Environ. Scl. Technol. 1983, 17, 371-373
heaters indoors. In any case, when these appliances are used in small rooms, increased ventilation or other pollution-control strategies should be considered. Registry No. CO, 630-08-0; COz, 124-38-9; NO, 10102-43-9; NO^, ioioi-44-0; HCHO, 50-00-0.
Literature Cited Traynor, G. W.; Anthon, D. W.; Hollowell, C. D. Atmos. Environ. 1982,16, 2979-2988. Girman, J. R.; Apte, M. G.; Traynor, G. W., Allen, J. R.; Hollowell, C. D. Enuiron. Int. 1982, 8, 213-221. U.S.Government, Code of Federal Regulations, Title 29, Section 1910.1000, 1979. State of California, California Administrative Code, Title 17, Subchapter 1.5, Section 70100, 1977.
(5) U.S. Government, Code of Federal Regulations, Title 40, Section 50.8, 1975. (6) Data provided by Chevron Research Co., Richmond, CA. (7) Yamanaka, S.; Hirose, H.; Takada, S. Atmos. Enuiron. 1979, 13. 407-412. ~-(8) Turk, A. ASHRAE J . 1963,5, 55-58. (9) Alonzo, J.; Cohen, B. L.; Rudolf, H.; Jow, H. N.; Frohliger, J. 0. Atmos. Enuiron. 1979. 13, 55-60. (10) Traynor, G. W.; Apte, M. G.; Dillworth, J. F.; Hollowell, C. D.; Sterling, E. M. Enuiron. Int. 1982, 8, 447-452. I
Received for review July 19,1982. Accepted December 27,1982. This work was supported by the Director, Office of Energy Research, Office of Health and Environmental Research, Human Health and Assessments Division of the U S . Department of Energy under Contract DE-AC03- 76SF00098.
Effects of Long-Term Ozone Exposure on Photosynthesis and Dark Respiration of Eastern White Pine Yaw-Shing Yang,*t John M. Skelly,? Borls I, Chevone,t and Jeffrey B. Birch$ Department of Plant Pathology and Physiology and Department of Statistics, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061
Net photosynthetic (PN) and dark respiratory rates (R,) of three eastern white pine (Pinus strobus L.) clones differing in sensitivity to ozone were determined during long-term O3exposure. Fumigations occurred for 4 h daily with 0.00, 0.10, 0.20, or 0.30 pL/L O3for 50 consecutive days. Every 10 days, PN and RD were measured. Ozone exposure adversely affected PN, and the magnitude of this effect was dependent upon the clone, O3concentration, and duration of O3exposure. Dark respiration of the 0,-treated sensitive clone was significantly decreased compared to controls, whereas RD of the other clones was unaffected at all O3concentrations. The implications of PY inhibition and decline of RD due to O3fumigation in relation to plant biomass production and foliar symptom expression are discussed. Introduction Exposure of plants to gaseous air pollutants induces various adverse effects such as foliar discoloration, premature defoliation, inhibition of photosynthesis, reduced growth rate, and biomass reduction (1-4). In the past, studies have focused on determining the types of foliar injury and the degree of growth reduction resulting from short-term pollutant exposure ( I , 2 , 5 , 6 ) . However, rarely has research emphasized the effects of long-term pollutant fumigation on plant assimilatory metabolism and subsequent biomass production (4, 6-8). In the area surrounding a pollutant point source (primarily oxides of nitrogen) in Virginia, a native population of eastern white pine (Pinus strobus L.) exhibited differential sensitivity to ambient pollutants and reduced increment growth during years of high pollutant concentrations (9). Frequently, in addition to oxides of nitrogen, this area is impacted by episodic ozone concentrations exceeding 0.08 pL/L during the summer season. Under controlled pollutant fumigations, white pine clones from this population showed differential sensitivity to O3(10, 11). Significant reduction of needle biomass production in the sensitive clones was observed after long-term O3 Department of Plant Pathology and Physiology. *Department of Statistics. 0013-936X/83/0917-0371$01.50/0
