ECOTOXICOLOGY
AND
Physiological
ENVIRONMENTAL
SAFETY
Responses
P. K. NANDI, MADHOOLIKA
19,64-7
1 (1990)
of Vicia faba Plants to Sulfur Dioxide AGRAWAL,’ S. B. AGRAWAL, AND D. N. RAO
Centre ofAdvanced Study in Botany, Banaras Hindu University, Varanasi - 221005. India Received January 31. 1989 Exposure of broad bean (Vicia faba L.) plants to 270 f 32 and 670 k 45 pg mm3 SO? for 1.5 hr daily between 40 and 85 days of their ages resulted in an increase in their transpiration rate, water saturation deficit, phenol content, and peroxidase activity and a decrease in protein content. With the increase in number of exposures of plants to SOz, chlorotic and brown, necrotic visible injury signs were also developed in leaves. It was further noted that the magnitude of undesirable biochemical changes, which possibly helped in the formation of new pigment characteristic of necrotic tissue of SOz-exposed plants, was not totally dependent on the pollutant concentration. 0 1990 Academic Press., Inc.
INTRODUCTION Plant responses to one of the widespread atmospheric pollutants, SOz, have been documented in detail (Malhotra and Khan, 1984). Several possibilities have also been suggested to understand the mechanism of SO2 phytotoxicity at the biochemical level (Rao and LeBlanc, 1966; Ziegler, 1973; Nandi et al., 1984; Shimazaki et al., 1984; Tanaka et al., 1982a,b). It is accepted that SOz-exposed plants accumulate sulfur, mainly in the form of SO:-, HSO;, and SO:-, which at subnecrotic levels of accumulation may cause physiological injury through a general increase in the capacity of the key enzymes of several metabolic pathways, such as respiration, organic acid synthesis, amino acid synthesis, and photorespiration (Pierre, 1977; Pierre and Quieroz, 198 1). But at higher levels, sulfur accumulation induces visible injury in plants (Khan and Malhotra, 1982). Peroxidase activity usually increases with the increase in sulfur content in SO,-exposed plants (Khan and Malhotra, 1982; Pierre and Queiroz, 1982). Further, Karolewski ( 1983) noted a maximum increase in the peroxidase activity in apparently healthy tissue adjacent to necrotic lesions. The activity of peroxidase could also be altered by many naturally occurring phenolic compounds (Balasimha, 1982). Since the rate of SO, uptake increases with transpiration rate and stomata1 opening (Heath, 1980), in the present study, the effects of SO2 on transpiration, water saturation deficit, peroxidase activity, and phenol and protein contents of Vicia faba plants were assessed, and one possible mechanism underlying SOz phytotoxicity was expressed. MATERIALS
AND
METHODS
Broad bean plants (V. faba L. cv. Local) were grown in sandy loam soil (organic carbon = 0.86%, pH 7.4-7.6, and cation exchange capacity = 15.4 meq. loo-’ g in ’ To whom correspondence should be addressed at Department of Botany, Banaras Hindu University, Varanasi - 221 005, India. 0147-6513/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
64
65
SO2 PHYTOTOXICITY
25-cm diameter plastic pots (two plants per pot). The cultivation and fumigation of plants were carried out in a greenhouse with an 1 I-hr photoperiod (6 AM-5 PM), day and night temperatures of 24.3 * 2.7 and 16.4 f 2.3”C, respectively, and a relative humidity of 65 f 5%. All plants received optimal watering conditions. Fumigation of plants (with eight to nine leaves) to 270 f 32 pg m-3 and 670 + 45 pg m-’ SOI for 1.5 hr daily (between 10 to 11:30 AM) for 45 days was started when the plants were 40 days old. During fumigation, plants in 25 pots for each treatment were placed within Perspex chambers of 1.5 X 1.5 X 1.5 m in size. The SO* gas was generated in a continuous manner by bubbling air through a 1.O- 1.5% aqueous solution of sodium metabisulfite (Agrawal et al., 1983), and its desired concentrations within the chambers were achieved by dilution with carrier air (56 liter set-‘). The gas was dispersed within the fumigation chambers through a network of perforated alkathene pipes arranged at the base. A small turbulance fan was used for good mixing and for a satisfactorily uniform gas distribution within the chambers. The control plants were also placed in identical chambers but were flushed with activated charcoal-filtered air. The SOz concentration within each chamber was continuously monitored using a conductimetric SO* analyzer (Kimoto Model 319, Japan). Fully expanded leaves devoid of injury symptoms were collected from randomly selected plants of all treatments (control (C); 270 + 32 pg m-3 SO1 treated (T,); and 670 + 5 pg me3 SO2 treated (T?) at 55, 70, 85, 100, and 115 days old. Controls were also collected at 40 days age prior to commencement of SOz fumigation. Plants were frequently examined for the presence of visible injuries. Leaf samples were quantified for transpiration rate, water saturation deficit, peroxidase activity, and protein and phenol contents. Water Saturation
Deficit (WSD)
The WSD was determined using the leaf disc method developed by Weatherley (1950). Ten punched fresh leaf discs of 0.5 cm in diameter were weighed (I,,,) and floated on distilled water kept in a covered petri dish. After 24 hr they were surface dried and weighed again (S,) and then their dry weight was determined after keeping them in an oven at 80°C for 24 hr (D,J. The percentage WSD was calculated by using the following formula: WSD (%) = s Transpiration
w
w
X 100.
