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Responses of subterranean clover and ryegrass to sulphur dioxide under field conditions
Environmental Pollution (Series .4) 36 (1984) 239-249
Responses of Subterranean Clover and Ryegrass to Sulphur Dioxide under Field Conditions Frank Murray Department of Biological Sciences, University of Newcastle, NSW 2308, Australia
A BS TRA C T Subterranean clover Trifolium subterraneum L. cv. Woogenellup and perennial ryegrass Lolium perenne L. cv. Tetralite were exposed to S O 2 m open-top chambers at mean ambient concentrations oJ 164, 70 or 19 tag m - 3j o r 48 days. Exposure to S 0 2 had no effect on calorific value or ash concentration in subterranean clover or ryegrass, but exposure to 164 lag m - 3 oJ S O 2 decreased lea,[ protein concentrations in both species. Chlorophyll concentrations were decreased by exposure to 164 and 70 tag m - 3 oJ S O 2 in subterranean clover but not in ryegrass. Peroxidase activity increased with increasing S O z concentration and duration oJ exposure in subterranean clover, but not in ryegrass. Specific peroxidase activity was increased in both species by exposure to 164 lag m - 3 oJ S O 2 only. R o o t and shoot dry weights were not affected by exposure to S O 2.
I N T R O D U C T I ON A considerable amount of evidence has recently been accumulated indicating that foliage yield of some grasses can be decreased by exposure to low concentrations of SO 2 for long durations. Differences exist between species, between cultivars, and within cultivars of a single species. The response of a plant to SO2 varies with exposure factors, including the concentration, duration and pattern of exposure to SO 2. It has been shown that light and temperature (Jones & Mansfield, 1982), relative humidity (McLaughlin & Taylor, 1980), soil moisture and mineral nutrition (Guderian, 1977) modify the response of plants to SO 2. Other factors which may increase the sensitivity of plants to SO 2 include 239 Environ. Pollut. Set..4. 0143-1471/84/$03.00 ,~('~Elsevier Applied Science Publishers Ltd,
England, 1984. Printed in Great Britain
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increased windspeed (Ashenden & Mansfield, 1977), early stage of development of plants (Bell, 1982; Whitmore & Mansfield, 1983), the presence of other air pollutants (Ormrod, 1982), factors responsible for slow growth (Bell et al., 1979; Davies, 1980; Jones & Mansfield, 1982) and sward development (Whitmore & Mansfield, 1983). Generally, plants appear to be particularly sensitive during winter conditions (Davies, 1980; Baker et al., 1982; Colvill et al., 1983). However, grasses also possess considerable physiological plasticity, enabling them to counteract the effects of an SO2-induced reduction in photosynthetic efficiency by compensatory growth mechanisms, possibly involving changes in assimilate partitioning (Whitmore & Mansfield, 1983). For these reasons, major discrepancies exist among studies, even when the same parameter is measured in the same species. For example, although decreased growth was found in Lolium perenne cv. $23 after exposure to 69/~g m - 3 of SO 2 for 84 days (Crittenden & Read, 1978) and 43/~gm-3 for 173 days (Bell et al., 1979), no effect on growth was found when the same cultivar was exposed to 400/~g m - 3 for 51 days (Cowling & Koziol, 1978). Consequently, attempts to define a threshold concentration of SO 2 for chronic injury in plants must be interpreted strictly in terms of the environmental and exposure conditions at the time of the experiment. Most experimental studies involving the exposure of forage and pasture crops to SO 2 have used high SO 2 concentrations in shortduration experiments, or, when realistic exposure conditions have been simulated, the climatic conditions have usually been cool-temperate. Very little information is available which enables the response to SO 2 of pasture and fodder crops grown in warm-temperate regions to be evaluated. Consequently, this study was initiated to characterise the effects of SO 2 on two important cultivars of TriJolium subterraneum and Lolium perenne, with particular attention to parameters of economic importance. In an attempt to ensure realistic experimental conditions, plants were exposed to low concentrations of SO 2 for relatively long periods in open-top chambers, which ensure near-ambient temperature, humidity, rainfall, light and soil conditions (Heagle et al., 1979). MATERIALS A N D METHODS Six open-top chambers (Heagle et al., 1973), each 3 m in diameter and 2.4 m tall, were operated at Tomago, New South Wales. Each chamber
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241
consisted of a rigid aluminium frame, the top half of which was covered by a single layer of PVC and the lower half by a double thickness PVC envelope with the inner layer perforated by holes 25 mm in diameter. Air was drawn by a fan through a dust filter, along a duct, into the chamber through the holes in the lower PVC envelope, and then out of the chamber through the open top. Dry air was mixed with bottled, anhydrous SO 2 from a temperaturecontrolled cylinder and the mixture was passed through a regulator and a series of needle valves into the inlet of the fumigated chambers. No SO 2 was added to control chambers. The concentration of SO 2 in each chamber was measured for 10 min each hour using a timer-controlled electrical sequencer in conjunction with solenoid valves. The SO 2 concentration was measured using a ThermoElectron series 43 Pulsed Fluorescent Ambient SO 2 analyser, calibrated using a ThermoElectron model 145 calibrator. Plants were exposed to SO 2 for 48 days from 16 May to 2 July 1983, incorporating the late autumn and early winter period. Plants were exposed to mean (_+ standard deviation) SO 2 concentrations of 164 ( _+82), 70 ( _+40) or 19 ( _+ 15) pg m - 3, designated high, low and control SO 2 treatment respectively, with each treatment duplicated. The values for 5, 50 and 95 percentiles, respectively, were 52, 174 and 275 pg m - 3 for the high SO 2 treatment, 23, 58 and 141 pg m - 3 for the low SO 2 treatment and 0.5, 17 and 44/~gm -3 for the control