exposure (8-11). This study was part of a series of pollutant fumigations designed to examine the causes of growth reduction and the mechanisms of differential plant sensitivity to pollutant exposure. With use of genetically identical ramets from different ortets, the specific objectives of this research were to investigate white pine photosynthetic and dark respiratory rates as affected by long-term O3exposure in relation to clonal sensitivity and biomass production. Materials and Methods Three clones of eastern white pine differing in 0, sensitivity (i.e., sensitive (clone 11-l),intermediately sensitive (clone 111-2),and insensitive (clone IV-2)) on the basis of controlled fumigation experiments (5,8,10,1I)were used for this study. Preparation and plant culture of 2-year-old white pine ramets with uniform needle age were reported previously (10). First-year needles were 21-25 days old at the beginning of the long-term O3 fumigation. Fumigations were conducted in continuously stirred tank reactors (CSTR) (22). Plants were exposed to 0.00, 0.10, 0.20, or 0.30 pL/L O3 4 h daily during 1400-1800 EST for 50 days consecutively. Plan& were fumigated at 28-32 OC, 60-70% relative humidity, 21-28 klux, and 360-410 peinsteins/ (m2 s) photosynthetically active radiation (400-700 nm). Except for daily O3fumigation, plants were maintained in a greenhouse supplied with charcoal-filtered air. Ozone was generated by a Welsbach Laboratory ozonator Model T-408 (Welsbach Ozone Systems Corp., Philadelphia) and monitored by a Bendix Model 8002 chemiluminescent ozone analyzer (Bendix Process Instruments Division, Lewisburg, WV). Ozone concentration in each CSTR was continuously sampled at 50 cm above the chamber floor and maintained within f0.01 pL/L of the desired concentration. The ozone monitor was calibrated every 2 weeks during the experiment with a Photocal3000 automated ozone calibrator (Columbia Scientific Industries, Austin, TX). Net photosynthetic and dark respiratory rates of current-year needles were measured at 10-day intervals during the 50-day experiment with an infrared gas analyzer (IRGA) Model AR 600R (Anarad, Inc., Santa Barbara,
0 1983 American Chemical Society
Environ. Sci. Technol., Vol. 17,No. 6, 1983 371
-0 0
Ulll
.......... 0 10 u l / l
n i t photo8ynthesls
~
._ ~_. 0.20 ~U l / l .
.
____ 0.30 u l / l
dark
respiration
DAYS AFTER BEGINNING OF EXPERIMENT
Figure 1. Effect of daily 4-h ozone exposure on net photosynthesis and dark respiration of sensitive (A, B), intermediately sensitive (C, D), and insensitive (E, F) clones of eastern white pine, respectlvely. Measurements on the same day in the same subfigure having the same letter are not significantly different (p I0.05) according to Duncan's multiple-range test.
CA). On each sampling day, the current-year needles of the main branchlet of each ramet were enclosed within a glass minichamber designed from a 1-L Pyrex beaker for through-flow measurement (8). The photosynthetic rate of each branchlet was measured just prior to daily 4-h 0, exposures. Dark respiratory rates were taken at 10-day intervals from the same branchlet but 5 days before photosynthesis measurement and were followed by using the same procedures. For dark respiration measurements, plants returned to the CSTR from the greenhouse were exposed to charcoal-filtered air in darkness. The rates of dark respiration were measured at 2200 EST at 20-22 "C and 60-70% relative humidity. Foliar injury, length, and dry weight of first-year needles were measured at the end of the study. The amount of foliar surface area injured was recorded at 5% increments (0-100% scale). Needle length of the five oldest fascicles of each ramet was measured to the nearest 1mm. Needle dry weight of the five oldest fascicles of each ramet was determined after freeze-drying fresh tissues for 24 h. The study was statistically constructed as a randomized block design. Treatments were randomly distributed among the CSTR chambers each day to minimize chamber effects. Three plants were used in each treatment. Results of clonal response to 0, exposure were analyzed by using Duncan's multiple-range test or Student's t test. Significant differences are expressed at the p I 0.05 probability level. Results The most evident adverse response of net photosynthesis to long-term Os exposure was the consistently lower rate compared to controls during the entire study, especially in the sensitive clone exposed to high 0, concentrations. In the sensitive clone (clone 11-1),the rate of net photosynthesis was significantly reduced by day 10 of exposure to 0.30 pL/L 0, (Figure 1A). By day 20, the rates of all 03-treated plants in the sensitive clone were significantly lower than controls and remained so for the duration of the experiment. The magnitude of reduction in this clone, after 50 days of exposure, was dependent upon 0, con372
Environ. Sci. Technol., Vol. 17, No. 6, 1983
