Influence of two growing seasons of experimental ozone fumigation on photosynthetic characteristics of white oak seedlings

Environmental Pollution 65 (1990) 371-380

/

Influence of Two Growing Seasons of Experimental Ozone Fumigation on Photosynthetic Characteristics of White Oak Seedlings Jeffrey R. Foster Holcomb Research Institute, Butler University, 4600 Sunset Avenue, Indianapolis, IN 46208, USA

Ken V. Loats Department of Biology, Denison University, Granville, OH 43023, USA

& Keith F. Jensen USDA Forest Service, Northeastern Forest Experiment Station, 359 Main Road, Delaware, OH 43015, USA (Received 6 April 1989; accepted 26 March 1990)

ABSTRACT White oak (Quercus alba L.) seedlings were exposed to charcoal-filtered air or to above-ambient ozone concentrations for 19-20 weeks during each of two growing seasons in continuously stirred tank reactors in greenhouses. Ozone treatments were 0.15 ppm (300 ltg m- 3) for 8 h day- 1, 3 days week- 1 in 1988, and continuous 15% above ambient in 1989. The seedlings were grown in forest soil watered twice weekly with simulated rain of pH 5.2. Responses of net photosynthesis to photosynthetically active radiation and intercellular C02 concentration were measured three times each year. There were no significant differences in light-saturated net photosynthesis or stomatal conductance, dark respiration, quantum or carboxylation efficiencies, and light or C02 compensation points on any date between control and ozoneexposed seedlings. 371 Environ. Pollut. 0269-7491/90/$03.50 O 1990 Elsevier Science Publishers Ltd, England. Printed in Great Britain

372

Jeffrey R. Foster, Ken V. goats, Keith F. Jensen

INTRODUCTION Exposure to ozone reduces net photosynthesis in a variety of crop and tree species (Reich & Amundson, 1985). The degree of reduction is strongly related to cumulative ozone dose, particularly when expressed as leaf uptake via the stomata (Reich, 1987). Recently, gas exchange techniques under controlled environment conditions have come into use to study specific physical and biochemical components of photosynthesis potentially influencedby ozone and other gaseous air pollutants (Atkinson & Winner, 1987; Kropf, 1987; Hanson et al., 1988; Mooney et al., 1988; RowlandBamford et al., 1989). For example, the response of photosynthesis to photosynthetic photon flux density (PPFD) provides information on dark respiration, quantum efficiency, light compensation point, and lightsaturated photosynthesis. The last attribute is limited by the concentration of ribulose biphosphate carboxylase (Rubisco). However, at any PPFD, reduced stomatal conductance may also limit photosynthesis. The relationship of photosynthesis to intercellular CO2 concentration reveals non-stomatal limitations on photosynthesis (Jones, 1985). The initial slope of the relationship (carboxylation efficiency) is proportional to the maximum activity of Rubisco, while CO2-saturated photosynthesis is determined by the rate of ribulose biphosphate regeneration (Farquhar et al., 1980; Farquhar & Sharkey, 1982). The objective of this study was to use diagnostic gas exchange techniques to examine the influence of two growing seasons of ozone exposure under greenhouse conditions on components of photosynthesis in white oak (Quercus alba L.) seedlings.

METHODS This experiment was part of a 2-year investigation of the effects of ozone and simulated acid rain on the growth, morphology, and physiology of four common forest species in the midwestern USA, and was conducted at the US Forest Service laboratory in Delaware, Ohio. Natural field soil (A and upper B horizons) was collected from a ridgetop oak-hickory forest on the Wayne National Forest, Ohio, in April 1988. This soil was a Berks silt loam (Typic Dystrochrept) with a mean pH of 5.0 (2:1 water: soil) and a high clay content. The soil was passed through a 2-mm mesh screen and mixed with an equal weight of washed silica sand to facilitate drainage. This treatment did not alter pH. One-year-old, bare-root seedlings of white oak (a mixture of various provenances) were acquired from the Ohio State Nursery in Zanesville,

