Effects of nitrogen dioxide on leaf chlorophyll and nitrogen content of soybean

Environmental Pollution 51 (1988) 113-120

Effects of Nitrogen Dioxide on Leaf Chlorophyll and Nitrogen Content of Soybean Sashikala Sabaratnam, G i a n G u p t a Department of Agricultural Sciences, University of Maryland, Eastern Shore, Princess Anne, Maryland 21853, USA

& Charles Mulchi Department of Agronomy, University of Maryland, College Park, Maryland 20742, USA

(Received 13 July 1987; revised version accepted 16 October 1987)

A BSTRA CT One-month-old soybean (Glycine max [L.] Merrill), cultivar 'Williams plants were exposed to nitrogen dioxide (0"1, 0"2, 0"3 and O.5 ppm ) and carbon filtered air (control), 7 h per day,for 5 days, under a controlled environment. L e a f chlorophyll content ( Ch a, Ch b, and total Ch content) and foliar nitrogen con ten t ( % N ) were determined beJore and after the exposure. The influence of NO2 treatments up to 0"3 ppm on leaf chlorophyll content was negligible although a stimulatoo, effect was evident in Ch a and total Ch content with 0.2ppm NOz. M a r k e d decline in Ch content was observed with 0"5 ppm treatment; the reductions in Ch a and total Ch were 45% and 47%, respectively. Foliar-N contents of plants treated with 0.2 and 0"3 ppm N O 2 were higher than the control; plants exposed to 0"5 ppm NO2 showed a 41% reduction in foliar-N compared to pre-exposure values.

INTRODUCTION Reduction in growth and yield are known to occur when plants are exposed to chronic levels o f air pollutants, yet m o r e i n f o r m a t i o n o n physiological 113 Environ. Pollut. 0269-7491/88/$03"50 ~') 1988 Elsevier Applied Science Publishers Ltd, England. Printed in Great Britain

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Sashikala Sabaratnam, Gian Gupta, Charles Mulchi

and biochemical responses of plants to air pollutants is needed (Heck & Brandt, 1977). Several metabolic processes are altered when plant tissue is subjected to pollutant stress (Craker & Starbuck, 1972; Leflter & Cherry, 1974). Fumigation with O a and SO2 has been shown to alter chlorophyll content in various plants which could reduce the photosynthetic capacity of affected plants (Knudson et al., 1977; Beckerson & Hofstra, 1979; Pratt & Krupa, 1981; Olszyk & Tibbitts, 1982). No studies have been reported on the effects of NO 2 on leaf chlorophyll levels. Changes in leaf protein levels have also been reported in response to pollutant treatments. Leaf protein in soybean (Tingey et al., 1973) and bean (Howell & Kremer, 1973) exposed to 0 3 (up to 0"5 ppm) was higher than that of the plants not exposed to these pollutants. Similar responses have been reported in plants exposed to SO 2 and NO z (Zeevart, 1976; Klarer et al., 1983). These experiments were undertaken to study the changes in chlorophyll and nitrogen contents of soybean leaves after exposure to 0" 1 to 0"5 ppm N O 2.

MATERIALS AND METHODS

Cultural conditions Soybean, cultivar 'Williams', plants were grown in 21 cm diameter plastic pots. The potting mixture was Cornell Tropical Plant Mix (Hummert Seed Co., St Louis, Missouri, USA), a mixture of perlite, vermiculite, and ProMix in 1 : 1 : 2 proportion with limestone and added fertiliser. Seeds were treated with commercial R h i z o b i u m j a p o n i c u m inoculant and planted, five per pot (in 25 pots) and the plants were thinned, based on uniformity of seedling size, to three per pot after emergence. Plants were watered twice a day (watered to excess and allowed to drain) by use of a 'spaghetti' watering system controlled by a time switch. These pots were fertilised biweekly with 300 ml of commercial 5" 11:26 fertiliser (Peter's professional). High pressure sodium lamps were used to supplement light in the greenhouse from 6 to 8 am and 5 to 7 pm, and on cloudy days. Environmental conditions in the non-filtered greenhouse were: day temperatures of 2 8 _ 2°C; night temperatures of 22 + 2°C; and relative humidity levels of 70 _+ 10%. Plants were moved to a growth chamber, Percival--Model PT 80, at the age of 3 weeks and acclimated for one week in carbon filtered air. The diurnal temperature change for the growth chamber was similar to that of the greenhouse. Light was provided from 6 a m to 7 p m simulating the summer daylight conditions using three separate timers. The incandescent lamps were turned on first, and the fluorescent lamps were turned on, as two

Effects of nitrogen dioxide on soybean

I 15

separate groups, at 30-min intervals each. Irradiance at canopy height was 350 + 20/rE m -2 s- 1 as measured by a quantum sensor attached to the LIC O R 6000 Portable Photosynthesis System (LI-COR Inc., Lincoln, Nebraska).