Rate
The transpiration rate was determined by the detached leaf method (Slavik, 1974) and expressed as grams of water lost per gram-’ dry weight per hour-‘. The determination of precise time between 10 AM and 12 AM for measuring transpiration rates was done on the basis of the maximum transpiration rate prevailing during these hours. Peroxidase Enzyme Assay For the determination of peroxidase activity, a lOO-mg leaf sample was homogenized with 10 ml of 0.1 M cold phosphate buffer containing 5 m&I cysteine, pH 6.8,
66
NAND1
ET
AL.
in a chilled mortar and pestle. The homogenate was centrifuged at 10,OOOg for 15 min at 0 to 4°C and the supematant was used for the enzyme assay. A 5-ml assay mixture for peroxidase activity, containing 125 PM phosphate buffer (pH 6.8), 50 PM pyrogallol, 50 jJ4 H202, and 1 ml of diluted enzyme extract was incubated at 25°C for 5 min after which the reaction was terminated by adding 0.5 ml of 5% H2S04. The colored end product (purpurogallin) was extracted in ether and the quantity was determined spectrophotometrically at 430 nm, where its extinction coefficient was 2.47 cm-’ m&-l (B&ton and Mehley, 1955). The activity was expressed as micromolar purpurogallin formed per minute per gram of fresh leaf.
Phenol Content A sample of a 0.2-g leaf was homogenized with 10 ml of 70% ethanol and centrifuged at 6000g for 10 min. The residue was given two washing with 70% ethanol, centrifuging each time. The pooled supernatant was concentrated in an evaporator under vacuum and the concentrate was partitioned through light petroleum to remove chlorophyll. The total phenol content of the concentrate was determined using the method of Bray et al. described by Bray and Thorpe ( 1954).
Protein A sample of a 0.5-g leaf was homogenized in 80% ethanol. The homogenate was boiled for 10 min and then centrifuged. The pellets were washed successively with 10% (w/v) cold trichloroacetic acid; ethanol-chloroform (3: 1, v/v); ethanol-ether (3: 1 v/v), and finally with ether. Pellets were evaporated to dryness and the protein was solubilized in 0.1 N NaOH for 15 min in a boiling water bath. After centrifugation, the protein content of the supematant was determined by following the method of Lowry et al. (195 1).
Statistics All analyses for each treatment were made with the sample taken from five separate plants. Data were analyzed using Students t test of significance and two-way analysis of variance. RESULTS In early stages of fumigation, i.e., between 40- and 55-day ages, the foliar injury was manifested as interveinal water-soaked areas which later turned into chlorotic and necrotic patches. The signs of injury first appeared in T2 plants, and at any given age, the level of injury in these plants was higher than T, plants. Between 85- and loo-day ages, younger leaves of T2 plants appeared to be relatively more sensitive, because at this age, the tips and margins of these leaves turned black. Injured leaves gradually became dry and dead and they were eventually shed off from the plants. Premature leaf fall was noted between 85- and 115-day ages in Tr and T2 plants. The transpiration rate of S02-exposed plants increased significantly (P < 0.0 1) with respect to the control (Tables l-6); the increase being most significant for T2 plants, rightfrom55-day(60.9%,P~O.Ol)to115-dayage(77.2%,P~0.001).Theincreases
67
SO2 PHYTOTOXICITY
TABLE 1 AGE-WISE CHANGES IN TRANSPIRATION RATE(g WATERLOST hr-’ g-’ DRY LEAF) OF C, T, , AND T2 PLANTS (MEAN + 1 SE)
Plant age (days)
C
T,
40 55 70 85 100 115
2.56 k 0.19 2.43 5 0.10 3.02 k 0.10 0.79 f 0.04 0.89 + 0.08 0.88 f 0.02
2.56 + 0.19 2.93 + 0.27” 3.42 t 0.34” 1.16+0.09’ 1.27+ 0.08’ 1.35k0.21U
Note.Significantlevel:%otsignificant: *P
< 0.05; ‘P i 0.0
T2
2.56 + 0.19“ 3.91 f 0.28’ 4.31 kO.17’ 1.35It 0.08’ 1.41+ 0.09” 1.56kO.13’
1.