Ozone and white oak photosynthesis

373

Ohio, and transplanted in early April 1988 into the soil/sand mix in 2-liter plastic pots. The seedlings were kept in a greenhouse (maximum clear-day PPFD ,~ 800 #mol m - 2 s- 1) with charcoal-filtered air to reduce ozone to less than 0.025 ppm (1 ppm = 1997 #g m - 3 at 20°C). Ozone fumigations occurred in continuously stirred tank reactors (CSTRs) located in a separate greenhouse. Different fumigation regimes were used during the two successive growing seasons of the study because of an upgrading in the capabilities of the CSTR system in the second year. Seedlings were randomly allocated to receive either charcoal-filtered air (<0.025 ppm ozone) or 'above-ambient' ozone (i.e., concentrations higher than those experienced in the field). There were four replicate seedlings (each in a separate CSTR) per treatment, and individual seedlings received the same treatment in both years. In 1988, seedlings in the above-ambient treatment received constant 0.15ppm (300#g m -3) ozone for 8h per day (1000-1800h) on three consecutive days per week (longer exposures were prevented by the simultaneous demands of several experiments on a limited number of CSTRs). Fumigations began on 23 May, coincident with budbreak, and continued for 19 weeks. Seedlings were left inside the CSTRs between successive fumigation days, but were kept in the original, charcoal-filtered greenhouse for the remaining 4 days each week. In 1989, all seedlings were fumigated continuously (24 h day- 1), starting on 24 May and continuing for 19½ weeks, with ozone concentrations in the above-ambient CSTRs changing every 2 h under computer control. The above-ambient treatment was equivalent to 1.15 times the 2-h mean ozone concentrations (i.e., ambient + 15%) measured in 1988 at a remote forested site in Parsons, West Virginia. Thus, each day had a unique pattern of ozone fumigation. Ozone was produced from pure oxygen with a corona discharge-type generator and ozone concentrations in each chamber were sequentially monitored (4 samples h - 1 chamber- 1) by a Thermoelectron model 49 ozone monitor, calibrated daily with a Monitor Labs model 8500 calibrator. At noon on clear days, PPFD inside the CSTRs was 400-500 #mol m - 2 s- 1. Mean daily air temperature inside the CSTRs ranged from 7.5 to 28.7°C (minimum 0.6°C, maximum 33.3°C) during 1989. Temperatures were not measured in 1988. Twice per week in both years, each pot was surfacewatered to saturation with simulated rain of pH 5.2 containing 2.1, 1.6, 0.3, 0.5, 0.5, 0-1, 0.1 and 0.2 mg liter-1, respectively, of sulfate, nitrate, chloride, ammonium, calcium, magnesium, potassium and sodium. The responses of net photosynthesis to PPFD and intercellular CO2 concentration were measured on three different, 2-day periods each growing season: 23-24 June, 11-12 August and 23-24 September in 1988; 8-9 June,

