Atmospheric Environment 43 (2009) 619–623
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Relationship between leaf antioxidants and ozone injury in Nicotiana tabacum ‘Bel-W3’ under environmental conditions in Sa˜o Paulo, SE – Brazil Marisia P. Esposito, Mauricio L. Ferreira, Silvia M.R. Sant’Anna, Marisa Domingos, Silvia R. Souza* ˆ nica, Seça ˜o de Ecologia, Caixa Postal 3005, 01061-970 Sa ˜o Paulo, SP, Brazil Instituto de Bota
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 May 2008 Received in revised form 25 August 2008 Accepted 2 October 2008
Previous studies have reported that the extent of leaf injury in Nicotiana tabacum ‘‘Bel-W3’’ exposed to environmental conditions in the city of Sa˜o Paulo is influenced by weather conditions. This influence may occur by means of antioxidant responses. Thus, this study aimed to evaluate whether daily antioxidant responses to environmental variations interfere on the progression of leaf injury on plants of this cultivar during their exposure in a state park of Sa˜o Paulo and to determine a linear combination of variables, among antioxidants and environmental factors, which mostly explain this visible response. Plants were exposed at the mentioned site for 14 days in four different experiments. During each experiment, three plants were daily sampled to determine the accumulated percentage of leaf area affected by necrosis and antioxidant responses (concentrations of total ascorbic acid (AA) and activity of superoxide dismutase (SOD) and peroxidases (POD)). Ozone concentrations and weather conditions were also daily measured. Pearson correlations and multivariate analyses assessed the relationship between biological and environmental variables. Leaf injury appeared between the 3rd and 6th days of exposure and increased over the exposure periods. The daily concentrations of AA tended to decrease with time of exposure in all experiments, but the activity of SOD and POD oscillated during plant exposure. Positive correlations were observed between AA or SOD and O3 concentrations, as well as negative correlations between AA and air temperature. The increasing percentage of leaf necrosis across the whole period was explained by decreasing levels of AA 2 days before injury estimation and by higher O3 concentrations 5 days before (R2 ¼ 0.36; p < 0.001). The use of N. tabacum Bel-W3 as a bioindicator can be restricted by leaf antioxidant responses to both atmospheric contamination and weather conditions. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Nicotiana tabacum ‘‘Bel-W3’’ Antioxidants Leaf injury Ozone
1. Introduction The troposphere of the Metropolitan Region of Sa˜o Paulo (MRSP) is strongly affected by the photochemical smog, which is formed by reactions between hydrocarbons and nitrogen oxides in the presence of sunlight, and results in the production of ozone (O3) and other oxidant species (Souza et al., 1999). In recent years, O3 levels at that region have been high enough to exceed several times the standard limits of air quality of 80 ppb for 1 h. Therefore, this pollutant may potentially cause serious environmental problems in the region (Molina and Molina, 2004). O3, a strong oxidant, is generally noxious to plants (Iriti and Faoro, 2008). The oxidative effects on plant ultrastructure and on physiological and metabolic processes in the cells are due to the increasing production of reactive oxygen species (ROS) as soon as it is taken by leaves via stomata (Puckette et al., 2007). Weather oscillations are other environmental factors that can impose
* Corresponding author. Tel.: þ55 11 5073 6300; fax: þ55 11 5073 3678. E-mail address:
[email protected] (S.R. Souza). 1352-2310/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.atmosenv.2008.10.006
