Ozone. Effect on Apparent Photosynthesis, Rate of Transpiration, and Stomatal Closure in Plants A. Clyde Hill and Neil Littlefield Center for Environmental Biology, University of Utah, Salt Lake City, Utah 84112
Little is known regarding the cause of plant growth suppression resulting from photochemical pollution. Studies with ozone indicate that elevated ozone concentrations may be involved. Ozone induced partial stomatal closure and a reduction in apparent photosynthesis and transpiration. High concentrations of ozone caused a temporary reduction in apparent photosynthesis of many species, often with little or no injury developing. The effect was reversible and plants frequently recovered within a few hours. Ozone concentrations as low as 10 p.p.h.m. for several hours caused partial stomatal closure of oats. A comparison of the effects of ozone under sunlight and artificial lighting showed that photosynthesis was reduced most in sunlight.
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zone causes visible leaf tissue destruction in many plant species throughout large sections of the country. Since necrotic tissue does not photosynthesize, the tissue destruction is usually believed to be associated with a proportional reduction in the rate of plant growth. The possibility that ozone might suppress plant growth in excess of that associated with tissue destruction, or reduce growth of plants that d o not show visible injury, must be considered when we evaluate ozone effects on plants. Growth suppression of plants exposed to natural photochemical smog has been reported for alfalfa, endive, sugar beet, and tomato (Hull and Went, 1952), Kentia palm (Todd, Middleton, et ul., 1956), tobacco (Menser, Crosso, et al., 1964), grapefruit (Taylor, 1958), and lemon (Thompson, Taylor, er nl.. 1967). The specific pollutant in the smog complex responsible for growth suppression in these studies was not identified. Erickson and Wedding (1956), Todd and Propst (1963), and Macdowall (1964) reported that ozone, a major photochemical reaction product: caused reduced photosynthesis which they associated with visible damage to the leaves or a reduction in chlorophyll. In connection with root oxygen diffusion studies, Stolzy, Taylor, et al. (1964) reported that photosynthesis of tomatoes was reduced by ozone without visible injury developing. Transpiration as well as apparent photosynthesis can be affected by smog (Taylor, 1958; Thompson, Taylor, et al., 1967), indicating the possibility of an effect on stomate closure. Dugger, Taylor, et al. (1962) reported that 70 p.p.h.m. of O3 for 30 minutes had no effect on stomatal closure of beans. Engle and Gabelman (1966), Lee (1965), Macdowall (1965), and Todd and Propst (1963) indicate that ozone may cause stomatal closure. Since the data available indicate that something in photochemical pollution causes reduced plant growth, studies were undertaken to determine the effects of ozone on apparent photosynthesis. After these studies showed that ozone could reduce apparent photosynthesis of many species, studies were conducted to determine if there was a relationship among stomatal opening, photosynthesis, and transpiration. Since light in the environmental chamber differed from sunlight in 52 Environmental Science & Technology
quality and intensity, studies were conducted to determine if the effect of ozone on growth was the same in artificial light as in sunlight. Materials and Methods
Because photosynthesis can be influenced by light, temperature, humidity, and wind velocity, the studies were conducted in environmental chambers designed to control these variables. Tests conducted on different days could then be compared without concern over differences caused by environmental variations during fumigation. Prior to fumigation the plants were grown in carbon-filtered greenhouses. Species used were barley (Hordeum tulgnre Gem), oat (Arena snrica Park), wheat (Triticum aesricum var. Lemhi), corn (Zen m i y s var. Golden Bantam), tobacco (Nicotiana tabacun~var. Bel B), sugar beet (Beta culgaris commercial hybrid ;42), Swiss chard (Bern culgnris Cicka var. Fordhook giant), pinto bean (P/7aseolus culgaris var. Pinto), lima bean (Phaseolus culgaris var. Henderson), bush bean (Plzaseolus vulgaris var. Tender Pod), cauliflower (Brassica oleracea)? potato (Sokanum tubernsum var. Russet Burbank), and tomato (Lycopersicon esculentum var. Moscow). During the growing season light, temperature, and relative humidity in the greenhouses were maintained near ambient in an attempt to grow plants that would respond in the same way as those grown in the field (Hill, Transtrum, et ai.,1959). Carbon-filtered and water-cooled air, circulated through the greenhouses at three changes per minute, permitted