in transpiration rates in 115-day-old T1 and T2 plants were 53.4 and 77.2%, respectively (Table 1). The WSD percentages of SOz-exposed leaves were also significantly higher (P < 0.001) than the control leaves (Tables 2 and 6). For example, the WSD values at the loo-day age were 25.90, 32.80, and 39.4% in C, T,, and T2 plants, respectively. In comparison to the control, the WSD peaks at the 85-day age in leaves of T1 and T2 plants were 28.9 and 53.2%, respectively. Relative to the control, the peroxidase activity in T1 and T2 plants increased with age (P < 0.00 1); the maximum increases of 63.5 and 8 1.3% were noted in 1 l$dayold T1 and T2 plants, respectively (Table 3). Such changes in peroxidase activity due to SO2 treatment, plant age, and their interaction were highly significant (P < 0.001) (Table 6). The phenol contents in SOz-exposed plants were higher than in the control throughout the experiment (Table 4). In comparison to the control, maximum increases in phenol contents of 85-day-old T1 and T2 plants were 46.6 and 60.0%, respectively. In contrast to the effects on phenol, SO2 decreased the protein content by 28.0 and 32.6% in treatments T, and TZ, respectively, at the age of 85 days (Table 5). A significant interaction (P < 0.05), due to SO2 treatment and plant age on the protein content, was also detected (Table 6). TABLE 2 AGE-WISECHANGESIN WATERSATURATION DEFICIT (%) IN LEAVES OF C, T, , AND T2 PLANTS (MEAN f 1 SE)
Note.
Plant age (days)
C
T,
40 55 70 85 100 115
17.75f 1.27 22.50 + 1.16 27.50 f 0.92 26.70+ 0.28 25.90+ 0.13 25.70f 3.02
17.75k 1.27’ 26.50f 0.13’ 30.60f 0.12’ 34.40* 1.50’ 32.80 + 0.47d 31.6OkO.14”
Significance level:“not significant; ‘P
< 0.05; ‘P
AND
Physiological
ENVIRONMENTAL
SAFETY
Responses
P. K. NANDI, MADHOOLIKA
19,64-7
1 (1990)
of Vicia faba Plants to Sulfur Dioxide AGRAWAL,’ S. B. AGRAWAL, AND D. N. RAO
Centre ofAdvanced Study in Botany, Banaras Hindu University, Varanasi - 221005. India Received January 31. 1989 Exposure of broad bean (Vicia faba L.) plants to 270 f 32 and 670 k 45 pg mm3 SO? for 1.5 hr daily between 40 and 85 days of their ages resulted in an increase in their transpiration rate, water saturation deficit, phenol content, and peroxidase activity and a decrease in protein content. With the increase in number of exposures of plants to SOz, chlorotic and brown, necrotic visible injury signs were also developed in leaves. It was further noted that the magnitude of undesirable biochemical changes, which possibly helped in the formation of new pigment characteristic of necrotic tissue of SOz-exposed plants, was not totally dependent on the pollutant concentration. 0 1990 Academic Press., Inc.