Exposure conditions Gaseous exposures began when the plants were one month old. Each individual treatment was given separately. Control plants were exposed to carbon filtered air (CF). For treatments, N O 2 w a s dispensed from a compressed (certified) gas cylinder (Matheson Gas Products, Baltimore, Maryland, USA) and diluted at the air inlet duct with carbon filtered air to the required concentration (0-1, 0"2, 0-3, 0"5/~g litre- 1). The flow rate of NO2 was regulated and maintained by use of a flow meter. The duration of exposure was 7h per day (9am to 4pm) for 5 days. Nitrogen dioxide concentration at the canopy level was continuously monitored using a NO/NO x Monitor, Model CSI-1600 (Columbia Scientific Instruments) and recorded on a Fisher Recordall Series 5000. The NO/NOx monitor was calibrated before the experiment using Columbia Scientific Instruments Gas Phase Titration Calibrator--CSI Model 1700 and Zero Air Supply--Model 205. The calibration drift was always less than 5%. Fluctuations in NO2 concentrations within treatments were _+5%.

Analyses Chlorophyll measurements were made before and after the exposure to NO2, using the extraction procedure described by Knudson et al. (1977). Chlorophyll a and b contents were calculated using the equations developed by Winterman & De Mots (1965). Total organic nitrogen of the leaves of individual plants was determined according to the method of Hach et al. (1985), using Kjeldahl Digestion A p p a r a t u s - - M o d e l 21400 (Hach Co., Pennsanken, New Jersey, USA). Each treatment in the experiment was replicated three times. Data were subjected to A N O V A for completely randomised design with three replicates that represented means of 10 + 2 observations per replicate. Mean separation was done using Duncan's Multiple Range Test.

RESULTS The differences in Ch a, Ch b, and total Ch between pre-exposure values and carbon filtered air treatments were not significant. There were significant

Sashikala Sabaratnam, Gian Gupta, Charles Mulchi

116

2G

Ch total

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Ch b

0

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0.1

0.2

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ppm Fig. 1.

Effect o f N O 2 level on c h l o r o p h y l l content (Ch) in soybean leaves.

TABLE 1 Ch a, Ch b and Total Ch of Soybean Leaves Before and After Exposure t o N O 2

NO 2 (ppm)

0 0"1 0-2 0"3 0"5

Ch a (mgg -1)

Ch b (mgg -1)

Total Ch (mgg -1)

Before

After

Before

After

Before

After

11.28 11.49 11'05 12.20 9'89

10.89a 10.66a 11'35a 10.99a 6.00b"

3"83 4"37 4"77 3"81 4"22

3"53ab 4-09a 4"04a 4"23a 2"1 lb

14.90 15'61 15"33 15.75 14'08

14.80a 14.78a 15" 14a 15-03a 7"80b"

Each mean represents the average of 30 + 4 observations. Values followed by the same letter are not significantly different from each other at P < 0.05 according to Duncan's Multiple Range Test and Waller-Duncan K-Ratio Tests. " Means are significantly different at P < 0"05.

Effects of nitrogen dioxide on soybean

117

TABLE 2 Total N Content of Soybean Leaves Before and After Exposure to NO2, 7 h per Day, for 5 Days

N Content (g per lOOg dr)' weight leaf)

NO 2 (pprn)

0 0.1 0'2 0.3 0'5

Before

After

Difference

3"12a 3-20a 3'08a 3'24a 3'13a

2.30c 2.78c 3.36b 3"88a 2-53c

-0.82 - 0-42 +0.28 + 0"64 -0.60

Each mean represents the average of 30 + 4 observations. Values followed by the same letter are not significantly different from each other at P _< 0.05.

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2.0 0

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0.1

9" 0.2

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0.3

0.5

ppm

Fig. 2.

Effect of

NO

2

level on foliar N content (FN) in soybean.

118

Sashikala Sabaratnam, G&n Gupta, Charles Mulchi

reductions in Ch a and total Ch with 0"5 ppm N O 2 treatment, of 45% and 47%, respectively. The changes in Ch a, Ch b, and total Ch of treated plants are shown in Fig. 1. Means separation for chlorophyll and foliar-N are given in Tables 1 and 2, respectively. Treatment differences with N O 2 w e r e significant at P < 0.05. Foliar nitrogen content was higher with 0-2 and 0.3 ppm NO2 treatments (46% and 69%, respectively) than the control. Figure 2 shows the changes in foliar-N before and after treatment with N O 2.