oxidative stress to plants. However, the extent of damage in plants depends on the efficiency of antioxidants, e.g., superoxide dismutase (SOD), peroxidases (POD), and ascorbic acid (AA), in maintaining the oxidant/antioxidant balance and increasing plant tolerance to stressful conditions of the polluted environment (Jones, 2006; Halliwell and Gutteridge, 2007). The disruption of this balance is an early response of sensitive plants growing in an O3-polluted environment, so that extensive physiological and metabolic disturbances, cell death and the occurrence of typical visible injury on leaves, such as chlorosis and necrosis, are rapidly observed (Faoro and Iriti, 2005). Due to these characteristics, such sensitive plants have been used as simple but effective indicators of O3 for biomonitoring purposes. The cultivar ‘Bel-W3’ of Nicotiana tabacum is the most frequently used for biomonitoring ambient O3 in European urban centers (Verge´ et al., 2002; Klumpp et al., 2006a,b; Calatayud et al., 2007). However, a recent study performed in the city of Sa˜o Paulo, SE Brazil, showed that the percentage of leaf area of N. tabacum ‘BelW3’ affected by necrosis was not strictly related to atmospheric ozone concentrations (Sant’Anna et al., 2008). In addition, a delay of 3–6 days in the appearance of the first typical visible symptom was
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observed, independent of the level of O3 in the atmosphere. Thus, we raised the hypothesis that leaf antioxidant responses in the plants of N. tabacum ‘Bel-W3’ during exposure to short-term variations on O3 concentrations and/or on weather conditions, which characterize the air contamination and the subtropical climate at the SE region of Brazil, may influence the occurrence and progression of leaf necrosis. The present study aimed to evaluate whether the daily antioxidant responses to environmental variations interfere on the progression of leaf injury on plants of this cultivar during their exposure in a state park of Sa˜o Paulo, SE – Brazil, and to determine a linear combination of variables, among antioxidants and environmental factors that mostly explain this visible response. 2. Materials and methods 2.1. Exposure site and sampling procedures The site used for plant exposure is located inside a state park in the South region of the city of Sa˜o Paulo (23 380 28.800 S; 46 370 15.800 W; 805 m above sea level). The air in the region is mainly contaminated by O3. Its precursors are predominantly emitted by a fragment of forest included the park and by vehicular emissions from the surrounding avenues that are situated around 3 Km faraway (Souza and Carvalho, 2001; Monteiro et al., 2002). Sa˜o Paulo is the largest city in area and in population of the Metropolitan Region of Sa˜o Paulo (MRSP). This region encompasses 39 municipalities and 8051 km2 of area and is the largest industrialized and urbanized region in Latin America (Molina and Molina, 2004). The MRSP has a complex topography surrounded by mountains that range from 650 to 1200 m.a.s.l., which facilitates the trapping of dust and of gaseous pollutants in inversion layers, increasing their concentrations in the air. The climate of the MRSP is subtropical, with a dry season during winter. Monthly average temperatures in the summer and winter reach 23 C (from December to February) and 16 C (from June to August), respectively. The rainy season normally begins in September and ends in March, with an annual average precipitation of 1200 mm. The local circulation of air is created by winds from both the southeast and northeast and is associated with the Atlantic Ocean breeze circulation (Andrade et al., 2004). The exposure of tobacco Bel-W3 to environmental conditions at the mentioned site occurred in four distinct periods: 31 August–13 September 2004, 14–27 October 2004, 21 November–4 December 2004, and 7–19 March 2005 (henceforth referred as exposure periods 1 through 4). Each exposure experiment stated with 28 plants. The plant cultivation and field exposure systems were those recommended by VDI (2000). During the period of exposure, the accumulated percentages of leaf area affected by typical necrosis were daily measured on the 4th–6th oldest leaves of all remaining plants. The concentrations of AA and the activity of POD and SOD were measured in a mixed sample per plant composed by fresh green portions the same three leaves. Three plants were randomly taken everyday of each period for these purposes. 2.2. Leaf injury assessment and analytical procedures The percentage of leaf area affected by typical necrosis was visually estimated in groups of 5% of affected leaf area, according to VDI (2000). The results were presented as average percentage of necrosis estimated on the 4th–6th oldest leaves. Collection of leaves and preparation of extracts for analysis of antioxidants always followed the same sequence in time to avoid diurnal variation. Ascorbic acid was measured in 0.5 g of fresh leaves and homogenized with 12 mL of EDTA-Na2 (0.07%) and