treatment. Measurements of temperature and relative humidity using thermohygrographs in Stevenson screens showed that during the experimental period the daily mean, maximum and minimum temperatures outside of the chambers were 13.4, 17.6 and 10.2°C, respectively, and that the daily mean, maximum and minimum relative humidities were 75.8, 91-3 and 57"4~0, respectively. Mean temperature was I°C higher, and relative humidity was 2Y/o lower, inside the chambers than outside. Rainfall, measured at Williamtown airport (12 km north east of the site), totalled 151 mm during the experimental period. Lolium perenne L. cv Tetralite and TriJolium subterraneum L. cv Woogenellup, which are important winter pasture crops, were grown from seed in jiffy pots containing a mixture of equal amounts of river loam and coarse sand. The Trifolium subterraneum seed was inoculated with a commercial inoculum (Root Nodules, Sydney). They were fertilized fortnightly with Hoagland's solution and maintained in a glasshouse from 16 November 1982 to 25 March 1983. They were then
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transplanted into the chambers with minimal root disturbance and allowed to establish for 52 days before fumigation commenced. Analysis of soil (twelve samples) within the chambers indicated mean pH (1:2 CaCI2, 0.01M) 6"0, conductivity 0 : 2 ) 7 m S c m -1, Bray phosphate 86 mg k g - 1, and the exchangeable cations (in me per 100 g) Ca, 5.5; Mg, 2.0; 0.15; and Na, 0.15. The plants were watered as required to prevent wilting. One newly mature leaf per plant from each of twenty-four plants was sampled and pooled for determinations of calorific value, peroxidase activity and ash, protein and chlorophyll concentrations, ensuring that no peripheral plants were used. For the measurement of calorific value, leaf samples were dried at 80 °C for 24 h, ground to pass a 40 mesh in a Wiley hammer mill and pressed into a pellet which was ignited in an adiabatic oxygen bomb calorimeter (Gallenkamp, London). Protein concentration was measured by the method of Lowry et al. (1951), chlorophyll concentration followed Bruinsma (1963) and the method for peroxidase activity was based on the technique of Horsman & Wellburn (1975). Ash concentration was measured by the procedures of the AOAC (1965) and all analyses used six replicates per treatment. At the end of the experiment, twenty plants from each treatment were harvested and the dry weights of roots and shoots were measured after washing to remove soil particles and drying at 80 °C for 24 h. Analysis of variance was performed on the data and significant differences between means were identified by Duncan's multiple range test (Steele & Torrie, 1960).
RESULTS Subterranean clover began to show chlorotic symptoms after about 25 days of exposure to the high SO 2 treatment and a few days later in the low SO 2 treatment, but SO2-induced leaf chiorosis was virtually absent in ryegrass in both SO 2 treatments. Exposure to SO 2 had no effect on the calorific value or ash concentration in subterranean clover or ryegrass but both SO 2 treatments decreased the leaf protein concentration of subterranean clover, and the high SO 2 treatment decreased leaf protein concentration in ryegrass (Table 1). Chlorophyll a and b concentrations were lowered by exposure
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243
TABLE 1
The Effect of Sulphur Dioxide on Calorific Value, Leaf Protein and Ash Concentration in Subterranean Clover and Ryegrass Calorific value ( k J g - 1 dry weight)
High SO 2 Low SO 2 Control
Ash concentration ( ° o)
Protein concentration (rag g - 1Jresh weight)
Clorer
Ryegrass
Clover
Ryegrass
Clorer
Ryegrass
16.9a'I" 17-0a 16.2a
17.3a 17.1 a 17-4a
16.5a 15.7a 21.8a
10.0a l l.5a 12.0a
25.3a 28.7a 35.1b
6-84a 8-05ab 9.04b
.I- Means separation in columns by Duncan's multiple range test, 5 ,°,,o probability level. to b o t h S O 2 t r e a t m e n t s in s u b t e r r a n e a n clover but neither t r e a t m e n t affected c h l o r o p h y l l c o n c e n t r a t i o n s in ryegrass ( T a b l e 2). G e n e r a l l y , p e r o x i d a s e activity was increased by e x p o s u r e to S O 2 in s u b t e r r a n e a n clover and the r e s p o n s e was e n h a n c e d by increases in S O 2 c o n c e n t r a t i o n and d u r a t i o n o f e x p o s u r e , but S O 2 had no significant effect on p e r o x i d a s e activity in ryegrass ( T a b l e 3). H o w e v e r , S O z increased specific p e r o x i d a s e activity in b o t h s u b t e r r a n e a n clover and ryegrass in the high S O 2 t r e a t m e n t . This a p p a r e n t d i s c r e p a n c y m a y be explained by the S O z - i n d u c e d decrease in the p r o t e i n c o n c e n t r a t i o n o f leaves o f s u b t e r r a n e a n clover a n d ryegrass. R o o t and s h o o t d r y weights were not affected by e x p o s u r e to S O 2 ( T a b l e 4). TABLE 2
The Effects of Sulphur Dioxide on Chlorophyll a and b Concentrations in Subterranean Clover and Ryegrass Chlorophyll concentration (rag g - 1Jresh weight) Clot,er
High SO_, Low SO z Control
R.vegrass
a
b
a
b
2"00a'l" 2-09a 2.34b
0.67a 0.69a 0.80b
1.19a 1.22a 1-16a
0.70a 0.71a 0'67a
•t- Means separation in columns by Duncan's multiple range test, 5 ..... ~o probability level.
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Frank Murray TABLE 3
The Effects of Sulphur Dioxide on Peroxidase Activity in Subterranean Clover and Ryegrass Peroxidase activity AA rain- 1 g- l Jresh weight
Clorer
Days after fumigation commenced High SO 2
Low SO 2 Control
26 726at 456b 368b
Specific peroxidase activity AA rain " l rag- 1 protein
Ryegrass
38 1 180a 758b 463c
30 166a 144a 143a
45 172a 155a 140a
Clover
Ryegrass
38 51.0a 21.6b 16.1b
45 25.1a 19.3b 16.6b
t Means separation in columns by Duncan's multiple range test, 5 ~o probability level. TABLE 4
The Effects of SO 2 on Root and Shoot Dry Weights in Subterranean Clover and Ryegrasst Dry weight (g) Clover Ryegrass
High SO 2 Low SO 2 Control
Root
Shoot
Root
Shoot
1"80 1.77 1.81
24"5 26"7 28"6
4'50 3"69 3'65
15'4 17'8 16.0
t No significant difference between treatments by Duncan's multiple range test, 5 ~,,,probability level.
