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Effects of nitrogen dioxide and its acid mist on reactive oxygen species production and antioxidant enzyme activity in Arabidopsis plants
JES-00362; No of Pages 7 J O U RN A L OF E N V I RO N ME N TA L S CI EN CE S X X (2 0 1 5 ) XX X–XXX
Available online at www.sciencedirect.com
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Xiaofang Liu1,⁎⁎, Fen Hou2,⁎⁎, Guangke Li1,⁎, Nan Sang1,⁎
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Effects of nitrogen dioxide and its acid mist on reactive oxygen species production and antioxidant enzyme activity in Arabidopsis plants
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1. College of Environment and Resource, Research Center of Environment and Health, Shanxi University, Taiyuan 030006, China 2. College of Environmental Science and Engineering, Nankai University, Tianjin 300071, China
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Article history:
Nitrogen dioxide (NO2) is one of the most common and harmful air pollutants. To analyze 16
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Received 27 October 2014
the response of plants to NO2 stress, we investigated the morphological change, reactive 17
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Revised 7 February 2015
oxygen species (ROS) production and antioxidant enzyme activity in Arabidopsis thaliana 18
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Accepted 3 March 2015
(Col-0) exposed to 1.7, 4, 8.5, and 18.8 mg/m3 NO2. The results indicate that NO2 exposure 19
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Available online xxxx
affected plant growth and chlorophyll (Chl) content, and increased oxygen free radical (O−2) 20
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Keywords:
peroxidation and protein oxidation, accompanied by the induction of antioxidant enzyme 22
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Nitrogen dioxide
activities and change of ascorbate (AsA) and glutathione (GSH) contents. Following this, we 23
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Nitric acid mist
mimicked nitric acid mist under experimental conditions, and confirmed the antioxidant 24
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Arabidopsis thaliana
mechanism of the plant to the stress. Our results imply that NO2 and its acid mist caused 25
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Reactive oxygen species
pollution risk to plant systems. During the process, increased ROS acted as a signal to 26
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Antioxidant system
induce a defense response, and antioxidant status played an important role in plant 27
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production rate in Arabidopsis shoots. Furthermore, NO2 elevated the levels of lipid 21
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protection against NO2/nitric acid mist-caused oxidative damage.
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© 2015 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. 29
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Introduction
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Nitrogen dioxide (NO2) is one of the most common and harmful air pollutants. Due to its massive discharge from motor vehicle exhaust and stationary sources such as electric utilities and industrial boilers, the concentration of NO2 in the atmosphere has gradually increased in many areas of the world during the past few decades. Therefore, NO2 has been a strong indicator in atmospheric environment monitoring and a potential risk factor in adverse effect exploration. As reported, peak levels of up to 0.8–8 mg/m3
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have been encountered in the outdoors, particularly along curbsides in downtown areas with heavy motor vehicular traffic (Pathmanathan et al., 2003). However, due to the development of industrialized production and the continuous rise of automobile exhaust emissions, NO2 concentration will be likely to further increase in the future. Following the increase of NO2 environmental concentration, another serious issue is the occurrence of nitric acid precipitation (nitric acid mist), which results from its combining with atmospheric moisture and causes adverse ecological effects.
⁎ Corresponding authors. E-mail: [email protected] (Guangke Li), [email protected] (Nan Sang). These authors contributed equally to this work.
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http://dx.doi.org/10.1016/j.jes.2015.03.011 1001-0742/© 2015 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.
Please cite this article as: Liu, X., et al., Effects of nitrogen dioxide and its acid mist on reactive oxygen species production and antioxidant enzyme activity in Arabidopsis..., J. Environ. Sci. (2015), http://dx.doi.org/10.1016/j.jes.2015.03.011
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Chl content was determined by the method reported by Bao (2005). Briefly, fresh shoots were pulverized with distilled water, and the homogenate was extracted by 80% acetone. Absorbance of the supernatant was measured at 663 and 645 nm using a spectrophotometer and Chl content was expressed as mg/g fresh weight (fw).
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1.2. Measurement of chlorophyll (Chl) content
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were disinfected with sodium hypochlorite at 1% (V/V, containing Triton-100 at 0.01%), washed three times with running water, and soaked in the water. After vernalization for 3 days at 4°C, they were seeded in seedling trays about 4.5 × 3.0 × 4.0 cm, 5 plants per tray, with peat imported from Germany as substrate (a kind of pure moss peat, with proportions of N, P and K of 14/16/18, and pH 5.5–6.5). Arabidopsis plants were grown in a controlled growth chamber at 22 ± 1°C with a 16 hr photoperiod per day, 70% relative humidity and a photosynthetic photon flux density of 140 μmol/(m2·sec). Four-week-old plants were exposed to 1.7, 4, 8.5, and 18.8 mg/m3 NO2 at 6 hr/day for 7 days, respectively, while a control group was placed in another identical chamber, which was continually flushed with filtered air for the same period of time. The NO2 gas was diluted with fresh air at the intake port of the chamber to yield the desired concentrations; then delivered to the plants through a tube positioned in the upper part of each chamber and distributed homogeneously via a fan. The NO2 concentration within the chambers was measured every 30 min by the Saltzman colorimetric method using a spectrometer calibrated at 545 nm (Kumie et al., 2009). The desired NO2 concentrations were controlled by the opening of a flow valve. For nitric acid mist treatment, four-week-old A. thaliana was exposed to nitric acid fog (pH 4.6–5.0), which was produced by misting HNO3 solution for 4, 6 and 8 hr/day for 7 days, respectively. To obtain the misting HNO3 solution, HNO3 solution was diluted by distilled water to the proper pH value, and then sprayed on the leaf surface with a sprayer in a mist flow. Meanwhile, a control group was placed in another identical chamber, which was continually exposed to water fog for the same period of time.