oxalic acid (0.5%). The mixture was centrifuged at 40 000 g for 30 min at 2 C. An aliquot of the supernatant was added to 2.5 mL of DCPiP (0.02%), and absorbance was measured with a spectrophotometer at 520 nm. After the addition of 0.05 mL of ascorbic acid (1%), a second absorbance measurement was taken. Both absorbance measurements were used to estimate the ascorbic acid content following Keller and Schwager (1977). Superoxide dismutase activity was measured in 0.35 g fresh leaves homogenized with 12 mL of potassium phosphate buffer (50 mM pH 7.5), EDTANa2 1 mM, NaCl 50 mM and ascorbic acid 1 mM in the presence of 0.4 g of PVPP 2%. This mixture was centrifuged at 22 000 g for 25 min at 2 C. The activity of SOD was assayed by measuring the SOD inhibition of the NBT photochemical reduction (Osswald et al., 1992). Each reaction mixture contained 0.5 mL of EDTA-Na2 0.54 mM, 0.8 mL of potassium phosphate buffer (0.1 M, pH 7.0), 0.5 mL of methionine 0.13 mM, 0.5 mL of NBT 0.44 mM, 0.2 mL of riboflavin 1 mM, and 0.2 mL of leaf extract. The samples were incubated for 20 min under a fluorescent lamp (80 W). The absorbance of the reaction mixture was measured at 560 nm. A similar mixture lacking the leaf extract was used as a control, and a dark control mixture served as a blank. The enzymatic activity was expressed as the amount of extract needed to inhibit the reduction of NBT by 50%. Peroxidase activity was determined in 0.3 g of leaves and homogenized with 12 mL potassium phosphate buffer (0.1 M, pH 7.0) in the presence of 0.4 g of PVPP 2%. The homogenate was centrifuged at 40 000 g for 30 min at 2 C. The peroxidases were determined in a reaction mixture of plant extracts using 0.1 M potassium phosphate buffer (pH 5.5) and fenylendiamine (1%), to which an aliquot of H2O2 (0.3%) was added. Unspecific POD activity was measured with a spectrophotometer following the increase in absorbance (DA) at 485 nm due to the formation of an H2O2-POD complex at two different times in the linear reaction curve (Klumpp et al., 1989). The O3 levels during all four exposure periods were continuously monitored by an EcotechÔ 9680 O3 Analyzer. Continuous data on irradiation, air temperature and relative humidity were provided by a meteorological station located in the state park and run by the Institute of Astronomy, Geophysics and Atmospheric Sciences from University of Sa˜o Paulo. 2.3. Statistical analysis A one-way Analyses of Variance and a Student-Newman–Keuls pairwise test identified significant differences among the exposure experiments. Correlation analyses (Pearson) were carried out to determine the interactions among environmental factors and among antioxidant responses in the leaves of plants analyzed during all experimental periods. The relations between antioxidants and environmental factors 1–5 days before leaf analyses were also established by analyses of Pearson. This procedure was adopted in order to find the strongest relations between pairs of these variables. Multiple regression analysis was performed to fit the most explicative linear model among accumulated percentage of leaf injury, O3 concentrations and antioxidant indicators during the 14 days of exposure (data from all four exposure periods included). In this case, we assumed that a period of time would be necessary between the loss of the antioxidative capacity in response to environmental conditions and the visualization of leaf injury induced by O3. Therefore, the daily average of leaf injury was widely related to daily values of O3 and of antioxidant responses observed 1–5 days before injury estimation. The most explicative linear combination was the one with the highest and significant coefficient of determination (R2). The data were analyzed by means of the stepwise backward method. The procedure started with all independent variables and, after successive new adjustments, only
M.P. Esposito et al. / Atmospheric Environment 43 (2009) 619–623 ozone (ppb)
Table 2 Pearson correlations between environmental variables and leaf antioxidants in plants of N. tabacum Bel-W3 measured during the four exposure periods and the linear combination of factors that mostly predicted the variations of leaf injury.