DISCUSSION T h e r e s p o n s e s o f s u b t e r r a n e a n clover a n d r y e g r a s s t o S O 2 were f o u n d to differ, as p e r o x i d a s e activity w a s increased, a n d leaf c h l o r o p h y l l c o n c e n t r a t i o n w a s d e c r e a s e d , by S O 2 in s u b t e r r a n e a n clover, b u t n o t in ryegrass. P r e v i o u s studies ( M a l h o t r a , 1977; L a u e n r o t h & D o d d , 1981) h a v e s h o w n t h a t leaf c h l o r o p h y l l c o n c e n t r a t i o n is sensitive to SO2, a n d a l t h o u g h there are i m p l i c a t i o n s for S O 2 effects on p h o t o s y n t h e s i s a n d
Responses oJ clover and ryegrass to SO 2
245
hence plant productivity, and SO 2- induced reductions in leaf chlorophyll concentration and photosynthesis have been found to be highly correlated (Saxe, 1983), other explanations of short-term responses of photosynthesis to SO 2 appear more likely (Black, 1982). The induction by SO 2 of increases in peroxidase activity in plants has been reported previously (e.g. Horsman & Wellburn, 1975; Khan & Malhotra, 1982; Pierre & Queiroz, 1982). The present study found that SO 2- induced increases in peroxidase activity were statistically significant (p < 0.05) in subterranean clover but not in ryegrass. However, SO2 induced statistically significant increases (p < 0-05) in specific peroxidase activity in both species, suggesting that specific peroxidase activity may represent a more sensitive indicator of subtle effects of SO 2 on plants. However, Saxe (1983) found that the opposite was the case in beans, as both soluble protein concentration and peroxidase activity increased with SO 2 concentration, making the effect of SO 2 on specific peroxidase activity insignificant. A number of studies have reported an SO2-induced increase in plant senescence which may be characterised by an increase in ethylene evolution (Bucher, 1981) or increased peroxidase activity (Khan & Malhotra, 1982; Pierre & Queiroz, 1982) before visible injury symptoms appear. Plant senescence is also associated with increases in the rate of breakdown of chlorophyll and proteins, and the accumulation of free amino acids (Birecka et al., 1979), processes which have been measured after SO 2 fumigation (Godzik & Linskens, 1974; Lauenroth & Dodd, 1981). Spinach plants accumulate hydrogen peroxide during SO 2 fumigation (Tanaka et al., 1982a) and hydrogen peroxide produced in chloroplasts may suppress photosynthesis during SO 2 fumigation by the reversible inhibition of chloroplast SH enzymes (Tanaka et al., 1982b). Thus, peroxidase activity may represent an induced protective reaction in delaying the effects of SO 2 by eliminating hydrogen peroxide. Peroxidase is thought to have a r61e in stabilising chlorophyll concentrations (Rudolph & Bukatsch, 1967) and it may be involved in the oxidation of SO ] - and HSO 3 to the less toxic SO 2- at sites other than the chloroplast (Horsman & Wellburn, 1975). The absence of any effects of SO 2 on calorific value or ash concentration in subterranean clover and ryegrass is in contrast with some other studies which have shown that SO 2 increased the ash concentration in western wheatgrass (Milchunas et al., 1981) and reduced the calorific value of soybean and wheat (Prasad & Rao, 1982). A decrease
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in foliar protein concentration after exposure to SO 2 has been observed in a number of studies (Godzik & Linskens, 1974; Prasad & Rao, 1982) although S~.rdi (1981) reported that exposure to 150/~gm -3 of SO 2 increased the protein content of both soybean and pea by the stimulation of the synthesis of amino acids containing sulphur, but exposure to 500 and 1000 ~g m - 3 of SO 2 had inhibitory effects on protein synthesis, and Saxe (1983) found that the soluble protein increased in bean plants exposed to SO 2. If leguminous plants are generally more sensitive to SO 2 than grasses, as Prasad & Rao (1982) suggested, the impact of SO 2 on mixed pastures may be a decrease in the ability of SO2-sensitive clover plants to compete with grasses, resulting in a shift of pasture dominance towards grasses. Such a vegetation change might be enhanced by the presence of other pollutants since SO 2 is rarely the only pollutant in the atmosphere, and the presence of 0 3 has been shown to induce clover decline under field conditions in white clover-tall fescue pastures (Blum et al., 1980; Montes et al., 1982). This would generally be viewed as detrimental to forage quality as carbohydrate, mineral and protein concentrations are usually higher in clover than grasses. However, in practical terms, the r61e of other environmental factors, including interactions with mineral nutrition, disease and sward structure, may modify or override the responses of mixed pastures to SO 2. In addition, plant tolerance to SO 2 may develop within 17-25 years in grasses (Ayazloo & Bell, 1981) and possibly in other plants (Taylor & Murdy, 1975). ACKN O W L E D G E M ENTS The assistance of John Clancy, Colin Freund, Marina Ivinskis and David Roshier is gratefully acknowledged. The Tomago Aluminium Company Pty Ltd provided a site, power and water for this project, and, in particular, the help of Graham Taylor is appreciated. Funding for the project was provided by the National Energy Research, Development and Demonstration Council of the Australian Department of Resources and Energy. REFERENCES AOAC (1965). 01~cial methods oJ analysis, 10th edn. Washington, DC, Association of Official Analytical Chemists.