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Recently, epidemiologic data and experimental results have linked NO2 exposure with a wide range of effects on human health, including increased mortality risk, increased rates of hospital admissions and emergency department visits, exacerbation of chronic respiratory conditions (e.g., asthma), and decreased lung function (Samet and Krewski, 2007). Therefore, it is expected that plants might be affected after exposure to atmospheric NO2 and its acid mist pollution. Some studies have demonstrated that NO2 could enter leaf mesophyll tissue through stomata, combine with water and then convert to nitric acid, which burns plant tissue. The symptom appears as lesions visible on both sides of the leaves, which initially occurs between leaf veins or along leaf edges (Li et al., 2007; Miao et al., 2008). A deeper level of toxicity was also studied for some plants such as Brassica campestris seedlings, Cinnamomum camphora seedlings and tomato plants (Ma et al., 2007a,b; Pandey and Agrawal, 1994; Teng et al., 2010). NO2 could induce change to growth and photosynthetic activity, causing oxidative damage accompanied by changes in the antioxidant defense system (Chen et al., 2010; Ma et al., 2007a; Takahashil et al., 2011). However, whether these toxic effects of NO2 apply to other plants or whether similar symptoms will occur under the stress of nitric acid mist is not yet clear. Hence, Arabidopsis thaliana, an internationally recognized model plant, was chosen in this study to evaluate the specific plant response to NO2 and nitric acid mist exposure, and the research results will be extended to other plants. There is ample evidence that reactive oxygen species (ROS) are crucial second messengers involved in the response to diverse abiotic and biotic stresses, and can confer a degree of cross-tolerance against distinct stresses (Apel and Hirt, 2004; Foyer and Noctor, 2005a). NO2 is an oxidant pollutant, and induces oxidative damage to cell membranes, resulting in the generation of ROS (Mustafa and Tierney, 1978; Pathmanathan et al., 2003). Increased ROS can attack biomacromolecules and result in oxidative damage to nucleic acids, proteins and lipids (Foyer and Noctor, 2005b; Mittler et al., 2004; Yi et al., 2005). However, plants can scavenge excess ROS by invoking the antioxidant defense system to avoid oxidative damage (Inzé and Van-Montagu, 1995; Mittler, 2002). The induction of antioxidant enzymes has been thought to be a protective reaction of plants against NO2 stress, but the exact defense mechanism was not clear. In the present study, we characterized the changes in morphological features and physiological indexes in response to NO2 and nitric acid mist in Arabidopsis shoots, and determined ROS production and antioxidant enzyme activities. Our research provides a particular insight into the capacity of NO2/ nitric acid mist to induce cellular ROS and antioxidant response in plant cells, and contributes to understanding the underlying mechanisms of toxicological reaction and plant adaptation to NO2/nitric acid mist stress.
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1.3. Measurement of oxygen free radical (O−2 ) production rate 160 O−2 production rate was determined by the hydroxylamine method (Zhang and Qu, 2003). Briefly, fresh leaf sample was homogenized in phosphate buffer (0.05 mol/L, pH 7.8), and the homogenate was centrifuged at 10,000 g for 20 min. The supernatant was mixed with hydroxylamine hydrochloride, and then kept at 25°C for 1 hr. α-Naphthylamine and aminobenzenesulfonic acid were used as chromogenic agents. Absorbance of the supernatant was measured at 530 nm using a spectrophotometer.
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1. Materials and methods
1.4. Estimation of ascorbate (AsA) and glutathione (GSH) pool 170
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1.1. Plant materials and NO2/nitric acid mist treatment
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Plants of A. thaliana (L.) ecotype Columbia (Col-0) were purchased from the Chinese Academy of Agricultural Sciences. The seeds
The content of reduced AsA was determined spectrophotometrically according to the method of Kampfenkel et al (1995). The amount of reduced GSH was examined according to the method of Sgherri and Navari-Izzo (1995).
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Please cite this article as: Liu, X., et al., Effects of nitrogen dioxide and its acid mist on reactive oxygen species production and antioxidant enzyme activity in Arabidopsis..., J. Environ. Sci. (2015), http://dx.doi.org/10.1016/j.jes.2015.03.011
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1.5. Measurement of lipid peroxidation level
1.8. Statistical analysis of data
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Experiments were repeated three times, and all values were expressed as mean ± SE. In the study, all experiment groups and control group were placed in several identical chambers separately, which were kept at the same temperature, photoperiod, relative humidity, etc. The exposure to NO2 and nitric acid mist were considered to be the single factor for NO2 treatment and nitric acid mist treatment, respectively, which were adopted to perform single factor analysis. Therefore, the statistical differences (0.05, 0.01 or 0.001) among the negative control and a series of treated groups were analyzed by one-way analysis of variance (ANOVA), using the Origin 7.0 software package.