374

Jeffrey R. Foster, Ken V. Loats, Keith F. Jensen

27-28 July and 21-22 September in 1989. Each 1988 sampling started on the day following completion of a 3-day fumigation period. In both years, half of the seedlings were randomly chosen for measurements on the first day; the rest were measured the following day. Gas exchange measurements were carried out with an LI-6200 portable gas exchange system (Li-Cor, Inc., Lincoln, Nebraska) using a stirred, l-liter cuvette incorporated in a closed loop. The equations of von Caemmerer & Farquhar (1981) were used to calculate gas exchange parameters. Prior to the experiments, leaf boundary layer conductance was determined using a wet-filter-paper leaf replica. A leak test with dry filter paper indicated negligible influx or efflux of CO2 at cuvette-to-ambient air concentration gradients as high as 713 ppm. The infrared gas analyzer was calibrated daily using bottled CO2 standards. Each seedling was preconditioned for 1 h under a high-intensity discharge (HID) lamp supplying approximately 650 gmol m - 2 s- ! P P F D at the top of the seedling. Then a leaf was clamped in the cuvette, which was illuminated by a second HID lamp in 1988 or by a General Electric 300-W PAR lamp (PPFD = 1400pmol m -2 s-1) in 1989. A waterbath between the lamp and the cuvette minimized cuvette heating. Within each year, one leaf(2nd to 5th internode below the shoot apex) on each seedling was tagged and this leaf was measured on successive sampling dates. All of the leaves on every seedling were fully expanded by the first sampling date (June) in both years. Comparisons among fully expanded leaves on each of the control and above-ambient seedlings on two different days indicated no significant effect of nodal position on light-saturated photosynthesis. All measurements occurred at a cuvette temperature of 25-32°C (leaf temperatures were within I°C of this) and a relative humidity of 40-55%. For light curves, CO2 concentration was maintained at 350 + 15 ppm and light levels were varied using fiberglass screens. Photosynthetic responses to intercellular CO2 concentration were measured with the technique of Davis et al. (1987). Briefly, this involved breathing into the cuvette to create a high CO2 concentration, then drawing down cuvette CO2 in steps by passing the cuvette air through soda lime in a bypass loop. Two minutes elapsed between successive measurements for both light and CO2 response curves to assure leaf equilibration (especially of stomatal conductance) with altered chamber conditions. Linear regressions were fit to the first 4-5 points on each light curve to estimate quantum efficiency, light compensation point, and dark respiration, and to the first 4-5 points of each intercellular CO 2 curve to estimate carboxylation efficiency and the CO 2 compensation point. Because experimental conditions differed between 1988 and 1989, comparisons

Ozone and white oak photosynthesis

375

among treatments were analyzed separately for each year's data using repeated-measures ANOVA (SYSTAT software; Wilkinson, 1986), with ozone fumigation concentration as the between-subjects factor and date of sampling as the within-subjects factor.

RESULTS During the 1988 fumigations, mean weekly ozone concentrations ranged from 0.108_+0.037 to 0.130_+ 0.019ppm (mean _+ 1 standard deviation) in the above-ambient CSTRs, less than the target value of 0.15 ppm. In 1989, seasonal averages for mean 8-h (1000-1800h) and mean 24-h ozone concentrations were 0.054-0.056 ppm and 0.040-0.043 ppm, respectively, in the above-ambient CSTRs, less than the target values of 0"070 and 0.048 ppm. Although the ozone exposures were less than expected, they still significantly exceeded ambient exposures. For example, 7-h (0900-1600 h) mean Oa concentration in the Ohio River Valley was 0.043 ppm for the growing seasons 1978-1985, with occasional hourly means >0.12ppm (Lefohn & Pinkerton, 1988). On the first sampling date in 1988 and the final sampling date in 1989, one control seedling had nearly closed stomata under high light, preventing measurement of response curves. Thus on these two dates, control treatment sample size was three. Most of the seedlings exhibited pronounced stomatal closure when cuvette CO2 concentrations exceeded 600 ppm. Because the calculation of intercellular CO2 is very sensitive to stomatal conductance, it was not possible to ascertain photosynthesis-saturating intercellular CO 2 concentrations. In 1988, there were no significant differences in any of the components of photosynthesis, or in light-saturated stomatal conductance to water vapor, between control plants and plants exposed to above-ambient ozone (repeatedmeasures ANOVA, P > 0.05; Figs 1, 2). Date of sampling influenced lightsaturated photosynthesis, quantum efficiency, light compensation point, CO 2 compensation point, and stomatal conductance (ANOVA, P < 0"05), but the temporal patterns differed in a complex fashion among these variables (Figs l, 2). There were no significant ozone x sampling date interactions. In 1989, there was no effect of ozone fumigation on any measured variable (ANOVA, P > 0"05; Figs 1, 2). The only effect of date of sampling was a higher CO2 compensation point in June than in August and September (ANOVA, P < 0.001; Fig. 2). With this exception, the significant effects of sampling date observed in 1988 were not apparent in 1989, suggesting either that leaf age did not influence photosynthesis once leaves were fully mature,