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Ashenden, T. W. & Mansfield, T. A. (1977). Influence of windspeed on the sensitivity of ryegras to SO 2. J. exp. Bot., 28, 729-35. Ayazloo, M. & Bell, J. N. B. (1981). Studies on the tolerance to sulphur dioxide of grass populations in polluted areas. 1. Identification of tolerant populations. New Phytol., 88, 203-22. Baker, C. K., Unsworth, M. H. & Greenwood, P. (1982). Leaf injury on wheat plants exposed in the field in winter to SO 2. Nature, Lond., 299, 149-51. Bell, J. N. B. (1982). Sulphur dioxide and the growth of grasses. In Effects oj gaseous air pollution in agriculture and horticulture, ed. by M. H. Unsworth and D. P. Ormrod, 225-46. London, Butterworth. Bell, J. N. B., Rutter, A. J. & Relton, J. (1979). Studies on the effects of low levels of sulphur dioxide on the growth of Loliurn perenne L. New Phytol., 83, 627-43. Birecka, H., Chaskes, M. J. & Goldstein, J. (1979). Peroxidase and senescence. J. exp. Bot., 30, 565-73. Black, V. J. (1982). Effects of sulphur dioxide on physiological processes in plants. In Effects of gaseous air pollution in agriculture and horticulture, ed. by M. H. Unsworth and D. P. Ormrod, 67-91. London, Butterworth. Blum, U., Heagle, A. S. & Burns, J. C. (1980). Effects ofO 3 on the yield of a tall fescue-ladino clover pasture. Bull. ecol. Soc. Am., 61, 125 (Abstract). Bruinsma, J. (1963). The quantitative analysis of chlorophylls a and b in plant extracts. Photochem. Photobiol., 2, 241-9. Bucher, J. B. (1981). S O2-induced ethylene evolution of forest tree foliage and its potential use as stress-indicator. Eur. J. Forest Pathol., 11, 369-73. Colvill, K. E., Bell, R. M., Roberts, T. M. & Bradshaw, A. D. (1983). The use of open-top chambers to study the effects of air pollutants, in particular sulphur dioxide, on the growth of ryegrass Lolium perenne L., Part II. The long-term effect of filtering polluted urban air or adding SO 2 to rural air. Era,iron. Pollut. Ser. A., 31, 35-55. Cowling, D. & Koziol, M. (1978). Growth of ryegrass (Lolium perenne L.) exposed to SO 2. 1. Effects on photosynthesis and respiration. J. exp. Bot., 29, 1029-36. Crittenden, P. D. & Read, D. J. (1978). The effects of air pollution on plant growth with special reference to sulphur dioxide. II. Growth studies with Loliurn perenne L. New Phytol., 80, 49-62. Davies, T. (1980). Grasses more sensitive to SO 2 pollution in conditions of low irradiance and short days. Nature, Lond., 284, 483-5. Godzik, S. & Linskens, H. F. (1974). Concentration changes of free amino acids in primary bean leaves after continuous and interrupted SO 2 fumigation and recovery. Environ. Pollut., 7, 25-38. Guderian, R. (1977). Air pollution. Berlin, Springer Verlag. Heagle, A, S., Body, D. E. & Heck, W. W. (1973). An open-top field chamber to assess the impact of air pollution on plants. J. environ. Qual., 2, 365-8. Heagle, A. S., Philbeck, R. B., Rogers, H. H. & Letchworth, M, B. (1979). Dispensing and monitoring ozone in open-top field chambers for planteffects studies. Phytopathology, 69, 15-20.
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Horsman, D. C. & Wellburn, A. R. (1975). Synergistic effect of SO z and NO 2 polluted air upon enzyme activity in pea seedlings. Environ. Pollut., 8, 123-33. Jones, T. & Mansfield, T. A. (1982). The effects of SO 2 on growth and development of seedlings of Phleum pratense under different light and temperature environments. Environ. Pollut. Ser. A., 27, 57-71. Khan, A. A. & Malhotra, S. S. (1982). Peroxidase activity as an indicator of SO 2 injury in jack pine and white birch. Biochem. Physiol. Pflanzen, 177, 643-50. Lauenroth, W. K. & Dodd, J. L. (1981). Chlorophyll reduction in western wheatgrass (Agropyron smithii Rybd.) exposed to sulfur dioxide. Water, Air Soil Pollut., 15, 309- 15. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). Protein measurement with the folin phenol reagent. J. biol. Chem., 193, 265-75. McLaughlin, S. B. & Taylor, G. E. (1980). Relative humidity--Important modifier of pollutant uptake by plants. Science, N.Y., 211, 167-9. Malhotra, S. S. (1977). Effects of aqueous sulphur dioxide on chlorophyll destruction in Pinus contorta. New Phytol., 78, 101-9. Milchunas, D. G., Lauenroth, W. K., Dodd, J. L. & McNary, T. J. (1981). Effects of SO 2 exposure with nitrogen and sulphur fertilization on the growth of Agropyron smithii. J. appl. Ecol., 18, 291-302. Montes, R. A., Blum, U. & Heagle, A. S. (1982). The effects of ozone and nitrogen fertilizer on tall fescue, ladino clover, and a fescue-clover mixture. 1. Growth, regrowth and forage production. Can. J. Bot., 60, 2745-52. Ormrod, D. P. (1982). Air pollutant interactions in mixtures. In Effects oj gaseous air pollution in agriculture and horticulture, ed. by M. H. U nsworth and D. P. Ormrod, 307-31. London, Butterworth. Pierre, M. & Queiroz, O. (1982). Modulation by leafage and SO 2 concentration of the enzymic response to subnecrotic SO 2 pollution. Ent'iron. Pollut. Set. A., 28, 209-17. Prasad, B. J. & Rao, D. N. (1982). Relative sensitivity of a leguminous and a cereal crop to sulphur dioxide pollution. Era'iron. Pollut. Set. A., 29, 57- 70. Rudolph, E. & Bukatsch, F. (1967). Die Bedeutung von Askorbins/iure, Katalase und Peroxidase fiir die Stabilisierung des Chlorophylls bei hohen Lichtintensit/iten. Flora, Jena, A158, 443-57. S/lrdi, K. (1981). Changes in the soluble protein content of soybean (Glycme max L.) and pea (Pisum sativum L.) under continuous SO 2 and soot pollution. Environ. Pollut. Ser. A., 25, 181-6. Saxe, H. (1983). Long-term effects of low levels of SO 2 on bean plants (Phaseolus vulgaris). II. Immission-response effects on biomass production: Quantity and quality. Physiologia PI., 57, 108-13. Steele, R. D. & Torrie, J. M. (1960). Principles and procedures oJ statistics. New York, McGraw-Hill. Tanaka, K., Kondo, N. & Sugahara, K. (1982a). Accumulation of hydrogen peroxide in chloroplasts of SOz-fumigated spinach leaves. Plant Cell Physiol., 23, 999-1007.
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Tanaka, K., Otsubo, T. & Kondo, N. (1982b). Participation of hydrogen peroxide in the inactivation of Calvin-cycle SH enzymes in SO 2 fumigated spinach leaves. Plant Cell Physiol., 23, 1009-18. Taylor, G. E. & Murdy, W. H. (1975). Population differentiation of an annual plant species, Geranium carolinianum, in response to sulfur dioxide. Bot. Gaz., 136, 212-5. Whitmore, M. E. & Mansfield, T. A. (1983). Effects of long-term exposure to SOz and N 0 2 on Poa pratensis and other grasses. Era'iron. Pollut. Set. A., 31, 217-35.