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The level of lipid peroxidation was estimated by measuring the concentration of malondialdehyde (MDA). MDA is a common product of lipid peroxidation and is a sensitive diagnostic index of oxidative injury (Janero, 1990; El-Moshaty et al, 1993). Fresh leaf sample was ground in 5% trichloroacetic acid (W/V) and the homogenate was centrifuged at 4000 g for 10 min. The supernatant fraction was added to an equal volume of 0.6% thiobarbituric acid (W/V). The mixture was heated at 100°C for 10 min and then centrifuged at 3000 g for 10 min after it was cooled. The absorbance of the supernatant was measured at 532 nm and lipid peroxidation level was expressed as μmol MDA/g fw. Protein carbonyl (PCO) content is the most general indicator and marker of protein oxidative damage. In the present study, PCO level was tested by 2,4-dinitrophenylhydrazine (DNPH) spectrophotometry (Levine et al., 1990). Briefly, fresh leaf sample (0.5 g) was ground in phosphate buffer (0.05 mol/L, pH 7.0) and the homogenate was centrifuged at 10,000 g for 5 min at 4°C. The supernatant was used for DNPH reactions. PCO content was calculated by the absorbance at 370 nm, and the result was expressed as nmol carbonyl/mg protein.
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1.6. Assay of antioxidant enzyme activities
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Superoxide dismutase (SOD) activity was measured by Nitro Blue tetrazolium (NBT) spectrophotometry (Zhu et al., 1990). Fresh leaf sample (0.5 g) was homogenized in phosphate buffer (0.05 M, pH 7.8) and the homogenate was centrifuged at 1000 g for 20 min at 4°C. The supernatant was added to a reaction mixture containing 130 mmol/L DL-methionine (Met), 750 μmol/L NBT, 100 μmol/L EDTA-Na2, and 20 μmol/L lactoflavin, in order to determine the absorbance at 560 nm. Inhibition of 50% of the reaction was defined as one unit of enzyme and the enzyme activity was expressed as U/g fw. Catalase (CAT) activity was assayed according to the method of Zhang (1990), with some modifications. Fresh leaf sample (0.25 g) was homogenized in phosphate buffer (0.2 mol/L, pH 7.8) containing 1% polyvinylpyrrolidone K30. The homogenate was centrifuged at 4000 g for 15 min at 4°C and the supernatant was used for the enzyme assay. CAT activity was examined by measuring the decrease of absorbance at 240 nm in a reaction mixture containing 0.3 mL H2O2 (0.1 mol/L) and 0.1 mL extract. Results were expressed as U/(min·g) fw. Peroxidase (POD) activity was determined using the guaiacol method (Maehly, 1955). Briefly, fresh leaf sample (0.1 g) was homogenized in phosphate buffer (0.1 mol/L, pH 6.0) and the homogenate was centrifuged at 4000 g for 15 min at 4°C. The supernatant was added to the reaction mixture, including 5 mmol/L guaiacol as the donor and 6.7 mmol/L H2O2 as the substrate. The rate of change in absorbance at 470 nm was measured and the activity was expressed as DA470/min/g fw.
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1.7. Determination of dissolved protein
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The amount of dissolved protein was determined by the Bradford method (Bradford, 1976). We used bovine serum albumin as the standard and results were expressed as mg/g fw.
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2.1. Effect of NO2 exposure on the morphology and Chl content 245 in Arabidopsis shoots 246
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To find the general toxic response, we first exposed Arabidopsis to NO2 at different concentrations (1.7, 4, 8.5 and 18.8 mg/m3) and observed their morphological changes. As shown in Fig. 1a, NO2 at lower concentrations affected the plant growth, and induced the occurrence of yellowing leaf after 4 and 8.5 mg/m3 exposure. However, when the NO2 concentration increased to 18.8 mg/m3, the plant appeared to have visibly acute injuries on the leaf and eventually died after treatment. Because of this, the 18.8 mg/m3 exposure was not considered in follow-up experiments. Following this morphological change, we further determined Chl content in Arabidopsis shoots, which in vivo was an important kind of pigment involved in the processes of photosynthesis. Fig. 1b indicates that Chl content was the lowest after 4 mg/m3 exposure, but there was no significant difference among the treated plants compared to the control.
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2.2. Effect of NO2 on ROS generation and antioxidants (AsA 263 and GSH) in Arabidopsis shoots 264 Exposure to NO2 caused an increase of ROS generation in Arabidopsis shoot cells. The O−2 generation rate enhanced with increasing NO2 concentration, and a statistical difference was observed at all exposure concentrations (Fig. 2a). AsA content tended to decrease with the enhancement of NO2 concentration, and a statistical difference occurred at the 8.5 mg/m3 group (Fig. 2b). On the contrary, GSH content showed an increasing tendency with increasing NO2 concentration, and was significantly induced after 1.7 and 8.5 mg/m3 exposure (Fig. 2c).
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2.3. Effect of NO2 on levels of lipid peroxidation and protein 275 oxidation in Arabidopsis shoots 276 The levels of lipid peroxidation and protein oxidation in NO2-treated Arabidopsis plants were measured from the contents of MDA and PCO. As presented in Fig. 3, MDA content and PCO level varied in a NO2 concentration-dependent manner and statistical increases were observed at 4 and 8.5 mg/m3,
Please cite this article as: Liu, X., et al., Effects of nitrogen dioxide and its acid mist on reactive oxygen species production and antioxidant enzyme activity in Arabidopsis..., J. Environ. Sci. (2015), http://dx.doi.org/10.1016/j.jes.2015.03.011
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indicating oxidative damage in plant cells after NO2 exposure at 282 higher concentrations. 283
2.4. Effect of NO2 on antioxidant enzyme activities in Arabidopsis 284 shoots 285 Fig. 4 indicates the changes of antioxidant enzyme activities in response to NO2 exposure. SOD activity increased as a function of NO2 concentration, and significant difference occurred at 4 and 8.5 mg/m3. POD activity remained unchanged after 1.7 mg/m3 treatment, and statistically increased when the NO2 concentration reached 4 and 8.5 mg/m3, despite a slight decline at 8.5 mg/m3. For CAT, it tended to be induced after the 8.5 mg/m3 treatment, but no statistical difference was observed at all exposure concentrations.