376

Jeffrey R. Foster, Ken V. Loats, Keith F. Jensen

t-

0

I

E

o

6

-6

Z

control ~

2

~:

E

c

c3

~

.06

w E

E

.04

¢o

C ~

O

E

o

~

t

+ozone

.02

45

30

L3

6-23/24

8-11/12 1988

9-23/2#

6-8/9

7-27/28

9-21/22

1989

Fig. 1. The response of light-saturated net photosynthesis, dark respiration, quantum efficiency, and light compensation point in white oak seedlings exposed to charcoal-filtered air (control) or to 0.15 ppm (1988) or 15 %-above-ambient (1989) ozone ( + ozone). Values are means + one standard deviation.

or that leaf age affected photosynthesis differently in each year. There were significant ozone x sampling date interactions for light-saturated photosynthesis (P < 0.05) and carboxylation efficiency (P < 0.01); both appeared associated with June-to-September trends from lower to higher values in control seedlings versus seedlings in the above-ambient treatment (Figs 1, 2). No visual symptoms of ozone injury appeared on any seedling at any time. However, in September of 1989, one seedling each in the control and aboveambient treatments showed moderate interveinal chlorosis on all leaves.

Ozone and white oak photosynthesis

377

IS7] control '0

+ozone

.06

W

.04

E

_o

.02 C)

150 0 (2-

6_ E E 0 r~

100

0

50

C 0

E

o

c 0

7

o}

S E o

E o CO

6-23/24

8-11/12

9-23/24

1988

6-8/9

7-27/28

9-21/22

1989

Fig. 2. The response of carboxylation efficiency, C02 compensation point, and lightsaturated stomatal conductance to water vapor in white oak seedlings exposed to charcoalfiltered air (control) or to 0-15 ppm (1988) or 15%-above-ambient (1989) ozone ( + ozone). Values are means + one standard deviation.

DISCUSSION White oak is a dominant overstory species within the oak-hickory forests that stretch across the lower midwestern USA from Arkansas to Tennessee. If ambient levels of ozone affect photosynthesis in this species, subsequent alterations in growth might occur, leading to changes in species composition and/or biomass of these forests. However, our experiments did not reveal any reductions in net photosynthesis or increases in dark respiration after two growing seasons of ozone exposure at above-ambient levels. In the absence of net changes in carbon uptake, ozone-induced growth reductions in white oak seem unlikely. In fact, Jensen & Dochinger (1989) found no influence of fumigation with 0.15 ppm ozone (3 days week- 1 for 16 weeks in CSTRs) on growth of white oak seedlings. However, alterations in stem or root respiration, which would be revealed by gas exchange analysis of whole