DISCUSSION Chlorophyll pigments are essential for photosynthesis. A decrease in Ch has been used as an indicator of air pollution injury (Gilbert, 1968). With 0.5 ppm N O 2 , there were reductions in Ch a, Ch b and total Ch. This reduction in Ch is in agreement with the effects of 03 and SO2 on soybean (Leffler & Cherry, 1974; Knudson et al., 1977; Beckerson & Hofstra, 1979; Pratt & Krupa, 1981; Pratt et al., 1983; Jones et al., 1985; Reich et al., 1986). The loss in chlorophyll due to 0 3 exposure was attributed to direct effects of 0 3 or due to blockage in new chlorophyll synthesis (Knudson et al., 1977). In the case of relatively higher concentrations of acidic pollutants (such as SO2), loss of chlorophyll may be explained by acidification (Grunwald, 1981; Koziol & Whatley, 1984) and subsequent loss of Mg + + from chlorophyll to form phaeophytin (Mudd & Kozlowski, 1975). Malhotra (1977) has proposed another mechanism for the reduction in leaf Ch following exposure to SO 2. Chlorophyll is stabilised by forming a complex with proteins and perhaps SO2 attacks this complex before the actual breakdown of chlorophyll occurs. It is also possible that with higher concentrations of N O 2 , a breakdown of this complex takes place. Another proposed mechanism for Ch destruction by SO2 is the oxidation (of Ch) by free radicals (Shimazaki et al., 1980). Slight increases in foliar-N were observed with exposures up to 0"3 ppm N O 2. Increase in leaf amino acids and proteins (which constitute the bulk of N in the leaves) have also been observed in response to 03 treatment (Craker & Starbuck, 1972; Tingey et al., 1973; Beckerson & Hofstra, 1979). The increase in protein-N may be explained by new protein synthesis as the plant attempts to recover from 03 injury (Craker & Starbuck, 1972). In the case of NO2, it is possible that it can act as a source of nitrogen and thus result in increased cellular protein (Koziol & Whatley, 1984). The normal scavenging mechanism for NO2 is reduction by nitrate reductase (observed in cytoplasm) and nitrite reductase (found in chloroplasts), that are usually present in sufficient amounts in cells, to form ammonia and

Effects of nitrogen dioxide on soybean

119

amino acids (Zeevart, 1976). This conversion is rapid in the presence of light because of the available reducing power from light reactions. The concurrent reduction in foliar-N (41% compared to pre-exposure values) and total Ch content (47%) at 0 ' 5 p p m suggests the failure of such conversion processes and supports the possible detrimental effects of N O 2 on enzymes and other cellular components (Leffler & Cherry, 1974). F r o m these changes in Ch and foliar-N, it appears that N O 2 up to 0.2 ppm has a stimulatory effect whereas 0-5 ppm N O 2 has an inhibitory effect.

ACKNOWLEDGEMENT This publication is a result of the RR-3 project of U S D A - C S R S and the financial support from this progam is appreciated.

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M udd, B. J. & Kozlowski, T. T. (1975). Responses of plants to air pollution. 9-51, New York, Academic Press. Olszyk, D. M. & Tibbitts, T. W. (1982). Evaluation of injury to expanded and expanding pea leaves exposed to SO2 and 03. J. Am. Soc. Hortic. Sci., 107, 266-71. Pratt, G. C. & Krupa, S. V. (1981). Soybean cv. 'Hodgson' response to 0 3. Can. J. Bot., 59, 1233-8. Pratt, G. C., Kromroy, K. W. & Krupa, S. V. (1983). Effects ofO 3 and SO2 on injury and foliar concentration of sulphur and chlorophyll in soybean. Environ. Pollut., Ser. A, 33, 91-9. Reich, P. S., Schoettle, A. W., Raba, R. M. & Amundson, R. G. (1986). Responses of soybean to low concentration of 0 3. J. Environ. QuaL, 15(1), 31-6. Shimazaki, K., Sasaki, T. & Sugahara, K. (1980). Active oxygen participation in chlorophyll destruction and lipid peroxidation in SO 2 fumigated leaves of spinach. Res. Rep. Natl. Environ. Stud.jpn., 11, 91-101. Tingey, D. T., Fites, R. C. & Wickliff, C. (1973). Ozone alteration of nitrate reductase in soybean. Physiol. Plant., 29, 33-8. Winterman, J. F. G. & De Mots, A. (1965). Spectrophotometric characteristics of chlorophylls a and b and their phaeophytin in ethanol. Biochem. Biophys. Acta, 109, 448-53. Zeevart, A. J. (1976). Some effects of fumigating plants for short periods with SO2. Environ. Pollut., Ser. A, I1, 97-108.