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To mimic the actually environmental exposure, we treated Arabidopsis with nitric acid mist for different exposure durations. Fig. 5a shows that with exposure to nitric acid mist, the Chl content significantly decreased with the increase of treatment time, and a statistical difference was observed after 6 and 8 hr/day exposure. Following the result, we observed the induction of MDA and PCO contents after nitric acid mist exposure for different durations (Fig. 5b). The MDA level tended to increase, but no statistical difference occurred, while the PCO content increased with the enhancement of treatment time, and showed significant difference when the daily exposure time reached 8 hr/day. Also, we further tested the change of SOD, POD and CAT activities (Fig. 5c). SOD activity changed slightly in all groups of A. thaliana, but no statistical difference occurred. POD activity significantly increased after exposure to nitric acid mist. CAT activity remained unchanged after 4 and 6 hr/day treatments for 7 days, and was slightly but statistically enhanced after the daily exposure time reached 8 hr/day.
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3. Discussion
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Fig. 1 – Effect of NO2 exposure on the morphology (a) and Chl content (b) in Arabidopsis shoots. Data represent the mean ± SE of three different experiments.
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2.5. Effect of nitric acid mist exposure on Chl content, lipid 295 peroxidation and protein oxidation, and antioxidant enzyme 296 activities in Arabidopsis shoots 297
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Fig. 2 – Effect of NO2 on ROS generation (a) and antioxidant contents (AsA (b) and GSH (c)) in Arabidopsis shoots. Data represent the mean ± SE of three different experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control. Please cite this article as: Liu, X., et al., Effects of nitrogen dioxide and its acid mist on reactive oxygen species production and antioxidant enzyme activity in Arabidopsis..., J. Environ. Sci. (2015), http://dx.doi.org/10.1016/j.jes.2015.03.011
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4 mg/m3 NO2 exposure. This finding suggests that NO2 at lower concentrations stimulated defense reactions, and these defense reactions might counteract ROS generated via elevated antioxidant defense systems in the tissue; whereas high concentration NO2 exposure induced the production of ROS, and the capacity of the antioxidant system was exceeded, or inhibited. As a result, the excessive ROS attacked lipids and proteins and the plant experienced substantial oxidative stress. Additionally, the activities of antioxidant enzymes (SOD, CAT and POD) were tested under the same treatment conditions. In this study, NO2 enhanced the activities of several antioxidant enzymes including SOD, POD and CAT associated with increased ROS generation, suggesting an induction of antioxidant defenses regulated by a ROS-mediated signaling pathway. SOD is a protein class that contains metals and catalyzes the dismutation of superoxide radical anions into H2O2 and molecular oxygen (Scandalios, 1993). The elevated SOD activity in Arabidopsis plants suggests that the chemical induced SOD to increase the generation of H2O2. CAT and POD are the primary H2O2-scavenging enzymes in plant cells. In this study, NO2 pronouncedly increased POD activity, whereas it had little effect on CAT activity. CAT has a high capacity but low affinity enzyme for H2O2, whereas POD has a high affinity for H2O2 (Mittler, 2002). After NO2 exposure and shorter daily exposure to nitric acid mist, POD activity was significantly
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Inhibited growth and Chl content in the present study might be attributed to the damaged defense system and consequently unbalanced metabolism. In plant cells, ROS are unavoidable by-products of aerobic metabolism. Under normal growth conditions, amounts of ROS are modest and cells experience only mild oxidative stress, whereas many stresses enhance ROS production (Inzé and Van-Montagu, 1995; Mittler et al., 2004). The results of our present study clearly show that NO2 triggered a rapid increase in O−2 generation rate in Arabidopsis shoots. The enhanced production of ROS under stress can pose a threat to cells, but it is also thought that ROS serve as signal molecules to activate the stress responses and defense pathways (Apel and Hirt, 2004; Knight and Knight, 2001; Mittler, 2002). Thus, NO2-induced ROS can be viewed as cellular indicators of stress and as secondary messengers involved in the stress-response signal transduction pathway. Since membrane lipids and proteins are the preferred targets of ROS in plants under environmental stress (Prassad, 1996), they are considered to be reliable indicators of controlled modulation of ROS levels and oxidative stress (Halliwell and Gutteridge, 1993). Therefore, we further investigated the levels of lipid peroxidation and protein oxidation by measuring MDA and PCO contents. In Arabidopsis shoots, the MDA level and PCO content increased, and oxidative stress occurred after
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Fig. 3 – Effect of NO2 on levels of lipid peroxidation (a) and protein oxidation (b) in Arabidopsis shoots. Data represent the mean ± SE of three different experiments.*p < 0.05, **p < 0.01, ***p < 0.001 vs. control.