leaf injury ( )
100 90 80 70 60 50 40 30 20 10 0
Variables
Environmental factors Ozone (O3)
humidity ( )
temperature (°C)
621
irradiation (MJ/m2)
100 90 80 70 60 50 40 30 20 10 0
Relative humidity Temperature Irradiation Biological variables Ascorbic acid (AA)# Superoxide dismutase# Peroxidase#
Correlation coefficients (r) Ozone
Relative humidity
Temperature
Irradiation
–
– –
– –
0.30 (p ¼ 0.02) 0.47 (p ¼ < 0.01) – –
0.34 (p ¼ 0.01)
–
0.33 (p ¼ 0.01) –
0.38 (p ¼ 0.01) 0.35 (p ¼ 0.01) 0.29 (ns)
0.01 (ns)
0.29 (ns)
0.23 (ns)
0.42 (p ¼ 0.01) 0.11 (ns)
0.01 (ns)
0.03 (ns)
0.19 (ns)
0.23 (ns)
0.73 (p ¼ < 0.01) 0.10 (ns) –
Sqrt(Leaf injury) ¼ 5.03 (0.64 ) AAA) þ (0.06 ) O3A); R2 ¼ 0.36 (p < 0.01)
1st exposure
2nd exposure
3th exposure
4th exposure
Fig. 1. Daily average values of leaf injury, ozone concentration, relative humidity, temperature and irradiation during the four exposure experiments with N. tabacum Bel-W3.
those that significantly contributed to explain the dependent variable (O3 concentrations or leaf necrosis) remained in the model. When necessary, data were transformed to reach normality and equal variances. Sigma Stat 3.5 Software was used for all tests. 3. Results The highest ozone values were observed during the 1st and 3rd exposure periods. Maximum ozone levels (above 80 ppb) were measured in the spring, especially in September, during the 1st exposure. Average temperature was significantly lower during the last period of plant exposure than during the other periods. Average irradiation did not significantly differ among periods (Fig. 1, Table 1). Pearson analyses indicated that these environmental variables were straightly correlated during the period of study (Table 2). In particular, the daily average of O3 concentrations during the four exposure experiments were negatively correlated with air temperature and relative humidity and positively correlated with global irradiation. Due to this interdependence of environmental factors, only O3 was included in the stepwise analysis that determined the linear model among leaf injury, environmental factors and antioxidant indicators. Table 1 Average values standard deviations of environmental factors and biological variables measured in leaves of N. tabacum ‘Bel-W3’ during the four exposure periods. Different letters show the significant differences among exposure experiments for each parameter analyzed (p < 0.05).
ns: no significance. # Correlations with environmental conditions 3 days before leaf analysis. > Average levels of AA 2 days before injury analysis. > Average levels of O3 5 days before injury analysis.
Leaf injury appeared between the 3rd and 6th days of exposure (Fig. 1). It occurred in almost all of the oldest leaves evaluated (data not shown). The leaf area affected by necrosis did not differ significantly among the exposure periods (Table 1). The average levels of AA were significantly higher during the 1st and 3rd exposure periods than during the 2nd and 4th periods. Average SOD activity did not differ among the exposure periods. In contrast, POD activity decreased significantly in plants from the 3rd experiment (Table 1). The AA concentrations in tobacco Bel-W3 tended to decrease with time of exposure in all experiments, but the daily SOD and POD activities oscillated during plant exposure. High activity of SOD was observed in plants sampled at the beginning of the 1st and 2nd and at the end of the 4th exposure, and high activity of POD was observed at the middle of the 2nd exposure (Fig. 2). Correlation analyses showed that these leaf antioxidants varied independently from each other over the exposure periods (data not shown in Table 2). These analyses also revealed the strongest correlations between AA or SOD and environmental conditions 3 days before leaf analyses. Positive correlations were observed between AA or SOD and O3 concentrations, as well as negative correlations between AA and air temperature. Activity of POD was not significantly related to oscillations on the environmental factors (Table 2). The multiple regression analysis showed that 36% of the increasing percentage of leaf necrosis across the whole period was explained by decreasing levels of AA 2 days before injury estimation and by higher O3 concentrations 5 days before (Table 2). This was the most explanatory linear model among others tested.