378

Jeffrey R. Foster, Ken Ix. Loats, Keith F. Jensen

seedlings, cannot be ruled out. Furthermore, mature trees in the field may respond differently than seedlings in the laboratory. Ozone effects on photosynthesis in other oak species have received limited attention. Carlson (1979) observed a 57% reduction in light-saturated photosynthesis of black oak (Q. velutina Lam.) seedlings exposed to a cumulative ozone dose (concentration × exposure time) of 38.5 to 60.5 ppmh (0.50 ppm, 7-11 h day- 1, 11 days total) in growth chambers. However, the ozone concentrations he used were exceptionally high and his apparent sample size was only two. Reich & Amundson (1985) and Reich et al. (1986) exposed red oak (Q. rubra L.) seedlings to filtered air ( < 0.025 ppm 03) and to 0.07 or 0.12 ppm ozone (7 h day- 1, 5 days week- 1, for 10 weeks) in growth chambers and in outdoor, open-top chambers (n > 10 per treatment). Mean reduction in light-saturated photosynthesis of ozone-exposed versus control seedlings declined linearly from 0% at < 7 ppm-h to about 10% at 32 ppm-h. In our experiments, mean (+ 1 SD) cumulative ozone doses in the aboveambient CSTRs were 46.2 (+ 4-4) ppm-h as of 22 September, 1988, and 53"8 (+__0"7) ppm-h (8-h, 1000-1800 h basis) or 121.3 (+__4.3) ppm-h (24-h basis) as of 21 September, 1989, but no reductions in photosynthesis occurred. Therefore, it appears that photosynthetic responses in white oak may be less sensitive to ozone than in red or black oaks. One reason for the lack of response by our white oaks may have been their low stomatal conductances (mostly < 5 mm s- 1). Species sensitive to ozone generally have higher conductances, and therefore absorb more ozone at a given ambient concentration, than do resistant species (Reich, 1987). However, the red oaks studied by Reich & Amundson (1985) had conductances < 3 mm s- 1. Another possible reason for lack of response was that our seedlings had 12-36 h in 1988 and 1 h in 1989 for possible recovery from ozone exposure to occur. In the studies cited above, photosynthesis was measured concurrent with ozone fumigation. Long-term ozone exposure at ambient or modestly above-ambient concentrations usually, but not always, reduces light-saturated photosynthesis in other tree species (Keller & H/isler, 1987; Reich, 1987; Reich et al., 1987; Chappelka et al., 1988; Hanson et al., 1988). Dark respiration either increases (Barnes, 1972), decreases (Reich, 1983; Yang et al., 1983), or is unaffected (Hanson et al., 1988). Decreased quantum efficiency, lower carboxylation efficiency, and increased light compensation points have also been reported for trees (Coyne & Bingham, 1982; Reich, 1983), with exceptions (Hanson et al., 1988). Stomatal responses to ozone in trees are quite variable, ranging from pronounced closure to moderate increases, and are affected by leaf age, time of day, and relative humidity (Coyne & Bingham, 1982; Reich & Lassoie, 1984; Reich & Amundson, 1985; Jensen & Roberts, 1986; Keller & H/isler, 1987; Chappelka et al., 1988; Tseng et al.,

Ozone and white oak photosynthesis

379

1988). N o n e o f these gas exchange parameters was affected by ozone in our experiments with white oak.

ACKNOWLEDGEMENTS The authors thank M a r y A n n Keyser for technical assistance, and Orie Loucks and T o m A r m e n t a n o for manuscript reviews. This research was supported by funds provided by the Northeastern Forest Experiment Station, Eastern H a r d w o o d s Research Cooperative, within the joint US Environmental Protection A g e n c y - U S D A Forest Service Forest Response Program. The Forest Response Program is part of the National Acid Precipitation Assessment Program. This paper has not been subject to EPA or Forest Service policy review and should not be construed to represent the policies o f either agency.

REFERENCES Atkinson, C. J. & Winner, W. E. (1987). Gas exchange characteristics of Heteromeles arbutifolia during fumigation with sulphur dioxide. New PhytoL, 106, 423-36. Barnes, R. L. (1972). Effects of chronic exposure to ozone on photosynthesis and respiration of pines. Environ. Pollut., 3, 133-8. Carlson, R. W. (1979). Reduction in the photosynthetic rate of Acer, Quercus and Fraxinus species caused by sulphur dioxide and ozone. Environ. Pollut., 18, 159-70. Chappelka, A. H., Chevone, B. I. & Seiler, J. R. (1988). Growth and physiological responses of yellow-poplar seedlings exposed to ozone and simulated acidic rain. Environ. Pollut., 49, 1-18. Coyne, P. I. & Bingham, G. E. (1982). Variation in photosynthesis and stomatal conductance in an ozone-stressed ponderosa pine stand: light response. For. Sci., 28, 257-73. Davis, J. E., Arkebauer, T. J., Norman, J. M. & Brandle, J. R. (1987). Rapid field measurement of the assimilation rate versus internal CO2 concentration relationship in green ash (Fraxinus pensylvanica Marsh.): The influence of light intensity. Tree Physiol., 3, 387-92. Farquhar, G. D. & Sharkey, T. D. (1982). Stomatal conductance and photosynthesis. Ann. Rev. Plant Physiol., 33, 317-45. Farquhar, G. D., von Caemmerer, S. & Berry, J. A. (1980). A Biochemical model of photosynthetic CO2 assimilation in leaves of C3 species. Planta, 149, 78-90. Hanson, P. J., McLaughlin, S. B. & Edwards, N. T. (1988). Net CO 2 exchange of Pinus taeda shoots exposed to variable ozone levels and rain chemistries in field and laboratory settings. Physiol. Plant., 74, 635-42. Jensen, K. F. & Dochinger, L. S. (1989). Response of eastern hardwood species to ozone, sulfur dioxide and acid precipitation. J. Air Pollut. Contr. Assoc., 39, 852-5.