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Fig. 4 – Effect of NO2 on antioxidant enzyme activities (including: SOD (a), POD (b), CAT(c)) in Arabidopsis shoots. Data represent the mean ± SE of three different experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control. Please cite this article as: Liu, X., et al., Effects of nitrogen dioxide and its acid mist on reactive oxygen species production and antioxidant enzyme activity in Arabidopsis..., J. Environ. Sci. (2015), http://dx.doi.org/10.1016/j.jes.2015.03.011
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enhanced and eliminated part of the generated H2O2. As a result, the remaining H2O2 might not be enough to induce CAT activity. This was not the case for the longest daily exposure, which accumulated more H2O2 and induced CAT activity. Thus, POD, but not CAT, was the most efficient scavenging enzyme to decrease the cellular levels of H2O2 in plant cells under NO2 stress. Moreover, the increase of POD activity can contribute to the overall cell resistance to NO2 stress since POD participates in many other cell processes involved in the plant defense reaction (Valério et al., 2004). POD can initiate cell-wall toughening events such as phenolic cross-linking and lignification, which can strengthen leaf and stem tissues against potential damage (Polle et al., 1994). The roles that POD can play in cell wall toughening and in the production of secondary metabolites, and its simultaneous oxidant and antioxidant capabilities, make it an important factor in the integrated defense response of plants to NO2 stress. Additionally the AsA– GSH redox system in chloroplasts is also an effective scavenger of O−2 and H2O2 in plant cells (Foyer and Halliwell, 1976; Jiang and Chen, 2008). Thus as NO2 concentrations increased, GSH content increased, but AsA content decreased, suggesting that the antioxidants also played an important role in plant defenses against NO2 stress.
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Fig. 5 – Effect of nitric acid mist exposure on Chl content (a), oxidative stress (b) and antioxidant enzyme activities (c) in Arabidopsis shoots. Data represent the mean ± SE of three different experiments.*p < 0.05, **p < 0.01, ***p < 0.001 vs. control.
4. Conclusions
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NO2 exposure affected plant growth and chlorophyll level, and increased O−2 production rate in Arabidopsis shoots. Furthermore, NO2 elevated the levels of lipid peroxidation and protein oxidation, accompanied by the induction of SOD, POD and CAT activities and the change of antioxidant contents. Following this, we mimicked nitric acid mist under experimental conditions, and confirmed antioxidant mechanisms of the plant in response to the stress. Our results imply that NO2 and its acid mist caused pollution risk to plant systems, increased ROS acted as a signal to induce a defense response, and antioxidant status played an important role in plant protection against NO2/nitric acid mist-caused oxidative damages.
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Acknowledgments
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This study was supported by the National Natural Science 411 Foundation of China (Nos. 21477070, 21377076), the Specialized 412 Research Fund for the Doctoral Program of Higher Education 413
Please cite this article as: Liu, X., et al., Effects of nitrogen dioxide and its acid mist on reactive oxygen species production and antioxidant enzyme activity in Arabidopsis..., J. Environ. Sci. (2015), http://dx.doi.org/10.1016/j.jes.2015.03.011
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(SRFDP) (Nos. 20121401110003, 20131401110005), the Project for Science and Technology Development of Shanxi Province (No. 20120313009-2), the Research Project supported by the Shanxi Scholarship Council of China (No. 2012-009), and the Program for the Top Young and Middle aged Innovative Talents of Higher Learning Institutions of Shanxi (TYMIT, No. 20120201).
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Please cite this article as: Liu, X., et al., Effects of nitrogen dioxide and its acid mist on reactive oxygen species production and antioxidant enzyme activity in Arabidopsis..., J. Environ. Sci. (2015), http://dx.doi.org/10.1016/j.jes.2015.03.011
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Available online at www.sciencedirect.com
ScienceDirect www.journals.elsevier.com/journal-of-environmental-sciences
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Xiaofang Liu1,⁎⁎, Fen Hou2,⁎⁎, Guangke Li1,⁎, Nan Sang1,⁎
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Effects of nitrogen dioxide and its acid mist on reactive oxygen species production and antioxidant enzyme activity in Arabidopsis plants
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1. College of Environment and Resource, Research Center of Environment and Health, Shanxi University, Taiyuan 030006, China 2. College of Environmental Science and Engineering, Nankai University, Tianjin 300071, China
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Article history:
Nitrogen dioxide (NO2) is one of the most common and harmful air pollutants. To analyze 16
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Received 27 October 2014
the response of plants to NO2 stress, we investigated the morphological change, reactive 17
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Revised 7 February 2015
oxygen species (ROS) production and antioxidant enzyme activity in Arabidopsis thaliana 18
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Accepted 3 March 2015
(Col-0) exposed to 1.7, 4, 8.5, and 18.8 mg/m3 NO2. The results indicate that NO2 exposure 19
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Available online xxxx
affected plant growth and chlorophyll (Chl) content, and increased oxygen free radical (O−2) 20
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Keywords:
peroxidation and protein oxidation, accompanied by the induction of antioxidant enzyme 22
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Nitrogen dioxide
activities and change of ascorbate (AsA) and glutathione (GSH) contents. Following this, we 23
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Nitric acid mist
mimicked nitric acid mist under experimental conditions, and confirmed the antioxidant 24
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Arabidopsis thaliana
mechanism of the plant to the stress. Our results imply that NO2 and its acid mist caused 25
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Reactive oxygen species
pollution risk to plant systems. During the process, increased ROS acted as a signal to 26
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Antioxidant system
induce a defense response, and antioxidant status played an important role in plant 27
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production rate in Arabidopsis shoots. Furthermore, NO2 elevated the levels of lipid 21
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protection against NO2/nitric acid mist-caused oxidative damage.
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Introduction
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Nitrogen dioxide (NO2) is one of the most common and harmful air pollutants. Due to its massive discharge from motor vehicle exhaust and stationary sources such as electric utilities and industrial boilers, the concentration of NO2 in the atmosphere has gradually increased in many areas of the world during the past few decades. Therefore, NO2 has been a strong indicator in atmospheric environment monitoring and a potential risk factor in adverse effect exploration. As reported, peak levels of up to 0.8–8 mg/m3
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have been encountered in the outdoors, particularly along curbsides in downtown areas with heavy motor vehicular traffic (Pathmanathan et al., 2003). However, due to the development of industrialized production and the continuous rise of automobile exhaust emissions, NO2 concentration will be likely to further increase in the future. Following the increase of NO2 environmental concentration, another serious issue is the occurrence of nitric acid precipitation (nitric acid mist), which results from its combining with atmospheric moisture and causes adverse ecological effects.