Variables
1st exposure 2nd exposure 3rd exposure 4th exposure
4. Discussion
Environmental factors Ozone (ppb) Relative humidity (%) Temperature ( C) Irradiation (MJ m2)
60.5 12.5 a 73.6 25.1 b 19.0 6.3 b 18.4 4.5 a
21.1 2.3 b 83.9 15.8 a 19.2 5.6 b 13.1 5.6 a
35.0 11.7 a 76.6 19.5 b 20.4 5.4 b 22.1 6.9 a
21.2 9.3 b 79.1 19.9 a 23.3 4.7 a 13.2 3.7 a
Biological variables Leaf injury (%) AA (mg g1 DM) SOD (102 U g1 DM) POD (102 DA min1 DM)
6.0 10.0 a 6.6 2.1 a 18.2 10.5 a –
2.0 3.0 a 4.6 1.8 b 12.5 18.8 a 15.9 17.1 a
7.0 10.0 a 5.9 2.9 a 7.8 3.6 a 5.8 4.2 b
18.8 23.0 a 4.9 2.3 b 14.4 20.8 a 14.1 8.0 a
Ascorbic acid (AA) seemed to be the most important antioxidant and a key indicator of environmental stress in plants of N. tabacum ‘Bel-W3’ exposed in the city of Sa˜o Paulo. Among its several functions, such as acting as co-factor for many enzymes, regulator of leaf senescence and defender against pathogen attack, this antioxidant contributes to increasing plant tolerance against oxidative stress by detoxifying hydrogen peroxide (Pastori et al., 2003; Conklin and Barth, 2004; Barth et al., 2004; Pavet et al., 2005; Barth et al., 2006; Burkey et al., 2006; Iriti and Faoro, 2008).
– Data not available.
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ascorbic acid
10 8 6 4 2
peroxidase
superoxide dismutase
0 60 50 40 30 20 10 0 80 70 60 50 40 30 20 10 0 1st exposure
2nd exposure
3th exposure
4th exposure
Fig. 2. Daily profile of ascorbic acid [mg g1 (DM)], superoxide dismutase [102 Uni g1 (DM)] and peroxidase [102 DA min1 (DM)] in leaves of N. tabacum Bel-W3 during the four exposure experiments.
It has also been suggested that ascorbic acid in the cell wall provides a first step of defense against O3 (Baier et al., 2005; Iriti and Faoro, 2008). In tolerant plants, O3 generally promotes increases in AA concentrations and the activity of enzymes from the metabolic route ascorbate-glutathione, so that damage on leaf cells and tissues is minimized (Castillo and Greppin 1988). Conversely, low concentrations of AA in the leaves of deficient mutants have been associated with growth restrictions and enhancement of ozone sensitivity (Conklin et al., 2000; Veljovic-Jovanovic et al., 2001). In the present study, the increased concentrations of O3 coincided with increased levels of AA in leaves of N. tabacum ‘BelW3’. However, the results suggest that the varying levels of leaf ascorbic acid among the experimental periods were one of the reasons for the distinct levels of leaf injury and then of O3 sensitivity verified in plants exposed in Sa˜o Paulo. The contrasting results obtained in the first and last exposure periods clearly illustrate this aspect. Burkey et al. (2006) observed similar relation in wild plants from the Great Smoky Mountains National Park (U.S.A.). The decrease of AA concentrations with time appeared to be a measurement of the increasing loss of antioxidative capacity in tobacco plants exposed at the experimental site in Sa˜o Paulo since this effect was linearly followed by the progression of injury, as also observed by Bulbovas et al. (2007) in plants of a sensitive Brazilian cultivar of soybean. In fact, the progression of leaf injury in the plants seemed to be delayed at least for 2 days of exposure by changes in the contents of AA, which in turn are altered in response to increases in the O3 concentrations in the atmosphere some days before. Therefore, the linear relations between O3 and leaf injury in plants of N. tabacum ‘Bel-W3’ at the monitored site seemed to be time-dependent. The stepwise analysis suggested that a significant part of the progression of leaf injury occurred in response to an increase of O3 levels 5 days before leaf analysis. In fumigation experiments, Sant’Anna et al. (2008) observed similar association between realistic concentrations of ozone and leaf contents of AA