380

Jeffrey R. Foster, Ken V. Loats, Keith F. Jensen

Jensen, K. F. & Roberts, B. R. (1986). Changes in yellow poplar stomatal resistance with SO 2 and 0 3 fumigation. Environ. Pollut., 41, 235-45. Jones, H. G. (1985). Partitioning stomatal and non-stomatal limitations to photosynthesis. Plant Cell Environ., 8, 95-104. Keller, T. & H/isler, R. (1987). Some effects of long-term ozone fumigations on Norway spruce. I. Gas exchange and stomatal response. Trees, 1, 129-33. Kropf, M. J. (1987). Physiological effects of sulphur dioxide. 1. The effect of SO2 on photosynthesis and stomatal regulation of Viciafaba L. Plant Cell Environ., 10, 753-60. Lefohn, A. S. & Pinkerton, J. E. (1988). High resolution characterization of ozone data for sites located in forested areas of the United States. J. Air Pollut. Contr. Assoc., 38, 1504-11. Mooney, H. A., Kiippers, M., Koch, G., Gorham, J., Chu, C. & Winner, W. E. (1988). Compensating effects to growth of carbon partitioning changes in response to SO2-induced photosynthetic reduction in radish. Oecologia, 75, 502-6. Reich, P. B. (1983). Effects of low concentrations of ozone on net photosynthesis, dark respiration and chlorophyll contents in aging hybrid poplar leaves. Plant Physiol., 73, 291-6. Reich, P. B. (1987). Quantifying plant response to ozone: a unifying theory. Tree Physiol., 3, 63-91. Reich, P. B. & Amundson, R. G. (1985). Ambient levels of ozone reduce net photosynthesis in tree and crop species. Science, 230, 566-70. Reich, P. B. & Lassoie, J. P. (1984). Effects of low level ozone exposure on leaf diffusive conductance and water-use efficiency in hybrid poplar. Plant Cell Environ., 7, 661-8. Reich, P. B., Schoettle, A. W. & Amundson, R. G. (1986). Effects of ozone and acidic rain on photosynthesis and growth in sugar maple and northern red oak seedlings. Environ. Pollut., 40, 1-15. Reich, P. B., Schoettle, A. W., Stroo, H. F., Troiano, J. & Amundson, R. G. (1987). Effects of ozone and acid rain on white pine (Pinus strobus) seedlings grown in five soils. I. Net photosynthesis and growth. Can. J. Bot., 65, 977-87. Rowland-Bamford, A. J., Coghlan, S. & Lea, P. J. (1989). Ozone-induced changes in CO 2 assimilation, 02 evolution and chlorophyll a fluorescence transients in barley. Environ. Pollut., 59, 129-40. Tseng, E. C., Seiler, J. R. & Chevone, B. I. (1988). Effects of ozone and water stress on greenhouse-grown Fraser fir seedling growth and physiology. Environ. Exper. Bot., 28, 37-42. von Caemmerer, S. & Farquhar, G. D. (1981). Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. Planta, 153, 376-87. Wilkinson, L. (1986). SYSTAT: The system for Statistics. Evanston, Illinois, SYSTAT, Inc. Yang, Y.-S., Skelly, J. M., Chevone, B. I. & Birch, J. B. (1983). Effects of long-term ozone exposure on photosynthesis and dark respiration of eastern white pine. Environ. Sci. Technol., 17, 371-3.