⁎ Corresponding authors. E-mail: [email protected] (Guangke Li), [email protected] (Nan Sang). These authors contributed equally to this work.
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http://dx.doi.org/10.1016/j.jes.2015.03.011 1001-0742/© 2015 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.
Please cite this article as: Liu, X., et al., Effects of nitrogen dioxide and its acid mist on reactive oxygen species production and antioxidant enzyme activity in Arabidopsis..., J. Environ. Sci. (2015), http://dx.doi.org/10.1016/j.jes.2015.03.011
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Chl content was determined by the method reported by Bao (2005). Briefly, fresh shoots were pulverized with distilled water, and the homogenate was extracted by 80% acetone. Absorbance of the supernatant was measured at 663 and 645 nm using a spectrophotometer and Chl content was expressed as mg/g fresh weight (fw).
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were disinfected with sodium hypochlorite at 1% (V/V, containing Triton-100 at 0.01%), washed three times with running water, and soaked in the water. After vernalization for 3 days at 4°C, they were seeded in seedling trays about 4.5 × 3.0 × 4.0 cm, 5 plants per tray, with peat imported from Germany as substrate (a kind of pure moss peat, with proportions of N, P and K of 14/16/18, and pH 5.5–6.5). Arabidopsis plants were grown in a controlled growth chamber at 22 ± 1°C with a 16 hr photoperiod per day, 70% relative humidity and a photosynthetic photon flux density of 140 μmol/(m2·sec). Four-week-old plants were exposed to 1.7, 4, 8.5, and 18.8 mg/m3 NO2 at 6 hr/day for 7 days, respectively, while a control group was placed in another identical chamber, which was continually flushed with filtered air for the same period of time. The NO2 gas was diluted with fresh air at the intake port of the chamber to yield the desired concentrations; then delivered to the plants through a tube positioned in the upper part of each chamber and distributed homogeneously via a fan. The NO2 concentration within the chambers was measured every 30 min by the Saltzman colorimetric method using a spectrometer calibrated at 545 nm (Kumie et al., 2009). The desired NO2 concentrations were controlled by the opening of a flow valve. For nitric acid mist treatment, four-week-old A. thaliana was exposed to nitric acid fog (pH 4.6–5.0), which was produced by misting HNO3 solution for 4, 6 and 8 hr/day for 7 days, respectively. To obtain the misting HNO3 solution, HNO3 solution was diluted by distilled water to the proper pH value, and then sprayed on the leaf surface with a sprayer in a mist flow. Meanwhile, a control group was placed in another identical chamber, which was continually exposed to water fog for the same period of time.
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Recently, epidemiologic data and experimental results have linked NO2 exposure with a wide range of effects on human health, including increased mortality risk, increased rates of hospital admissions and emergency department visits, exacerbation of chronic respiratory conditions (e.g., asthma), and decreased lung function (Samet and Krewski, 2007). Therefore, it is expected that plants might be affected after exposure to atmospheric NO2 and its acid mist pollution. Some studies have demonstrated that NO2 could enter leaf mesophyll tissue through stomata, combine with water and then convert to nitric acid, which burns plant tissue. The symptom appears as lesions visible on both sides of the leaves, which initially occurs between leaf veins or along leaf edges (Li et al., 2007; Miao et al., 2008). A deeper level of toxicity was also studied for some plants such as Brassica campestris seedlings, Cinnamomum camphora seedlings and tomato plants (Ma et al., 2007a,b; Pandey and Agrawal, 1994; Teng et al., 2010). NO2 could induce change to growth and photosynthetic activity, causing oxidative damage accompanied by changes in the antioxidant defense system (Chen et al., 2010; Ma et al., 2007a; Takahashil et al., 2011). However, whether these toxic effects of NO2 apply to other plants or whether similar symptoms will occur under the stress of nitric acid mist is not yet clear. Hence, Arabidopsis thaliana, an internationally recognized model plant, was chosen in this study to evaluate the specific plant response to NO2 and nitric acid mist exposure, and the research results will be extended to other plants. There is ample evidence that reactive oxygen species (ROS) are crucial second messengers involved in the response to diverse abiotic and biotic stresses, and can confer a degree of cross-tolerance against distinct stresses (Apel and Hirt, 2004; Foyer and Noctor, 2005a). NO2 is an oxidant pollutant, and induces oxidative damage to cell membranes, resulting in the generation of ROS (Mustafa and Tierney, 1978; Pathmanathan et al., 2003). Increased ROS can attack biomacromolecules and result in oxidative damage to nucleic acids, proteins and lipids (Foyer and Noctor, 2005b; Mittler et al., 2004; Yi et al., 2005). However, plants can scavenge excess ROS by invoking the antioxidant defense system to avoid oxidative damage (Inzé and Van-Montagu, 1995; Mittler, 2002). The induction of antioxidant enzymes has been thought to be a protective reaction of plants against NO2 stress, but the exact defense mechanism was not clear. In the present study, we characterized the changes in morphological features and physiological indexes in response to NO2 and nitric acid mist in Arabidopsis shoots, and determined ROS production and antioxidant enzyme activities. Our research provides a particular insight into the capacity of NO2/ nitric acid mist to induce cellular ROS and antioxidant response in plant cells, and contributes to understanding the underlying mechanisms of toxicological reaction and plant adaptation to NO2/nitric acid mist stress.