and leaf injury in plants of N. tabacum ‘Bel-W3’. Significant reductions in AA were followed by evident increases in the leaf area affected by necrosis in plants exposed to over 40 ppb of ozone for 4 days. This may be one the possible explanations for the weak relation between average percentages of leaf area of N. tabacum ‘Bel-W3’ affected by necrosis and average O3 concentrations in a biomonitoring study also performed in the city of Sa˜o Paulo by Sant’Anna et al. (2008) and might help to understand similar weak linear relations observed by several other authors (e.g., Ribas and ˜ uelas, 2003; Yuska et al., 2003; Klumpp et al., 2006a,b; CalaPen tayud et al., 2007). Numerous studies have observed that the activity of SOD, POD and other enzymatic antioxidants can oscillate in plants growing in a stressing environment (Klumpp et al., 1989; Baier et al., 2005; Bulbovas et al., 2007; Sant’Anna et al., 2008, Iriti and Faoro, 2008). This effect may depend on the plant species, stage of development, and/or intrinsic metabolic rhythms (Kuk et al., 2003). This effect was observed in plants of N. tabacum Bel-W3 during our field experiments only for SOD. Increased concentrations of O3 happened together with high activity of this enzyme in the plants exposed at the site of study, as verified by Scebba et al. (2003). On the other hand, activity of SOD was reduced in Lycopersicon esculentum cv. Tiny Tim treated with ozone, indicating a disruption of proxidant–antioxidant equilibrium (Calatayud and Barreno, 2001). However, changes in the SOD activity did not affect significantly the progression of visible leaf disturbances in the plants of the present study. As suspected, ozone was not the only environmental factor to cause oscillations in antioxidant responses during the experimental periods. Air temperature appeared to be at least indirectly related to decreased leaf concentrations of AA, as evidenced during the 4th exposure for instance. It should be considered that the O3 flux into the leaves via stomata may be modified by various environmental factors such as air temperature, relative humidity and solar radiation (Karlsson et al., 2004; Filella et al., 2005; Hassan, 2006). Thus, it is plausible to assume that the meteorological conditions during the last field experiment were more appropriate for the O3 uptake than during the other periods, even though the concentrations of this pollutant were comparatively low. This might result in stronger oxidative stress, decreased levels of AA, and, consequently, more intense leaf injury in such experimental period. Some authors, such as Payton et al. (2001) and Ding et al. (2007), also found that plants are increasingly sensitive to oxidants when exposed to chilling temperatures. In conclusion, the employment of the percentage of leaf area affected by necrosis for biomonitoring O3 under the environmental conditions of Sa˜o Paulo must be carefully evaluated. Acknowledgements Authors are gratefully acknowledged to Fundaça˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP; 02/04751-6) and to Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) for the continuous financial support and to the specialists from the American Journal Experts (http://www.journalexperts. com) for the English revision. References Andrade, M.F., Ynoue, R.Y., Harley, R., Miguel, A.H., 2004. Air quality model simulating photochemical formation of pollutants: the Sa˜o Paulo Metropolitan Area, Brazil. International Journal of Environment and Pollution 22 (4), 460–475. Baier, M., Kandlbinder, A., Golldack, D., Dietz, K.-J., 2005. Oxidative stress and ozone: perception, signalling and response. Plant, Cell & Environment 28 (8), 1012–1020. Barth, C., Moeder, W., Klessig, D.F., Conklin, P.L., 2004. The timing of senescence and response to pathogens is altered in the ascorbate-deficient Arabdopsis Mutant vitamin c-11. Plant Physiology 134, 1784–1792.
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