INTRODUCTION Plant responses to one of the widespread atmospheric pollutants, SOz, have been documented in detail (Malhotra and Khan, 1984). Several possibilities have also been suggested to understand the mechanism of SO2 phytotoxicity at the biochemical level (Rao and LeBlanc, 1966; Ziegler, 1973; Nandi et al., 1984; Shimazaki et al., 1984; Tanaka et al., 1982a,b). It is accepted that SOz-exposed plants accumulate sulfur, mainly in the form of SO:-, HSO;, and SO:-, which at subnecrotic levels of accumulation may cause physiological injury through a general increase in the capacity of the key enzymes of several metabolic pathways, such as respiration, organic acid synthesis, amino acid synthesis, and photorespiration (Pierre, 1977; Pierre and Quieroz, 198 1). But at higher levels, sulfur accumulation induces visible injury in plants (Khan and Malhotra, 1982). Peroxidase activity usually increases with the increase in sulfur content in SO,-exposed plants (Khan and Malhotra, 1982; Pierre and Queiroz, 1982). Further, Karolewski ( 1983) noted a maximum increase in the peroxidase activity in apparently healthy tissue adjacent to necrotic lesions. The activity of peroxidase could also be altered by many naturally occurring phenolic compounds (Balasimha, 1982). Since the rate of SO, uptake increases with transpiration rate and stomata1 opening (Heath, 1980), in the present study, the effects of SO2 on transpiration, water saturation deficit, peroxidase activity, and phenol and protein contents of Vicia faba plants were assessed, and one possible mechanism underlying SOz phytotoxicity was expressed. MATERIALS
AND
METHODS
Broad bean plants (V. faba L. cv. Local) were grown in sandy loam soil (organic carbon = 0.86%, pH 7.4-7.6, and cation exchange capacity = 15.4 meq. loo-’ g in ’ To whom correspondence should be addressed at Department of Botany, Banaras Hindu University, Varanasi - 221 005, India. 0147-6513/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
64
65
SO2 PHYTOTOXICITY
25-cm diameter plastic pots (two plants per pot). The cultivation and fumigation of plants were carried out in a greenhouse with an 1 I-hr photoperiod (6 AM-5 PM), day and night temperatures of 24.3 * 2.7 and 16.4 f 2.3”C, respectively, and a relative humidity of 65 f 5%. All plants received optimal watering conditions. Fumigation of plants (with eight to nine leaves) to 270 f 32 pg m-3 and 670 + 45 pg m-’ SOI for 1.5 hr daily (between 10 to 11:30 AM) for 45 days was started when the plants were 40 days old. During fumigation, plants in 25 pots for each treatment were placed within Perspex chambers of 1.5 X 1.5 X 1.5 m in size. The SO* gas was generated in a continuous manner by bubbling air through a 1.O- 1.5% aqueous solution of sodium metabisulfite (Agrawal et al., 1983), and its desired concentrations within the chambers were achieved by dilution with carrier air (56 liter set-‘). The gas was dispersed within the fumigation chambers through a network of perforated alkathene pipes arranged at the base. A small turbulance fan was used for good mixing and for a satisfactorily uniform gas distribution within the chambers. The control plants were also placed in identical chambers but were flushed with activated charcoal-filtered air. The SOz concentration within each chamber was continuously monitored using a conductimetric SO* analyzer (Kimoto Model 319, Japan). Fully expanded leaves devoid of injury symptoms were collected from randomly selected plants of all treatments (control (C); 270 + 32 pg m-3 SO1 treated (T,); and 670 + 5 pg me3 SO2 treated (T?) at 55, 70, 85, 100, and 115 days old. Controls were also collected at 40 days age prior to commencement of SOz fumigation. Plants were frequently examined for the presence of visible injuries. Leaf samples were quantified for transpiration rate, water saturation deficit, peroxidase activity, and protein and phenol contents. Water Saturation
Deficit (WSD)
The WSD was determined using the leaf disc method developed by Weatherley (1950). Ten punched fresh leaf discs of 0.5 cm in diameter were weighed (I,,,) and floated on distilled water kept in a covered petri dish. After 24 hr they were surface dried and weighed again (S,) and then their dry weight was determined after keeping them in an oven at 80°C for 24 hr (D,J. The percentage WSD was calculated by using the following formula: WSD (%) = s Transpiration
w
w
X 100.
Rate
The transpiration rate was determined by the detached leaf method (Slavik, 1974) and expressed as grams of water lost per gram-’ dry weight per hour-‘. The determination of precise time between 10 AM and 12 AM for measuring transpiration rates was done on the basis of the maximum transpiration rate prevailing during these hours. Peroxidase Enzyme Assay For the determination of peroxidase activity, a lOO-mg leaf sample was homogenized with 10 ml of 0.1 M cold phosphate buffer containing 5 m&I cysteine, pH 6.8,
66
NAND1
ET
AL.
in a chilled mortar and pestle. The homogenate was centrifuged at 10,OOOg for 15 min at 0 to 4°C and the supematant was used for the enzyme assay. A 5-ml assay mixture for peroxidase activity, containing 125 PM phosphate buffer (pH 6.8), 50 PM pyrogallol, 50 jJ4 H202, and 1 ml of diluted enzyme extract was incubated at 25°C for 5 min after which the reaction was terminated by adding 0.5 ml of 5% H2S04. The colored end product (purpurogallin) was extracted in ether and the quantity was determined spectrophotometrically at 430 nm, where its extinction coefficient was 2.47 cm-’ m&-l (B&ton and Mehley, 1955). The activity was expressed as micromolar purpurogallin formed per minute per gram of fresh leaf.