maintenance of near ambient temperatures without shading. Cold water coil heat exchangers prevented temperature rise without affecting relative humidity. The plants were grown in 1-gallon cans or 13-gallon boxes (16 X 22 X 9 inches) filled with a soilpeat-vermiculite mixture. The plants were exposed to ozone in environmental chambers designed specifically for these studies (Hill, 1967). The chambers are airtight, and during these studies a 100% recirculating air system was used. C o n was maintained at 325 p.p.m. by adding Cor to the chamber at the same rate that the plants were removing it. The rate of CO? addition required to control the concentration was equal to and a measure of the apparent rate of photosynthesis. Respiration was measured by turning the lights off and determining the rate of CO? buildup. Transpiration was determined by maintaining humidity and temperature within narrow limits and measuring the rate of water condensation on the cold water coils. To simulate typical wind conditions during high ozone episodes, the air was moved across the chamber above the plants at a velocity of 2 miles per hour. Light at the plant height ranged from 40 to 50 klux, dep2nding on the age of the bulbs. Temperature was maintained at 24" C. and relative humidity at 50 %. Ozone was generated electrostatically from tank oxygen and the concentration controlled by the automatic system previously described (Hill, 1967). Ozone concentrations were measured with Mast ozone analyzers. Ozone-induced leaf injury was determined by estimating the amount of injury that developed on 100 leaves selected at random. An attempt was made to develop a plant canopy typical of
field conditions in the environmental chambers and, depending on the species and size, the canopy ranged from 9 to 450 plants. The rate of gaseous exchange was expressed in grams (for H?O) or milligrams (for CO,) per cubic meter of ground area per minute. The net rate of CO? assimilation was measured in each chamber for several hours, and then the plants in one chamber were subjected to ozone while the other chamber was maintained as a control. When a reduction in apparent photosynthesis was measured, ozone addition was terminated and measurement of COSassimilation was continued. I n a few studies, conducted while the second chamber was being built, control tests were conducted in the same chamber on different days to measure COS uptake in the absence of ozone. Apparent photosynthesis could be measured over at least 5 or 6 hours before the rate in the control chamber began to decLine. In the initial studies, stomate width measurements were made with a microscope using the silicone rubber method (Lee, 1965; Zelitch. 1961). This method did not accurately measure stomatal openings in the range 0 to 1 micron, where effects o n photosynthesis were most critical and the correlation between stomatal opening and photosynthesis was poor. I n all studies reported here the epidermis strip method was used (Lloyd, 1908; Loftfield, 1921). To facilitate measurement, the image was projected with a Leitz microscope projector. A mirror mounted on the ceiling reflected the image back on the projector table, allowing fine focusing of the projector on each stomate. Stomate width was measured with a micrometer, after marking the two sides of the projected stomate opening with a pencil on a sheet of white paper. The projector was mounted in a vertical position, allowing the slide t o be horizontal, so that the tissue could be kept in alcohol. The height was adjusted so that 1 micron equaled 2 mm., which gave a good, sharp image and adequate precision of measurement
Table I. Effects of Ozone on Apparent Photosynthesis Apparent Duration PhotoOzone of synthesis, Leaf Age, Concn., Treatment, of Injury, Species Days P.P.H.M. Minutes Control Oat 50 40 30 67 1 48 45 30 60 1 47 50 60 55 1 50 60 60 69 2 Barley 36 62 30 42 0 47 60 60 62 0 Wheat 56 70 60 50 4 Tobacco 99 40 90 22 0 110 45 75 38 1 69 50 45 67 4 70 48 35 62 2 118 48 80 50 1 Pinto bean 56 45 90 50 10 Lima bean 26 45 120 52 1 39 70 45 36 5 Bush bean 51 50 80 65 3 Chard 78 60 70 55 3 Corn 49 50 90 68 3 Cauliflower 77 75 50 38 0 Sugar beet 74 65 90 80 4 80 90 90 49 7 Potato 39 60 60 50 3 Tomato 72 60 60 57 1