Responses of Subterranean Clover and Ryegrass to Sulphur Dioxide under Field Conditions Frank Murray Department of Biological Sciences, University of Newcastle, NSW 2308, Australia
A BS TRA C T Subterranean clover Trifolium subterraneum L. cv. Woogenellup and perennial ryegrass Lolium perenne L. cv. Tetralite were exposed to S O 2 m open-top chambers at mean ambient concentrations oJ 164, 70 or 19 tag m - 3j o r 48 days. Exposure to S 0 2 had no effect on calorific value or ash concentration in subterranean clover or ryegrass, but exposure to 164 lag m - 3 oJ S O 2 decreased lea,[ protein concentrations in both species. Chlorophyll concentrations were decreased by exposure to 164 and 70 tag m - 3 oJ S O 2 in subterranean clover but not in ryegrass. Peroxidase activity increased with increasing S O z concentration and duration oJ exposure in subterranean clover, but not in ryegrass. Specific peroxidase activity was increased in both species by exposure to 164 lag m - 3 oJ S O 2 only. R o o t and shoot dry weights were not affected by exposure to S O 2.
I N T R O D U C T I ON A considerable amount of evidence has recently been accumulated indicating that foliage yield of some grasses can be decreased by exposure to low concentrations of SO 2 for long durations. Differences exist between species, between cultivars, and within cultivars of a single species. The response of a plant to SO2 varies with exposure factors, including the concentration, duration and pattern of exposure to SO 2. It has been shown that light and temperature (Jones & Mansfield, 1982), relative humidity (McLaughlin & Taylor, 1980), soil moisture and mineral nutrition (Guderian, 1977) modify the response of plants to SO 2. Other factors which may increase the sensitivity of plants to SO 2 include 239 Environ. Pollut. Set..4. 0143-1471/84/$03.00 ,~('~Elsevier Applied Science Publishers Ltd,
England, 1984. Printed in Great Britain
240
Frank Murray
increased windspeed (Ashenden & Mansfield, 1977), early stage of development of plants (Bell, 1982; Whitmore & Mansfield, 1983), the presence of other air pollutants (Ormrod, 1982), factors responsible for slow growth (Bell et al., 1979; Davies, 1980; Jones & Mansfield, 1982) and sward development (Whitmore & Mansfield, 1983). Generally, plants appear to be particularly sensitive during winter conditions (Davies, 1980; Baker et al., 1982; Colvill et al., 1983). However, grasses also possess considerable physiological plasticity, enabling them to counteract the effects of an SO2-induced reduction in photosynthetic efficiency by compensatory growth mechanisms, possibly involving changes in assimilate partitioning (Whitmore & Mansfield, 1983). For these reasons, major discrepancies exist among studies, even when the same parameter is measured in the same species. For example, although decreased growth was found in Lolium perenne cv. $23 after exposure to 69/~g m - 3 of SO 2 for 84 days (Crittenden & Read, 1978) and 43/~gm-3 for 173 days (Bell et al., 1979), no effect on growth was found when the same cultivar was exposed to 400/~g m - 3 for 51 days (Cowling & Koziol, 1978). Consequently, attempts to define a threshold concentration of SO 2 for chronic injury in plants must be interpreted strictly in terms of the environmental and exposure conditions at the time of the experiment. Most experimental studies involving the exposure of forage and pasture crops to SO 2 have used high SO 2 concentrations in shortduration experiments, or, when realistic exposure conditions have been simulated, the climatic conditions have usually been cool-temperate. Very little information is available which enables the response to SO 2 of pasture and fodder crops grown in warm-temperate regions to be evaluated. Consequently, this study was initiated to characterise the effects of SO 2 on two important cultivars of TriJolium subterraneum and Lolium perenne, with particular attention to parameters of economic importance. In an attempt to ensure realistic experimental conditions, plants were exposed to low concentrations of SO 2 for relatively long periods in open-top chambers, which ensure near-ambient temperature, humidity, rainfall, light and soil conditions (Heagle et al., 1979). MATERIALS A N D METHODS Six open-top chambers (Heagle et al., 1973), each 3 m in diameter and 2.4 m tall, were operated at Tomago, New South Wales. Each chamber
Responses oJ clorer and ryegrass to SO 2
241
consisted of a rigid aluminium frame, the top half of which was covered by a single layer of PVC and the lower half by a double thickness PVC envelope with the inner layer perforated by holes 25 mm in diameter. Air was drawn by a fan through a dust filter, along a duct, into the chamber through the holes in the lower PVC envelope, and then out of the chamber through the open top. Dry air was mixed with bottled, anhydrous SO 2 from a temperaturecontrolled cylinder and the mixture was passed through a regulator and a series of needle valves into the inlet of the fumigated chambers. No SO 2 was added to control chambers. The concentration of SO 2 in each chamber was measured for 10 min each hour using a timer-controlled electrical sequencer in conjunction with solenoid valves. The SO 2 concentration was measured using a ThermoElectron series 43 Pulsed Fluorescent Ambient SO 2 analyser, calibrated using a ThermoElectron model 145 calibrator. Plants were exposed to SO 2 for 48 days from 16 May to 2 July 1983, incorporating the late autumn and early winter period. Plants were exposed to mean (_+ standard deviation) SO 2 concentrations of 164 ( _+82), 70 ( _+40) or 19 ( _+ 15) pg m - 3, designated high, low and control SO 2 treatment respectively, with each treatment duplicated. The values for 5, 50 and 95 percentiles, respectively, were 52, 174 and 275 pg m - 3 for the high SO 2 treatment, 23, 58 and 141 pg m - 3 for the low SO 2 treatment and 0.5, 17 and 44/~gm -3 for the control treatment. Measurements of temperature and relative humidity using thermohygrographs in Stevenson screens showed that during the experimental period the daily mean, maximum and minimum temperatures outside of the chambers were 13.4, 17.6 and 10.2°C, respectively, and that the daily mean, maximum and minimum relative humidities were 75.8, 91-3 and 57"4~0, respectively. Mean temperature was I°C higher, and relative humidity was 2Y/o lower, inside the chambers than outside. Rainfall, measured at Williamtown airport (12 km north east of the site), totalled 151 mm during the experimental period. Lolium perenne L. cv Tetralite and TriJolium subterraneum L. cv Woogenellup, which are important winter pasture crops, were grown from seed in jiffy pots containing a mixture of equal amounts of river loam and coarse sand. The Trifolium subterraneum seed was inoculated with a commercial inoculum (Root Nodules, Sydney). They were fertilized fortnightly with Hoagland's solution and maintained in a glasshouse from 16 November 1982 to 25 March 1983. They were then
242
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transplanted into the chambers with minimal root disturbance and allowed to establish for 52 days before fumigation commenced. Analysis of soil (twelve samples) within the chambers indicated mean pH (1:2 CaCI2, 0.01M) 6"0, conductivity 0 : 2 ) 7 m S c m -1, Bray phosphate 86 mg k g - 1, and the exchangeable cations (in me per 100 g) Ca, 5.5; Mg, 2.0; 0.15; and Na, 0.15. The plants were watered as required to prevent wilting. One newly mature leaf per plant from each of twenty-four plants was sampled and pooled for determinations of calorific value, peroxidase activity and ash, protein and chlorophyll concentrations, ensuring that no peripheral plants were used. For the measurement of calorific value, leaf samples were dried at 80 °C for 24 h, ground to pass a 40 mesh in a Wiley hammer mill and pressed into a pellet which was ignited in an adiabatic oxygen bomb calorimeter (Gallenkamp, London). Protein concentration was measured by the method of Lowry et al. (1951), chlorophyll concentration followed Bruinsma (1963) and the method for peroxidase activity was based on the technique of Horsman & Wellburn (1975). Ash concentration was measured by the procedures of the AOAC (1965) and all analyses used six replicates per treatment. At the end of the experiment, twenty plants from each treatment were harvested and the dry weights of roots and shoots were measured after washing to remove soil particles and drying at 80 °C for 24 h. Analysis of variance was performed on the data and significant differences between means were identified by Duncan's multiple range test (Steele & Torrie, 1960).
