the plants were treated with acidic rain and the other half were exposed to O3 from 1200 to 1500 h. Twenty-five plants received no treatment. The experiment was conducted twice. Seventy-two hours after the treatment with simulated acidic rain, the first and second leaf on two plants in each pot were evaluated for injury. Ozone injury was rated on a 1 to 10 scale with 1 = 1 to 10% injury...and 10 = 91 to 100% injury (De Vos et al, 1983). The number of leaf spots induced by acidic rain were counted on the first two leaves as well. Determination of ethylene One hour after an experiment was completed the first and second leaf from one plant in each pot were removed at the stem axis. Both leaves were placed in a 75 ml test tube with cut surface submerged in 1 ml of deionized water. Test tubes were sealed with rubber serum bottle stoppers and plastic laboratory film, and the foliage was incubated for 18 h in a growth chamber in the dark at 21 C. Ethylene concentration was determined with an HP 5840A gas chromatograph (Hewlett Packard, 3000 Hanover, Palo Alto, CA 94304). A 0-9 ml air sample was injected into a Poropak-QS column maintained at 65 C. The injection port and detector temperatures were 65 and 175 C, respectively. Ethylene concentration in the air space was adjusted according to test tube volume, and the final concentration was expressed as nanolitres per dry weight of leaf tissue (Arny & Pell, 1985). Statistical analysis An analysis of variance was conducted for each experiment using the treatment by replicate interaction as the error term to test for treatment effects and the Waller-Duncan i^-ratio T-test for separation of means (General Linear Models Procedure of SAS Institute, Inc., Box 8000, Cary, NC 27511). RESULTS Expression of symptoms Ozone injury was always more severe on the first leaf while acidic rain induced a greater response on the second leaf. In two replicates of Experiment I and in both replicates of Experiments II and HI, O3 induced 1 to 10% injury on 70 to 90% of the older leaf tissue on each plant; under 10% of the leaf tissue was asymptomatic and 10 to 30% exhibited more intensive symptoms. In the first replicate of Experiment I, 55% of the older leaves exhibited injury between 30 and 80%. Acidic rain induced white lesions 0-5 mm wide and 0-5 to 2-0 mm in length. Foliage treated with simulated acidic rain ph 2-8 exhibited 0 to 25 lesions often located at the leaf margin; lesions were observed on 10 to 89% of the younger leaves evaluated in individual replicates of each experiment. Simulated acidic rain of ph 3-8 induced 0 to 3 lesions per leaf; 0 to 19% of the younger leaves in each of the replicates of Experiment I exhibited injury which was ascribed to acidic rain.

4 712 E. J. PELL AND M. PuENTE Table 1. Ethylene production of oat plant foliage exposed to 588 fig m^^ {030 ppm) ozone for 3 h and/or a subsequent 1 h treatment with simulated acidic rain {Experiment T) (a) Analysis of variance table Source Replicate Error (treatment x replicate) * Significant at the 0 05 level, d.f M.S * * (b) Mean ethylene concentrations Ozone ph Ethylene emission (nl g-') -I A'' B B B B A -I A -I A A B ' Means represent 30 observations pooled from three replicates of the experiment. '' Means followed by different letters are significantly different at the 0-05 level based on a Waller-Duncan K-ratio T-test. Emission of ethylene Experiment I. An analysis of variance pooled across replicates revealed significant treatment and replicate effects [Table l(a)]. Mean separation indicated that oat plants exposed to ozone produced significantly more ethylene than those not exposed to the gas [Table l(b)]. Acidic rain, alone or following an exposure to Og, had no effect on ethylene emission. Experiment IL The high coefficient of variation (44-8 %) for ethylene emission did not preclude detection of the effect of O3. To ensure that potentially smaller effects of acidic rain were not being masked by variability inherent in the assay, we conducted a second experiment, in which there were 25 plants per treatment and only simulated rain at ph 2-8 was used. The results were similar to those observed in Experiment I. effects were significant [Table 2(a)] and only O3 induced significant increases in ethylene emissions [Table 2(b)]. Experiment IIL In order to determine whether acidic rain could predispose plants to the O3 response, plants were first treated with simulated acidic rain and then O3. The treatment elicited a significant ethylene response [Table 3(a)]. Mean separation revealed that O3 stimulated the ethylene response but simulated acidic rain ph 2-8 did not enhance the effect [Table 3(b)].