centrations. At the end of the 50-day study, the net photosynthetic rate of 0.10,0.20, and 0.30 pL/L 0,-treated plants in the sensitive clone was 24%, 42%, and 51% lower than controls, respectively. Net photosynthetic rate of Os-treated trees in the intermediately sensitive clonal group (clone 111-2) were inhibited less than those in the sensitive clone. However, the magnitude of inhibition varied and was not proportional to 0, concentrations (Figure IC). At the end of the experiment, net photosynthesis in the intermediately sensitive clone was 94%, 86%, and 80% of the controls for 0.10, 0.20, and 0.30 pL/L O3 exposure, respectively. Among these, only the last two treatments were significantly different from controls. The patterns of long-term net photosynthetic rate of Os-free and O,-treated plants in the insensitive clone (clone IV-2) were very similar and not always different from one another. Significant reduction of photosynthetic rate in the insensitive clone due to 0, exposure was observed only in the middle of the fumigation period at 20, 30, and 40 days of exposure. At day 50, such treatment effects became insignificant although photosynthetic rates of 0,treated plants remained below that of control plants (Figure 1E). It is interesting to note that although the rates of net photosynthesis of control plants varied with white pine clone and the state of needle development, the patterns of net photosynthesis of sensitive, intermediately sensitive, and insensitive clones in 03-free environments were very similar during the study (Figure 1); rated as 100% on an arbitrary scale at the beginning of long-term exposure, the net photosynthetic rate steadily increased during the first 20-30 days of the experiment to its peak. The peak photosynthetic rate varied among clones, 176%,165%, and 182% being the highest rates observed in the sensitive, intermediately sensitive, and insensitive clones, respectively. Following the maximum rate of net photosynthesis, as needles aged, the net photosynthetic rate gradually decreased for the remainder of the experiment. The only pronounced 0, effect on dark respiration of white pine plants was found in the sensitive clone, where significant reductions were observed after day 15 of the experiment (Figure 1B). Thereafter, the rates of dark respiration in the sensitive clone were found to steadily decrease at all 0, concentrations for the rest of the experiment. At the end of the study, the rate of respiration was 89%, 73%, and 61% of control value for 0.10, 0.20, and 0.30 pL/L 0, exposure, respectively. These rates were all significantly different from controls and different from each other. During long-term 0, fumigation, the patterns of dark respiration rates of Os-treated intermediately sensitive and insensitive clones were similar to those of control plants and, in general, were not significantly different from controls (Figure 1D and 1F). The Os-induced foliar symptoms included chlorosis, pigmented mottling, necrotic banding, and necrotic tipburn (Table I). For each 0, concentration used, the amount of foliar injury in the sensitive clone was always the highest followed by the intermediately sensitive and insensitive clones. Within each clone, the amount of foliar area injured was not always distinguish_ably different among different 0, concentrations. By the end of the study, 0.20 and 0.30 pL/L 0, exposure had significantly reduced needle length in the sensitive clone, whereas only 0.30 pL/L Os exposure resulted in reduced needle length in the intermediately sensitive clone (Table I). None of the 0, treatments was found to affect needle length in the insensitive clone.
Table I. Percentage of Foliar Injury, Needle Length, and Dry Weight of Eastern White Pine First-Year Needles after Exposure to 0.00, 0.10, 0.20,.or 0.30 bL/L Ozone for 4 h Daily for 50 Days Consecutively plant response/clonea needle dry weight, mg/mm fascicle needle length, m m foliar symptomb, % surface area ozone -_.__ concn, b L / L 11-1 111-2 IV-2 11-1 111-2 IV-2 11-1 111-2 IV-2 450 94 102 0 89 0 0 96 43OC 76 15c 0 84 2 oc 99 430* 0 7 gc 80 3 0‘ 15c 0.20 381‘ 103 5 66c 64c 15c 0.30 4 Oc a Clone 11-1= sensitive t o 0,, 111-2 = intermediately sensitive to O,?,and IV-2 = insenstive t o 0,. Significantly different from surface included general chlorosis, pigmented mottling, and necrosis. on the basis of Student’s t test. 0.00
0.10
Ozone exposure at all three concentrations reduced needle dry weight in the sensitive and intermediately sensitive clones, whereas only 0.20 and 0.30 pL/L 0, exposure reduced needle biomass in the insensitive clone (Table I).