RESULTS Subterranean clover began to show chlorotic symptoms after about 25 days of exposure to the high SO 2 treatment and a few days later in the low SO 2 treatment, but SO2-induced leaf chiorosis was virtually absent in ryegrass in both SO 2 treatments. Exposure to SO 2 had no effect on the calorific value or ash concentration in subterranean clover or ryegrass but both SO 2 treatments decreased the leaf protein concentration of subterranean clover, and the high SO 2 treatment decreased leaf protein concentration in ryegrass (Table 1). Chlorophyll a and b concentrations were lowered by exposure
Responses o,I clo~'er and ryegrass to SO 2
243
TABLE 1
The Effect of Sulphur Dioxide on Calorific Value, Leaf Protein and Ash Concentration in Subterranean Clover and Ryegrass Calorific value ( k J g - 1 dry weight)
High SO 2 Low SO 2 Control
Ash concentration ( ° o)
Protein concentration (rag g - 1Jresh weight)
Clorer
Ryegrass
Clover
Ryegrass
Clorer
Ryegrass
16.9a'I" 17-0a 16.2a
17.3a 17.1 a 17-4a
16.5a 15.7a 21.8a
10.0a l l.5a 12.0a
25.3a 28.7a 35.1b
6-84a 8-05ab 9.04b
.I- Means separation in columns by Duncan's multiple range test, 5 ,°,,o probability level. to b o t h S O 2 t r e a t m e n t s in s u b t e r r a n e a n clover but neither t r e a t m e n t affected c h l o r o p h y l l c o n c e n t r a t i o n s in ryegrass ( T a b l e 2). G e n e r a l l y , p e r o x i d a s e activity was increased by e x p o s u r e to S O 2 in s u b t e r r a n e a n clover and the r e s p o n s e was e n h a n c e d by increases in S O 2 c o n c e n t r a t i o n and d u r a t i o n o f e x p o s u r e , but S O 2 had no significant effect on p e r o x i d a s e activity in ryegrass ( T a b l e 3). H o w e v e r , S O z increased specific p e r o x i d a s e activity in b o t h s u b t e r r a n e a n clover and ryegrass in the high S O 2 t r e a t m e n t . This a p p a r e n t d i s c r e p a n c y m a y be explained by the S O z - i n d u c e d decrease in the p r o t e i n c o n c e n t r a t i o n o f leaves o f s u b t e r r a n e a n clover a n d ryegrass. R o o t and s h o o t d r y weights were not affected by e x p o s u r e to S O 2 ( T a b l e 4). TABLE 2
The Effects of Sulphur Dioxide on Chlorophyll a and b Concentrations in Subterranean Clover and Ryegrass Chlorophyll concentration (rag g - 1Jresh weight) Clot,er
High SO_, Low SO z Control
R.vegrass
a
b
a
b
2"00a'l" 2-09a 2.34b
0.67a 0.69a 0.80b
1.19a 1.22a 1-16a
0.70a 0.71a 0'67a
•t- Means separation in columns by Duncan's multiple range test, 5 ..... ~o probability level.
244
Frank Murray TABLE 3
The Effects of Sulphur Dioxide on Peroxidase Activity in Subterranean Clover and Ryegrass Peroxidase activity AA rain- 1 g- l Jresh weight
Clorer
Days after fumigation commenced High SO 2
Low SO 2 Control
26 726at 456b 368b
Specific peroxidase activity AA rain " l rag- 1 protein
Ryegrass
38 1 180a 758b 463c
30 166a 144a 143a
45 172a 155a 140a
Clover
Ryegrass
38 51.0a 21.6b 16.1b
45 25.1a 19.3b 16.6b
t Means separation in columns by Duncan's multiple range test, 5 ~o probability level. TABLE 4
The Effects of SO 2 on Root and Shoot Dry Weights in Subterranean Clover and Ryegrasst Dry weight (g) Clover Ryegrass
High SO 2 Low SO 2 Control
Root
Shoot
Root
Shoot
1"80 1.77 1.81
24"5 26"7 28"6
4'50 3"69 3'65
15'4 17'8 16.0
t No significant difference between treatments by Duncan's multiple range test, 5 ~,,,probability level.