Phenol Content A sample of a 0.2-g leaf was homogenized with 10 ml of 70% ethanol and centrifuged at 6000g for 10 min. The residue was given two washing with 70% ethanol, centrifuging each time. The pooled supernatant was concentrated in an evaporator under vacuum and the concentrate was partitioned through light petroleum to remove chlorophyll. The total phenol content of the concentrate was determined using the method of Bray et al. described by Bray and Thorpe ( 1954).
Protein A sample of a 0.5-g leaf was homogenized in 80% ethanol. The homogenate was boiled for 10 min and then centrifuged. The pellets were washed successively with 10% (w/v) cold trichloroacetic acid; ethanol-chloroform (3: 1, v/v); ethanol-ether (3: 1 v/v), and finally with ether. Pellets were evaporated to dryness and the protein was solubilized in 0.1 N NaOH for 15 min in a boiling water bath. After centrifugation, the protein content of the supematant was determined by following the method of Lowry et al. (195 1).
Statistics All analyses for each treatment were made with the sample taken from five separate plants. Data were analyzed using Students t test of significance and two-way analysis of variance. RESULTS In early stages of fumigation, i.e., between 40- and 55-day ages, the foliar injury was manifested as interveinal water-soaked areas which later turned into chlorotic and necrotic patches. The signs of injury first appeared in T2 plants, and at any given age, the level of injury in these plants was higher than T, plants. Between 85- and loo-day ages, younger leaves of T2 plants appeared to be relatively more sensitive, because at this age, the tips and margins of these leaves turned black. Injured leaves gradually became dry and dead and they were eventually shed off from the plants. Premature leaf fall was noted between 85- and 115-day ages in Tr and T2 plants. The transpiration rate of S02-exposed plants increased significantly (P < 0.0 1) with respect to the control (Tables l-6); the increase being most significant for T2 plants, rightfrom55-day(60.9%,P~O.Ol)to115-dayage(77.2%,P~0.001).Theincreases
67
SO2 PHYTOTOXICITY
TABLE 1 AGE-WISE CHANGES IN TRANSPIRATION RATE(g WATERLOST hr-’ g-’ DRY LEAF) OF C, T, , AND T2 PLANTS (MEAN + 1 SE)
Plant age (days)
C
T,
40 55 70 85 100 115
2.56 k 0.19 2.43 5 0.10 3.02 k 0.10 0.79 f 0.04 0.89 + 0.08 0.88 f 0.02
2.56 + 0.19 2.93 + 0.27” 3.42 t 0.34” 1.16+0.09’ 1.27+ 0.08’ 1.35k0.21U
Note.Significantlevel:%otsignificant: *P
< 0.05; ‘P i 0.0
T2
2.56 + 0.19“ 3.91 f 0.28’ 4.31 kO.17’ 1.35It 0.08’ 1.41+ 0.09” 1.56kO.13’
1.
in transpiration rates in 115-day-old T1 and T2 plants were 53.4 and 77.2%, respectively (Table 1). The WSD percentages of SOz-exposed leaves were also significantly higher (P < 0.001) than the control leaves (Tables 2 and 6). For example, the WSD values at the loo-day age were 25.90, 32.80, and 39.4% in C, T,, and T2 plants, respectively. In comparison to the control, the WSD peaks at the 85-day age in leaves of T1 and T2 plants were 28.9 and 53.2%, respectively. Relative to the control, the peroxidase activity in T1 and T2 plants increased with age (P < 0.00 1); the maximum increases of 63.5 and 8 1.3% were noted in 1 l$dayold T1 and T2 plants, respectively (Table 3). Such changes in peroxidase activity due to SO2 treatment, plant age, and their interaction were highly significant (P < 0.001) (Table 6). The phenol contents in SOz-exposed plants were higher than in the control throughout the experiment (Table 4). In comparison to the control, maximum increases in phenol contents of 85-day-old T1 and T2 plants were 46.6 and 60.0%, respectively. In contrast to the effects on phenol, SO2 decreased the protein content by 28.0 and 32.6% in treatments T, and TZ, respectively, at the age of 85 days (Table 5). A significant interaction (P < 0.05), due to SO2 treatment and plant age on the protein content, was also detected (Table 6). TABLE 2 AGE-WISECHANGESIN WATERSATURATION DEFICIT (%) IN LEAVES OF C, T, , AND T2 PLANTS (MEAN f 1 SE)
Note.
Plant age (days)
C
T,
40 55 70 85 100 115
17.75f 1.27 22.50 + 1.16 27.50 f 0.92 26.70+ 0.28 25.90+ 0.13 25.70f 3.02
17.75k 1.27’ 26.50f 0.13’ 30.60f 0.12’ 34.40* 1.50’ 32.80 + 0.47d 31.6OkO.14”
Significance level:“not significant; ‘P
< 0.05; ‘P