Discussion Results indicate that there is a substantial variation of white pine clonal sensitivity to 0, on the basis of plant photosynthetic and respiratory responses. Ozone-induced changes in photosynthesis and dark respiration were found to be closely correlated with the clonal sensitivity to 0, as determined by other indexing parameters such as visible foliar injury and needle biomass production. The more tolerant the white pine clone, the less repressed was the rate of net photosynthesis and the less affected the rate of dark respiration. Results suggest that the differential white pine clonal sensitivity to 0, and the amount of the reduction in biomass production could be based on the differential response of these assimilatory processes to 0, exposure. In general, the occurrence of the initial significant reduction in net photosynthesis and dark respiration in the sensitive clone due to 0, exposure, as observed in this study, was closely related to the timing of the appearance of foliar injury reported in a previous study (11). Present results indicate that there was a significant reduction in photosynthetic rate and dark respiratory rate after needles have been visibly injured. Yang et al. (8,lO)further reported that a reduction in net photosynthesis due to 4-h O3exposure occurred in white pine plants and that 0,treated sensitive plants frequently experienced premature senescence. These findings suggest that the greater biomass reductions of the sensitive plants to 0, exposure can be attributed to the reduced photosynthetic areas as evidenced by foliar injury, the complete loss of assimilatory apparatus as indicated by premature defoliation in the sensitive clone, and/or the prolonged reduced rates of photosynthesis as observed in this study. After comparing the amount of foliar injury and the degree of photosynthetic inhibition resulting from 0, exposure on intermediately sensitive and insensitive eastern white pines, it appears that changes in these two responses are not necessarily coincidental with each other. This is especially evident in biomass production observed in the O,-treated insensitive clone where inhibition of photosynthesis occurred without concurrent significant visible foliar injury. Reductions in various parameters of plant growth resulting from long-term exposures to 0, concentrations below 0.25 pL/L have been reported in agricultural and forest plant species. Various studies have shown that prolonged inhibition of net photosynthesis due to 0, exposure could result in decreased photosynthetic efficiency and reduction of plant biomass production ( 4 , 7, 13,1.4).
478 414 4 54c 478 45OC 44 ZC 46gC 46 ZC Symptoms o n injured control at p Q 0.05 level
Manning et al. (13)exposed emerging pinto bean seedlings to 0.10-0.15 pL/L 0, for 8 h daily for 28 days. They reported that 0,-treated plants began to exhibit height reductions as early as 7 days after initial exposure and that plants in filtered air were 2-3 times taller and produced leaves twice as large as those exposed to 0,. Also, fresh and dry weights of shoots of 0,-treated plants were found to be significantly reduced compared to the control plants. In field conditions, pollutant-sensitive trees have been observed with less annual increment growth than pollutant-insensitive trees (9,15,16). These findings are all in agreement with the results of this study. The differential biomass production of eastern white pine in response to 0, exposure observed in this study and in previous studies could be summarized as resulting from one or more of the following mechanisms: (1)different sensitivity of photosynthesis and dark respiration to 0, exposure; (2) different degree of recovery of affected plant metabolisms from 0, stress (8); (3) different amount of injury on assimilatory apparatus induced by 0, fumigation. Registry No. Os, 10028-15-6.
Literature Cited (1) (2) (3) (4)
Berry, C. R. Can. J . For. Res. 1973, 3, 543-547. Heagle, A. S. Environ. Pollut. 1979, 19, 1-10, Heck, W. W. Annu. Rev. Phytopathol. 1968,6, 165-188. Mann, L. K.; McLaughlin, S. B.; Shriner, D. S. Environ. Exp. Bot. 1980,20, 99-105. (5) Nicholson, C. R. M.S. Thesis, Virginia Polytechnic Institute and State University, Blacksburg, VA, 1977. (6) Tingey, D. T. Am. Chem. SOC.Symp. Ser. 1974,3,40-57. (7) Coyne, P. I.; Bingham, G. E. For. Sci. 1980,26, 114-119. (8) Yang, Y. S.; Skelly, J. M.; Chevone, B. I.; Birch, J. B. Enviorn. Int., in press. (9) Phillips, S. 0.; Skelly, J. M.; Burkhart, H. E. Phytopathology 1977, 67, 721-725. (10) Yang, Y. S.; Skelly, J. M.; Chevone, B. I. Can. J . For. Res. 1982,12, 803-806. (11) Yang, Y. S.; Skelly, J. M.; Chevone, B. I. Phytopathology, in press. (12) Heck, W. W.; Philbeck, R. B.; Dunning, J. A. Agricultural Research Series, No. 181. U.S. Government Printing Office. Washington, DC, 1978. (13) Manning, W. J.; Feder, W. A.; Papia, P. M.; Perkins, I. Environ. Pollut. 1971, 1 , 305-312. (14) Taylor, 0. C. “Photochemical Oxidant Air Pollution Effects on a Mixed Conifer Forest Ecosystem”; final report, US EPA, Corvallis, OR, 1977. (15) Benoit, L. F. M.S. Thesis, Virginia Polytechnic Institute and State University, Blacksburg, VA, 1981. (16) Vins, B.; Mrkva, R. Acta. Univ. Agric. Ser. C. (Brno)1973, 42, 25-46. Received for review June 4,1982. Revised manuscript received January 17,1983. Accepted February 23,1983. We thank the United States Army Research Officefor funds provided to this project (Grant DAAG 29-78-G-0151). Environ. Sci. Technol., Vol. 17, No. 6, 1983
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