DISCUSSION T h e r e s p o n s e s o f s u b t e r r a n e a n clover a n d r y e g r a s s t o S O 2 were f o u n d to differ, as p e r o x i d a s e activity w a s increased, a n d leaf c h l o r o p h y l l c o n c e n t r a t i o n w a s d e c r e a s e d , by S O 2 in s u b t e r r a n e a n clover, b u t n o t in ryegrass. P r e v i o u s studies ( M a l h o t r a , 1977; L a u e n r o t h & D o d d , 1981) h a v e s h o w n t h a t leaf c h l o r o p h y l l c o n c e n t r a t i o n is sensitive to SO2, a n d a l t h o u g h there are i m p l i c a t i o n s for S O 2 effects on p h o t o s y n t h e s i s a n d
Responses oJ clover and ryegrass to SO 2
245
hence plant productivity, and SO 2- induced reductions in leaf chlorophyll concentration and photosynthesis have been found to be highly correlated (Saxe, 1983), other explanations of short-term responses of photosynthesis to SO 2 appear more likely (Black, 1982). The induction by SO 2 of increases in peroxidase activity in plants has been reported previously (e.g. Horsman & Wellburn, 1975; Khan & Malhotra, 1982; Pierre & Queiroz, 1982). The present study found that SO 2- induced increases in peroxidase activity were statistically significant (p < 0.05) in subterranean clover but not in ryegrass. However, SO2 induced statistically significant increases (p < 0-05) in specific peroxidase activity in both species, suggesting that specific peroxidase activity may represent a more sensitive indicator of subtle effects of SO 2 on plants. However, Saxe (1983) found that the opposite was the case in beans, as both soluble protein concentration and peroxidase activity increased with SO 2 concentration, making the effect of SO 2 on specific peroxidase activity insignificant. A number of studies have reported an SO2-induced increase in plant senescence which may be characterised by an increase in ethylene evolution (Bucher, 1981) or increased peroxidase activity (Khan & Malhotra, 1982; Pierre & Queiroz, 1982) before visible injury symptoms appear. Plant senescence is also associated with increases in the rate of breakdown of chlorophyll and proteins, and the accumulation of free amino acids (Birecka et al., 1979), processes which have been measured after SO 2 fumigation (Godzik & Linskens, 1974; Lauenroth & Dodd, 1981). Spinach plants accumulate hydrogen peroxide during SO 2 fumigation (Tanaka et al., 1982a) and hydrogen peroxide produced in chloroplasts may suppress photosynthesis during SO 2 fumigation by the reversible inhibition of chloroplast SH enzymes (Tanaka et al., 1982b). Thus, peroxidase activity may represent an induced protective reaction in delaying the effects of SO 2 by eliminating hydrogen peroxide. Peroxidase is thought to have a r61e in stabilising chlorophyll concentrations (Rudolph & Bukatsch, 1967) and it may be involved in the oxidation of SO ] - and HSO 3 to the less toxic SO 2- at sites other than the chloroplast (Horsman & Wellburn, 1975). The absence of any effects of SO 2 on calorific value or ash concentration in subterranean clover and ryegrass is in contrast with some other studies which have shown that SO 2 increased the ash concentration in western wheatgrass (Milchunas et al., 1981) and reduced the calorific value of soybean and wheat (Prasad & Rao, 1982). A decrease
246
Frank Murray
in foliar protein concentration after exposure to SO 2 has been observed in a number of studies (Godzik & Linskens, 1974; Prasad & Rao, 1982) although S~.rdi (1981) reported that exposure to 150/~gm -3 of SO 2 increased the protein content of both soybean and pea by the stimulation of the synthesis of amino acids containing sulphur, but exposure to 500 and 1000 ~g m - 3 of SO 2 had inhibitory effects on protein synthesis, and Saxe (1983) found that the soluble protein increased in bean plants exposed to SO 2. If leguminous plants are generally more sensitive to SO 2 than grasses, as Prasad & Rao (1982) suggested, the impact of SO 2 on mixed pastures may be a decrease in the ability of SO2-sensitive clover plants to compete with grasses, resulting in a shift of pasture dominance towards grasses. Such a vegetation change might be enhanced by the presence of other pollutants since SO 2 is rarely the only pollutant in the atmosphere, and the presence of 0 3 has been shown to induce clover decline under field conditions in white clover-tall fescue pastures (Blum et al., 1980; Montes et al., 1982). This would generally be viewed as detrimental to forage quality as carbohydrate, mineral and protein concentrations are usually higher in clover than grasses. However, in practical terms, the r61e of other environmental factors, including interactions with mineral nutrition, disease and sward structure, may modify or override the responses of mixed pastures to SO 2. In addition, plant tolerance to SO 2 may develop within 17-25 years in grasses (Ayazloo & Bell, 1981) and possibly in other plants (Taylor & Murdy, 1975). ACKN O W L E D G E M ENTS The assistance of John Clancy, Colin Freund, Marina Ivinskis and David Roshier is gratefully acknowledged. The Tomago Aluminium Company Pty Ltd provided a site, power and water for this project, and, in particular, the help of Graham Taylor is appreciated. Funding for the project was provided by the National Energy Research, Development and Demonstration Council of the Australian Department of Resources and Energy. REFERENCES AOAC (1965). 01~cial methods oJ analysis, 10th edn. Washington, DC, Association of Official Analytical Chemists.