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1.3. Measurement of oxygen free radical (O−2 ) production rate 160 O−2 production rate was determined by the hydroxylamine method (Zhang and Qu, 2003). Briefly, fresh leaf sample was homogenized in phosphate buffer (0.05 mol/L, pH 7.8), and the homogenate was centrifuged at 10,000 g for 20 min. The supernatant was mixed with hydroxylamine hydrochloride, and then kept at 25°C for 1 hr. α-Naphthylamine and aminobenzenesulfonic acid were used as chromogenic agents. Absorbance of the supernatant was measured at 530 nm using a spectrophotometer.
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1. Materials and methods
1.4. Estimation of ascorbate (AsA) and glutathione (GSH) pool 170
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Plants of A. thaliana (L.) ecotype Columbia (Col-0) were purchased from the Chinese Academy of Agricultural Sciences. The seeds
The content of reduced AsA was determined spectrophotometrically according to the method of Kampfenkel et al (1995). The amount of reduced GSH was examined according to the method of Sgherri and Navari-Izzo (1995).
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Please cite this article as: Liu, X., et al., Effects of nitrogen dioxide and its acid mist on reactive oxygen species production and antioxidant enzyme activity in Arabidopsis..., J. Environ. Sci. (2015), http://dx.doi.org/10.1016/j.jes.2015.03.011
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1.5. Measurement of lipid peroxidation level
1.8. Statistical analysis of data
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Experiments were repeated three times, and all values were expressed as mean ± SE. In the study, all experiment groups and control group were placed in several identical chambers separately, which were kept at the same temperature, photoperiod, relative humidity, etc. The exposure to NO2 and nitric acid mist were considered to be the single factor for NO2 treatment and nitric acid mist treatment, respectively, which were adopted to perform single factor analysis. Therefore, the statistical differences (0.05, 0.01 or 0.001) among the negative control and a series of treated groups were analyzed by one-way analysis of variance (ANOVA), using the Origin 7.0 software package.
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The level of lipid peroxidation was estimated by measuring the concentration of malondialdehyde (MDA). MDA is a common product of lipid peroxidation and is a sensitive diagnostic index of oxidative injury (Janero, 1990; El-Moshaty et al, 1993). Fresh leaf sample was ground in 5% trichloroacetic acid (W/V) and the homogenate was centrifuged at 4000 g for 10 min. The supernatant fraction was added to an equal volume of 0.6% thiobarbituric acid (W/V). The mixture was heated at 100°C for 10 min and then centrifuged at 3000 g for 10 min after it was cooled. The absorbance of the supernatant was measured at 532 nm and lipid peroxidation level was expressed as μmol MDA/g fw. Protein carbonyl (PCO) content is the most general indicator and marker of protein oxidative damage. In the present study, PCO level was tested by 2,4-dinitrophenylhydrazine (DNPH) spectrophotometry (Levine et al., 1990). Briefly, fresh leaf sample (0.5 g) was ground in phosphate buffer (0.05 mol/L, pH 7.0) and the homogenate was centrifuged at 10,000 g for 5 min at 4°C. The supernatant was used for DNPH reactions. PCO content was calculated by the absorbance at 370 nm, and the result was expressed as nmol carbonyl/mg protein.
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Superoxide dismutase (SOD) activity was measured by Nitro Blue tetrazolium (NBT) spectrophotometry (Zhu et al., 1990). Fresh leaf sample (0.5 g) was homogenized in phosphate buffer (0.05 M, pH 7.8) and the homogenate was centrifuged at 1000 g for 20 min at 4°C. The supernatant was added to a reaction mixture containing 130 mmol/L DL-methionine (Met), 750 μmol/L NBT, 100 μmol/L EDTA-Na2, and 20 μmol/L lactoflavin, in order to determine the absorbance at 560 nm. Inhibition of 50% of the reaction was defined as one unit of enzyme and the enzyme activity was expressed as U/g fw. Catalase (CAT) activity was assayed according to the method of Zhang (1990), with some modifications. Fresh leaf sample (0.25 g) was homogenized in phosphate buffer (0.2 mol/L, pH 7.8) containing 1% polyvinylpyrrolidone K30. The homogenate was centrifuged at 4000 g for 15 min at 4°C and the supernatant was used for the enzyme assay. CAT activity was examined by measuring the decrease of absorbance at 240 nm in a reaction mixture containing 0.3 mL H2O2 (0.1 mol/L) and 0.1 mL extract. Results were expressed as U/(min·g) fw. Peroxidase (POD) activity was determined using the guaiacol method (Maehly, 1955). Briefly, fresh leaf sample (0.1 g) was homogenized in phosphate buffer (0.1 mol/L, pH 6.0) and the homogenate was centrifuged at 4000 g for 15 min at 4°C. The supernatant was added to the reaction mixture, including 5 mmol/L guaiacol as the donor and 6.7 mmol/L H2O2 as the substrate. The rate of change in absorbance at 470 nm was measured and the activity was expressed as DA470/min/g fw.
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The amount of dissolved protein was determined by the Bradford method (Bradford, 1976). We used bovine serum albumin as the standard and results were expressed as mg/g fw.