with the micrometer. I n this position an auxiliary blower was required for cooling the projector. Epidermis strips were taken from the lower surface of 10 middle-aged leaves and 50 stomata were selected a t random from the strips and measured. To correct for errors caused by nonvertical alignment of oat stomates, representative strips were also examined under a universal stage microscope. The long axis of each stomate was aligned so that the stage could be rotated vertically at right angles to the long axis. By measuring the width of a large number of stomates when the stage was flat and again after the stage had been rotated to the point of maximum observable opening, correction factors were obtained for different degrees of opening and were used to adjust average values measured by the projection technique. Since opening the chamber doors to take stomate samples disrupted measurement of transpiration and C 0 2assimilation, only three or four stomate samples were taken while apparent photosynthesis was being measured. I n one study duplicate fumigations were conducted in which apparent photosynthesis was not measured, to allow more frequent stomate sampling during the fumigation. To compare the response of plants to ozone under sunlight and artificial lighting, a chamber constructed of 3/,-inch ultraviolet-transmitting Plexiglas was placed in the sun and connected to the conditioning unit of one of the environmental chambers by air ducts. In this way the plant area, temperature, relative humidity, ozone concentration, and wind velocity could be maintained the same in both chambers and the response of plants under the two light conditions compared. Results Numerous fumigations were conducted to measure the effects of ozone on apparent photosynthesis. The plants were subjected to ozone until a substantial reduction in apparent photosynthesis was measured and then the ozone treatment was terminated. Typical results (Table I) show that COZ assimilation was usually reduced to 40 to 7 0 z of the control within 30 to 90 minutes by the ozone concentrations used. All species responded to ozone in a similar manner. Tobacco, for example, was fumigated 24 times in the concentration range 50 to 70 p.p.h.m. of 03.From 20 to 120 minutes were required to reduce photosynthesis and the per cent reduction ranged from 24 to 78. Since it was desired to study effects unrelated to visible leaf destruction, relatively resistant varieties were usually selected and the most sensitive stages of growth were avoided. Occasionally large amounts of injury developed ; these fumigations were omitted from Table I because the reduction in apparent photosynthesis could have been associated with the developing injury. Usually from 0 to 4 z of the total leaf area developed injury within 24 hours after fumigation, but the reduction in apparent photosynthesis during ozone treatment was largely independent of visible leaf injury. Following termination of the ozone treatment apparent photosynthesis normally began to increase within 30 minutes. Usually recovery was only partial within 3 or 4 hours and complete or nearly complete by the next morning. Occasionally photosynthesis equaled the control within 2 or 3 hours. When visible injury was extensive, apparent photosynthesis frequently continued to drop during the time measurements were made. A typical curve showing the reduction in apparent photosynthesis of 36-day-old barley is shown in Figure 1 . No visible injury developed on these plants in any of the four replications. Respiration measured prior to and following ozone treatment in a few studies showed no differences due to the treatment. Volume 3, Number 1, January 1969 53
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Figure 1. Effect of ozone on apparent photosynthesis of barley 60 p.p.h.m. of 03 for 45 minutes
To attempt to determine if stomatal closure was responsible for the temporary suppression of apparent photosynthesis, stomate width measurements were made during fumigations of oats. Apparent photosynthesis and transpiration were measured for 3 hours, after which the plants in one chamber were subjected to 60 p.p.h.m. of ozone for 65 minutes. Stomate samples were taken prior to and following the ozone treatment and after a 21/2-hourrecovery period. Fumigations were conducted on two days and the data averaged. Since it was not possible to take more frequent stomate samples while measuring apparent photosynthesis and transpiration, six additional ozone fumigations were conducted in which apparent photosynthesis and transpiration were not measured and stomate samples were taken every 20 minutes. The results are shown in Figure 2. Ozone caused a 40 decrease in apparent photosynthesis, but after the ozone treatment was terminated the rate returned almost to the control. A 48 reduction in transpiration was produced and transpiration also tended to recover. Ozone treatment resulted in a rapid reduction in the average width of stomatal opening. Over half of the stomates were closed completely by the termination of the ozone treatment, and the width of the remainder ranged from 0.2 to 1.9 microns and averaged 1.3 microns. All stomates were open in the control chamber. About 15 minutes after the treatment was discontinued the stomata apparently began to reopen, but data