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Ashenden, T. W. & Mansfield, T. A. (1977). Influence of windspeed on the sensitivity of ryegras to SO 2. J. exp. Bot., 28, 729-35. Ayazloo, M. & Bell, J. N. B. (1981). Studies on the tolerance to sulphur dioxide of grass populations in polluted areas. 1. Identification of tolerant populations. New Phytol., 88, 203-22. Baker, C. K., Unsworth, M. H. & Greenwood, P. (1982). Leaf injury on wheat plants exposed in the field in winter to SO 2. Nature, Lond., 299, 149-51. Bell, J. N. B. (1982). Sulphur dioxide and the growth of grasses. In Effects oj gaseous air pollution in agriculture and horticulture, ed. by M. H. Unsworth and D. P. Ormrod, 225-46. London, Butterworth. Bell, J. N. B., Rutter, A. J. & Relton, J. (1979). Studies on the effects of low levels of sulphur dioxide on the growth of Loliurn perenne L. New Phytol., 83, 627-43. Birecka, H., Chaskes, M. J. & Goldstein, J. (1979). Peroxidase and senescence. J. exp. Bot., 30, 565-73. Black, V. J. (1982). Effects of sulphur dioxide on physiological processes in plants. In Effects of gaseous air pollution in agriculture and horticulture, ed. by M. H. Unsworth and D. P. Ormrod, 67-91. London, Butterworth. Blum, U., Heagle, A. S. & Burns, J. C. (1980). Effects ofO 3 on the yield of a tall fescue-ladino clover pasture. Bull. ecol. Soc. Am., 61, 125 (Abstract). Bruinsma, J. (1963). The quantitative analysis of chlorophylls a and b in plant extracts. Photochem. Photobiol., 2, 241-9. Bucher, J. B. (1981). S O2-induced ethylene evolution of forest tree foliage and its potential use as stress-indicator. Eur. J. Forest Pathol., 11, 369-73. Colvill, K. E., Bell, R. M., Roberts, T. M. & Bradshaw, A. D. (1983). The use of open-top chambers to study the effects of air pollutants, in particular sulphur dioxide, on the growth of ryegrass Lolium perenne L., Part II. The long-term effect of filtering polluted urban air or adding SO 2 to rural air. Era,iron. Pollut. Ser. A., 31, 35-55. Cowling, D. & Koziol, M. (1978). Growth of ryegrass (Lolium perenne L.) exposed to SO 2. 1. Effects on photosynthesis and respiration. J. exp. Bot., 29, 1029-36. Crittenden, P. D. & Read, D. J. (1978). The effects of air pollution on plant growth with special reference to sulphur dioxide. II. Growth studies with Loliurn perenne L. New Phytol., 80, 49-62. Davies, T. (1980). Grasses more sensitive to SO 2 pollution in conditions of low irradiance and short days. Nature, Lond., 284, 483-5. Godzik, S. & Linskens, H. F. (1974). Concentration changes of free amino acids in primary bean leaves after continuous and interrupted SO 2 fumigation and recovery. Environ. Pollut., 7, 25-38. Guderian, R. (1977). Air pollution. Berlin, Springer Verlag. Heagle, A, S., Body, D. E. & Heck, W. W. (1973). An open-top field chamber to assess the impact of air pollution on plants. J. environ. Qual., 2, 365-8. Heagle, A. S., Philbeck, R. B., Rogers, H. H. & Letchworth, M, B. (1979). Dispensing and monitoring ozone in open-top field chambers for planteffects studies. Phytopathology, 69, 15-20.
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Horsman, D. C. & Wellburn, A. R. (1975). Synergistic effect of SO z and NO 2 polluted air upon enzyme activity in pea seedlings. Environ. Pollut., 8, 123-33. Jones, T. & Mansfield, T. A. (1982). The effects of SO 2 on growth and development of seedlings of Phleum pratense under different light and temperature environments. Environ. Pollut. Ser. A., 27, 57-71. Khan, A. A. & Malhotra, S. S. (1982). Peroxidase activity as an indicator of SO 2 injury in jack pine and white birch. Biochem. Physiol. Pflanzen, 177, 643-50. Lauenroth, W. K. & Dodd, J. L. (1981). Chlorophyll reduction in western wheatgrass (Agropyron smithii Rybd.) exposed to sulfur dioxide. Water, Air Soil Pollut., 15, 309- 15. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). Protein measurement with the folin phenol reagent. J. biol. Chem., 193, 265-75. McLaughlin, S. B. & Taylor, G. E. (1980). Relative humidity--Important modifier of pollutant uptake by plants. Science, N.Y., 211, 167-9. Malhotra, S. S. (1977). Effects of aqueous sulphur dioxide on chlorophyll destruction in Pinus contorta. New Phytol., 78, 101-9. Milchunas, D. G., Lauenroth, W. K., Dodd, J. L. & McNary, T. J. (1981). Effects of SO 2 exposure with nitrogen and sulphur fertilization on the growth of Agropyron smithii. J. appl. Ecol., 18, 291-302. Montes, R. A., Blum, U. & Heagle, A. S. (1982). The effects of ozone and nitrogen fertilizer on tall fescue, ladino clover, and a fescue-clover mixture. 1. Growth, regrowth and forage production. Can. J. Bot., 60, 2745-52. Ormrod, D. P. (1982). Air pollutant interactions in mixtures. In Effects oj gaseous air pollution in agriculture and horticulture, ed. by M. H. U nsworth and D. P. Ormrod, 307-31. London, Butterworth. Pierre, M. & Queiroz, O. (1982). Modulation by leafage and SO 2 concentration of the enzymic response to subnecrotic SO 2 pollution. Ent'iron. Pollut. Set. A., 28, 209-17. Prasad, B. J. & Rao, D. N. (1982). Relative sensitivity of a leguminous and a cereal crop to sulphur dioxide pollution. Era'iron. Pollut. Set. A., 29, 57- 70. Rudolph, E. & Bukatsch, F. (1967). Die Bedeutung von Askorbins/iure, Katalase und Peroxidase fiir die Stabilisierung des Chlorophylls bei hohen Lichtintensit/iten. Flora, Jena, A158, 443-57. S/lrdi, K. (1981). Changes in the soluble protein content of soybean (Glycme max L.) and pea (Pisum sativum L.) under continuous SO 2 and soot pollution. Environ. Pollut. Ser. A., 25, 181-6. Saxe, H. (1983). Long-term effects of low levels of SO 2 on bean plants (Phaseolus vulgaris). II. Immission-response effects on biomass production: Quantity and quality. Physiologia PI., 57, 108-13. Steele, R. D. & Torrie, J. M. (1960). Principles and procedures oJ statistics. New York, McGraw-Hill. Tanaka, K., Kondo, N. & Sugahara, K. (1982a). Accumulation of hydrogen peroxide in chloroplasts of SOz-fumigated spinach leaves. Plant Cell Physiol., 23, 999-1007.
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Tanaka, K., Otsubo, T. & Kondo, N. (1982b). Participation of hydrogen peroxide in the inactivation of Calvin-cycle SH enzymes in SO 2 fumigated spinach leaves. Plant Cell Physiol., 23, 1009-18. Taylor, G. E. & Murdy, W. H. (1975). Population differentiation of an annual plant species, Geranium carolinianum, in response to sulfur dioxide. Bot. Gaz., 136, 212-5. Whitmore, M. E. & Mansfield, T. A. (1983). Effects of long-term exposure to SOz and N 0 2 on Poa pratensis and other grasses. Era'iron. Pollut. Set. A., 31, 217-35.