5 Pollutants and ethylene emissions by oat plants 713 Table 2. Ethylene production of oat plant foliage exposed to 588 fig m~^ {0-30 ppm) ozone for 3 h and/or a subsequent 1 h treatment with simulated acidic rain {Experiment IT) (a) Analysis of variance table Source Replicate Error (treatment x replicate) * Significant at the 0-05 level, d.f M.S * (b) Mean etbylene concentration Ozone Acid rain Ethylene emission (nl g-') A' B A 84-9 B ' Mean represents.so observations pooled from two replicate experiments. ^ Means followed by different letters are significantly different at the 0-05 level based on a Waller-Duncan /f-ratio T-test. Table 3. Ethylene production of oat plant foliage treated with simulated acidic rain ph 2-8 and/or a subsequent exposure to 588 /ig m~^ {0-30 ppm) ozone for 3 li {Experiment III) (a) Analysis of variance table Source d.f. M.S. Replicate Error (treatment X replicate) * * Significant at tbe 0-05 level. (b) Mean ethylene concentration Acid rain Ozone Ethylene emission (nl g-') A' B B A ' Mean represents 42 observations pooled over two replicate experiments. ' Means followed by different letters are significantly different at the 0-05 level based on a Waller-Duncan /C-ratio T-test.

6 714 E. J. PELL AND M. PUENTE DISCUSSION Ozone induced a highly significant increase in emission of ethylene hy oat foliage in all experiments [Tables l(b), 2(b) and 3(b)]. These results are consistent with previous reports for many plants species (Tingey et al., 1976; Adepipe & Tingey, 1978; Stan & Schicker, 1982) Simulated acidic rain did not elicit ethylene production at any of the ph values utilized [Tables l(b), 2(b) and 3(b)]. Simulated acidic rain treatments of ph 2-8 and 3-8 induced foliar lesions, but the symptoms were not associated with production of ethylene. It has been shown previously that potato, radish and soybean foliage did not emit ethylene in response to simulated acidic rain (Arny & Pell, 1985). Chia et al. (1984) did, however, report an increase in the emission of ethylene from the foliage of Phaseolus vulgaris following 2 h treatment with rain at ph 3-0 on four consecutive days. It is possible that the species used and/or the longer period of rain treatment may explain the difference between the results of Chia et al. (1984) and those observed in this study, and in our previous experiments (Arny & Pell, 1985). A complete evaluation of the experiment of Chia et al. (1984) was difficult because of the absence of a detailed description of the experimental design. Enzymes responsible for ethylene production are associated with the plasma membrane (Mattoo & Lieberman, 1977). Arny & Pell (1985) have suggested that injury induced by acidic rain is probably not associated directly with the cell membrane, but perhaps with cuticular erosion, in which case, emission of ethylene might not be expected. It has been suggested that acidic rain may exacerbate foliar injury induced by O3 (Shriner, 1978; Troiano et al., 1983; Prinz, 1984). Prinz (1984) postulates that O3 elicits a membrane effect resulting in ion leakage; acidic rain exacerbates this effect by more effectively leaching the ions, thereby, perhaps, reducing the chances for membrane repair. An increase in ethylene emission might be a predicted interactive plant response to O3 and acidic rain if the scheme described above were correct, but we found no evidence that an interaction to O3 and acidic rain was expressed in the ethylene emissions of oat plants. A different plant species or experimental protocol might, however, have allowed detection of such an interaction. We also looked for an interaction in which acidic rain would predispose plants to injury by O3. There is some evidence that acidic rain can partially erode the cuticle, particularly in the area of stomatal depressions (Evans, Gmur & Da Costa, 1978). Such erosion could reduce leaf resistance and allow greater uptake of O3 by the plant. We found no evidence that acidic rain predisposed plants to O3 injury as measured by the emission of ethylene. It is, of course, possible that if the plants had received a different pretreatment to acidic rain, either of longer duration or multiple treatments, uptake of O3 and subsequent injury would have been greater. Our experiments do not support the usefulness of ethylene as a measure of the sensitivity of plants to acidic rain. Acidic rain does not appear to be as toxic to plants as O3. Before scientists formulate lasting conclusions, however, careful consideration must be given to the protocol employed in experiments such as those we have just described. Additional experimentation may be appropriate in order fully to explore an interaction which, if it were a factor in determining the injury to plants, might be of great consequence.