are inadequate to draw the stomatal width curve in any detail during the recovery period. After about 21/2hours in the light on the next day, visible leaf injury became evident in the ozone chamber. An average of 4% of the total leaf area was damaged. In subsequent fumigations with oats, occasionally the stomata in both chambers were only partially open (about 1 micron average) at the beginning of the ozone treatment. Under these conditions the reduction in apparent photosynthesis and stomatal closure were more rapid than usual and no injury developed. In such instances closure of stomata caused by the ozone treatment appeared to protect the plants from ozone injury. To see if stomatal closure occurred with other species, potato, oats, barley, tomato, bean, and tobacco were exposed to 60 p,p.h,m. of ozone for 1 hour and the reduction in apparent photosynthesis and degree of stomatal closure were 54 Emironmental Science & Technolog:
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measured. The results (Table 11) show partial stomatal closure and reduced photosynthesis with each species. Caution is necessary in comparing the relative sensitivity of the different species from this limited study because the response within the same species varies considerably when plants vary in age and pretreatment growing conditions.
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Table 11. Stomatal Closure and Reduction in Apparent Photosynthesis Caused by 60 P.P.H.M. of O3for 1 Hour Average Stomatal Width, Reduction, Microns Apparent Species Control Ozone Photosynthesis Potato 3.9 1.5 35 Barley 1.9 0.6 35 Tomato 2.8 0.6 25 Oat 3.6 0.8 27 Bean 2.1 1.1 29 Tobacco 2.9 2.1 14 Since apparent photosynthesis is greatly influenced by light, a question arose as to the relative response of plants to ozone when exposed to direct sunlight as compared to artificial light. Demand for CO.?and stomatal opening could both be affected by increased light. To evaluate this, oats were subjected to 50 p.p.h.m. of ozone for 1 hour with one group of plants under artificial lighting at 50 klux and the other group in sunlight averaging 96 klux. The 1.4-sq. meter light bank consisted of fluorescent (3465 watts) and incandescent (1200 watts) lights. The incandescent lights had little effect on the light measured but they affected photosynthesis. The data (Figure 3) are averages of three fumigations of 37-day-old, 18-inch-high oat plants. The control curves were obtained in the same chambers on different days in which ozone was not added. Photosynthesis in the sun was over twice as high as under artificial lighting. Under the higher intensity and different spectral quality of sunlight, restriction of C o r diffusion into the leaf by partial stomatal closure had a greater effect on apparent photosynthesis. Ozone reduced apparent photosynthesis 5 mg. of COSper minute in artificial light compared to 21 mg. in the sun. Per cent reductions were 21 and 33 under artificial light and sunlight, respectively. A greater effect on apparent photosynthesis in the sun would be expected because COS in the intercellular spaces would become rate-limiting more readily with the higher photosynthetic rate. Eleven per cent of the leaf area developed injury when fumigated in the sun compared to 5W in artificial light. The greater injury in sunlight may be due to more rapid ozone uptake, since the stomata were open wider in the sun both before and following fumigation. Our measurements of ozone uptake by plants have consistently been higher in the sunlight. Sunlight might also affect the content of sugars and various other cell constituents in the leaf tissues and thereby affect sensitivity to injury. Subsequent studies conducted to evaluate apparent photosynthetic rate and injury development under the two light conditions resulted in the addition of quartz iodide lights (2000 watts) to the light bank which overcame much of the difference in photosynthetic rate and all of the differences in injury development. Stomatal width but not apparent photosynthesis measurements were made with oats subjected to 10 and 20 p.p.h.m. of ozone for 6 hours and 60 p.p.h.m. of ozone for 1 hour (Figure 4). The dosage (time X concentration) required to cause stomatal closure appears to be about the same for each of the three concentrations. Ten and 20 p.p.h.m. of 0 8 caused about the same degree of stomatal closure as 60 p.p.h.m. when the exposure time was increased to give the same total exposure. Reopening of the stomata under the 10- and 20-p.p.h.m. treatments was more rapid. Similar measurements made in connection with ozone uptake studies showed an opening of 5.2 microns for the control compared to 2.5 microns for the ozone treatment after a 4-hour exposure to 5 p.p.h.m. of ozone.
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Figure 3. Effect of ozone on apparent photosynthesis and stomate width of oats under sunlight and artificial lighting 50 p.p.h.m. for 1 hour
Discussion Stomatal closure, reduced apparent photosynthesis, and reduced transpiration occur simultaneously during ozone fumigation, indicating that suppression of transpiration and apparent photosynthesis may have resulted from the stomatal closure. This explanation appears best to fit the data available. It is possible, although unlikely, that these three effects are not directly related, ozone affecting each process directly and independent of the other. A third possibility is that the initial effect of ozone is on C o nutilization, resulting in a rapid increase in the CO, content of the guard cells with resultant stomatal closing. Again this is unlikely. In a number of fumigations at lower concentrations a definite but relatively small reduction in stomatal opening occurred without measurable effect o n apparent photosynthesis or transpiration. Studies we have conducted with pollutants that affect apparent photosynthesis directly indicate that the stomatal response differs considerably from the results presented in Figure 2. A relatively large reduction in apparent photosynthesis is required to produce a small reduction in stomatal opening. Closure of stomata in response to toxic pollutants may not be uncommon. Woltz and Leonard (1964) reported stomatal closure of orange leaves dipped in hydrofluoric acid to simulate atmospheric fluoride accumulation. Stomatal closure has been produced by sulfur dioxide (Katz and Ledingham, 1939) and the reaction products of ozone and hexene (Koritz and Went, 1953). Engle and Gabelman (1966) showed that ozone caused stomatal closure of onion leaf disks and related the resistance of certain varieties to ozone injury to protection by stomatal closure. Volume 3, Number 1, January 1969 55
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Figure 4. Effect of three concentrations of ozone on stomatal width of oats The average stomatal width is convenient to use to indicate the degree of closure. When relating it to gaseous exchange, however, we must remember that over half of the stomates were closed when the width averaged 0.6 to 0.7 micron. Since there is some tendency for closed stomates to group, photosynthesis may be reduced in numerous small portions of the leaf. Our estimateof stomatal width below 1-micron is probably about 50z higher than reported by others. When the width is narrow, a slight deviation from vertical in stomatal orientation can make a big difference in the opening measured because the stomatal depth is large in comparison to the width. It is necessary for light to travel vertically up through the opening before the opening can be measured. Some partially open stomates even appear closed when the stage is horizontal, but rotation allows the opening to be observed and measured. The silicone rubber technique did not provide a reliable measure of stomatal width after partial closure had occurred. The rubber forms a V-shaped notch over the stomate and the notch differs little in shape or size in the range 0 to 1.5 microns. A reduction from several microns to this range can easily be detected, but changes in the lower range cannot. When closed, guard cells usually make contact from 1 to several microns below the outer surface, and the V-shaped notch that forms over the closed stomate does not indicate closing. Zelitch (1961) called all stomata measuring less than 2 microns closed, and Lee (1965) called those less than 0.8 micron closed, but we were unable to get a good correlation with gaseous exchange with either approach. Dugger, Taylor, et al. (1962) may not have reported an effect of ozone on stomatal opening because they were using a pressure porometer. Unless the stomates were almost closed, this porometer may tend to force air through the small openings and could give a false