7 Pollutants and ethylene emissions by oat plants 715 ACKNOWLEDGEMENTS The authors thank Kathleen Evensen for the use of the gas chromatograph. This is Pennsylvania Agricultural Experiment Station Journal Series Paper No. 7331, Department of Plant Pathology No. 1557, and Center for Air Environment Studies No Funds were provided in part by U.S. Department of Agriculture Grant No. 84-CSRS REFERENCES ADEPIPE, N. O. & TiNGEY, D. T. (1978). Ozone phytotoxicity in relation to stress ethylene evolution and stomatal resistance in cowpea cultivars. Zeitschrift fiir Pfianzensphysiologie, 93, ARNY, C. J. & PELL, E. J. (1985). Ethylene production by potato, radish, and soybean leaf tissue treated with simulated acid rain. Environmental arid Experimental Botany, 26, CHIA, L. S., MAYFIELD, C. 1. & THOMPSON, J. E. (1984). Simulated acid rain induces lipid peroxidation and membrane damage in foliage. Plant, Cell and Environment, 7, DE Vos, N. E., PELL, E. J., HILL, R. R., JR. & COLE, R. H. (1983). Laboratory versus field response of potato genotypes to oxidant stress. Plant Disease, 67, EVANS, L. S. (1984). Acidic precipitation effects on terrestrial vegetation. Annual Review of Phytopathology, 22, EVANS, L. S., GMUR, N. F. & DA COSTA, F. (1978). Foliar response of six clones of hybrid poplar to simulated acid rain. Phytopathology, 68, MARSHALL, H. G. & KOLB, F. L. (1982). Individual crown selection for resistance to freezing stress in winter oats. Crop Science, 22, MATTOO, A. K. & LiEBERMAN, M. (1977). Localization of ethylene-synthesizing systems in apple tissue. Plant Physiology, 60, PRINZ, B. (1984). Recent forest decline in the Federal Republic of Germany and contribution of the Landesanstalt fur Imissionschutz for its explanation. Landesanstalt fur Imissionschutz des Lnndes Nordrhein-Westfalen. Essen, Federal Republic of Germany. SHRINER, D. S. (1978). Interactions between acidic precipitation and SOj or Oji Effects on plant response. Phytopathology News, 12, 153. STAN, H. J. & SCHICKER, S. (1982). EflFect of repetitive ozone treatment on bean plants, stress ethylene production and necrosis. Atmospheric Environment, 16, TiNGEY, D. T., STANDLEY, C. & FIELD, R. W. (1976). Stress ethylene evolution; a measure of ozone eflfects on plants. Atomospheric Environment, 10, TROIANO, J., COLAVITO, L., HELLER, L., MCCUNE, D. C. & JACOBSON, J. S. (1983). EflFects of acidity of simulated rain and its joint action with ambient ozone on measures of biomass and yield in soybean. Environmental and Experimental Botany, 23, US Environmental Protection Agency. (1978). Air quality criteria for ozone and other photochemical oxidants. EPA report no. EPA-600/ Research Triangle, NC.

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