impression of closure. The ozone concentrations used in the apparent photosynthesis studies are higher than are normally measured in the ambient air, and exposure times were relatively short in comparison to ambient exposures. The higher concentrations were used to produce a relatively large reduction in apparent photosynthesis in a short time to facilitate measurement. Studies with lower concentrations and longer times are planned. The data presented in Figure 4 indicate that exposure to relatively low ozone concentrations for several hours could cause partial 56 Environmental Science & Technology
stomatal closure and the data in Figures 2 and 3 indicate that this degree of closure could reduce apparent photosynthesis. In the field, if photosynthesis is limited by light, temperature, or factors other than C o r in the intercellular spaces, a reduction in stomata openings may occur with little effect on photosynthesis. When the stomates are open wide, some closing may occur with little effect. In areas where elevated ozone concentrations persist for relatively long periods, however, ozone may reduce photosynthesis frequently and thereby suppress plant growth. Stomatal closure by ozone appears to be a possible explanation for the growth suppression associated with photochemical smog that has been reported. It may also partially explain the reduction in photosynthesis and water use by citrus in ambient air reported by Thompson, Taylor, et a/. (1967). As they pointed out, ozone was not removed from the air in their low ozone treatments until the concentration exceeded 10 p.p.h.m., so it is possible that ozone caused stomatal closure when concentrations were below 10 p.p.h.m., which would be much of the time. Leaf drop and possibly other factors including other pollutants could have caused the reduced photosynthesis and water loss observed in their studies, however. The reduction in photosynthesis of tomato leaves exposed to 17 p.p.h.m. of ozone reported by Stolzy, Taylor, et al. (1964) may be explained by stomatal closure. Acknowledgment
The authors express appreciation to Warren Monson, Pamela Hutchinson, and Karma Anderson for their technical assistance. Literature Cited
Dugger, W. M., Jr., Taylor, 0. C., Cardiff, E., Thompson, C. R., Plant Physiol. 37, 487-91 (1962). Engle, R. L.. Gableman, W. H., Proc. A m . SOC.Hort. Sci. 89, 423-30 (1966). Erickson, L. C.. Wedding, R. T., Am. J. Bot., 43, 32-6 (1956). Hill. A. C., J . Air Pollution Control Assoc. 17, 743-8 (1967). Hill, A. C., Transtrum, L. G., Pack, M. R., Holloman, A., J . Air Pollution Control Assoc. 9, 22-7 (1959). Hull, H. F., Went, F. W., Proceedings of Second National Air Pollution Symposium, Pasadena, Calif., pp. 122-8, 1952. Katz, M., Ledingham, G. A., “Effect of Sulfur Dioxide on Vegetation,” National Research Council of Canada, Ottawa, Canada, 1939. Koritz, H. G., Went, F. W., Plant Physiol. 28,50-62 (1953). Lee. T. T., Can. J . Plant Sci. 43, 677-85 (1965). Lloyd, F. E., Carnegie Inst. Wash., Publ. 82 (1908). Loftfield, J. V. G., Carnegie Inst. Wash., Publ. 314 (1921). Macdowall, F. D. H., Can. J. Bot. 43, 419-27 (1964). Macdowall, F. D. H., Can. J. PIant Sci. 45, 1-12 (1965). Menser, H. A., Crosso, J. J., Heggestad, H. E., Street, 0. E., Plant Physiol. Suppl. 39, 58 (1964). Stolzy, L. H., Taylor, 0. C., Dugger, W. M., Jr., Mersereau, J. D., Proc. SoilSci. SOC.Am. 28, 305-8 (1964). Taylor, 0. C., Agron. J. 50, 556-8 (1958). Thomuson, C. R.. Taylor. 0. C., Thomas, M. D., Ivie, J. O., ENGIRON. SCI. TECHNOL. 1,644-50 (1967). Todd. G. W.. Middleton. J. T.. Brewer. R. F.,I Calif Aa. 10._ “ 7-8 (1956).’ Todd, G. W., Propst, B., Physiol. Plant. 16,57-65 (1963). Woltz, S. S., Leonard, C. D.. Proc. Florida State Hort. SOC. 77,9-15 (1964). Zelitch, I., Proc. Natl. Acad. Sci. 47, 1423-33 (1961). Received for reciew April 15, 1968. Accepted September 25, 1968. Work supported by Grant AP00452 from the Research Grants Branch, National Air Pollution Control Administration.