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2.1. Effect of NO2 exposure on the morphology and Chl content 245 in Arabidopsis shoots 246
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To find the general toxic response, we first exposed Arabidopsis to NO2 at different concentrations (1.7, 4, 8.5 and 18.8 mg/m3) and observed their morphological changes. As shown in Fig. 1a, NO2 at lower concentrations affected the plant growth, and induced the occurrence of yellowing leaf after 4 and 8.5 mg/m3 exposure. However, when the NO2 concentration increased to 18.8 mg/m3, the plant appeared to have visibly acute injuries on the leaf and eventually died after treatment. Because of this, the 18.8 mg/m3 exposure was not considered in follow-up experiments. Following this morphological change, we further determined Chl content in Arabidopsis shoots, which in vivo was an important kind of pigment involved in the processes of photosynthesis. Fig. 1b indicates that Chl content was the lowest after 4 mg/m3 exposure, but there was no significant difference among the treated plants compared to the control.
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2.2. Effect of NO2 on ROS generation and antioxidants (AsA 263 and GSH) in Arabidopsis shoots 264 Exposure to NO2 caused an increase of ROS generation in Arabidopsis shoot cells. The O−2 generation rate enhanced with increasing NO2 concentration, and a statistical difference was observed at all exposure concentrations (Fig. 2a). AsA content tended to decrease with the enhancement of NO2 concentration, and a statistical difference occurred at the 8.5 mg/m3 group (Fig. 2b). On the contrary, GSH content showed an increasing tendency with increasing NO2 concentration, and was significantly induced after 1.7 and 8.5 mg/m3 exposure (Fig. 2c).
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2.3. Effect of NO2 on levels of lipid peroxidation and protein 275 oxidation in Arabidopsis shoots 276 The levels of lipid peroxidation and protein oxidation in NO2-treated Arabidopsis plants were measured from the contents of MDA and PCO. As presented in Fig. 3, MDA content and PCO level varied in a NO2 concentration-dependent manner and statistical increases were observed at 4 and 8.5 mg/m3,
Please cite this article as: Liu, X., et al., Effects of nitrogen dioxide and its acid mist on reactive oxygen species production and antioxidant enzyme activity in Arabidopsis..., J. Environ. Sci. (2015), http://dx.doi.org/10.1016/j.jes.2015.03.011
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indicating oxidative damage in plant cells after NO2 exposure at 282 higher concentrations. 283
2.4. Effect of NO2 on antioxidant enzyme activities in Arabidopsis 284 shoots 285 Fig. 4 indicates the changes of antioxidant enzyme activities in response to NO2 exposure. SOD activity increased as a function of NO2 concentration, and significant difference occurred at 4 and 8.5 mg/m3. POD activity remained unchanged after 1.7 mg/m3 treatment, and statistically increased when the NO2 concentration reached 4 and 8.5 mg/m3, despite a slight decline at 8.5 mg/m3. For CAT, it tended to be induced after the 8.5 mg/m3 treatment, but no statistical difference was observed at all exposure concentrations.
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To mimic the actually environmental exposure, we treated Arabidopsis with nitric acid mist for different exposure durations. Fig. 5a shows that with exposure to nitric acid mist, the Chl content significantly decreased with the increase of treatment time, and a statistical difference was observed after 6 and 8 hr/day exposure. Following the result, we observed the induction of MDA and PCO contents after nitric acid mist exposure for different durations (Fig. 5b). The MDA level tended to increase, but no statistical difference occurred, while the PCO content increased with the enhancement of treatment time, and showed significant difference when the daily exposure time reached 8 hr/day. Also, we further tested the change of SOD, POD and CAT activities (Fig. 5c). SOD activity changed slightly in all groups of A. thaliana, but no statistical difference occurred. POD activity significantly increased after exposure to nitric acid mist. CAT activity remained unchanged after 4 and 6 hr/day treatments for 7 days, and was slightly but statistically enhanced after the daily exposure time reached 8 hr/day.
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Fig. 1 – Effect of NO2 exposure on the morphology (a) and Chl content (b) in Arabidopsis shoots. Data represent the mean ± SE of three different experiments.
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Fig. 2 – Effect of NO2 on ROS generation (a) and antioxidant contents (AsA (b) and GSH (c)) in Arabidopsis shoots. Data represent the mean ± SE of three different experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control. Please cite this article as: Liu, X., et al., Effects of nitrogen dioxide and its acid mist on reactive oxygen species production and antioxidant enzyme activity in Arabidopsis..., J. Environ. Sci. (2015), http://dx.doi.org/10.1016/j.jes.2015.03.011
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4 mg/m3 NO2 exposure. This finding suggests that NO2 at lower concentrations stimulated defense reactions, and these defense reactions might counteract ROS generated via elevated antioxidant defense systems in the tissue; whereas high concentration NO2 exposure induced the production of ROS, and the capacity of the antioxidant system was exceeded, or inhibited. As a result, the excessive ROS attacked lipids and proteins and the plant experienced substantial oxidative stress. Additionally, the activities of antioxidant enzymes (SOD, CAT and POD) were tested under the same treatment conditions. In this study, NO2 enhanced the activities of several antioxidant enzymes including SOD, POD and CAT associated with increased ROS generation, suggesting an induction of antioxidant defenses regulated by a ROS-mediated signaling pathway. SOD is a protein class that contains metals and catalyzes the dismutation of superoxide radical anions into H2O2 and molecular oxygen (Scandalios, 1993). The elevated SOD activity in Arabidopsis plants suggests that the chemical induced SOD to increase the generation of H2O2. CAT and POD are the primary H2O2-scavenging enzymes in plant cells. In this study, NO2 pronouncedly increased POD activity, whereas it had little effect on CAT activity. CAT has a high capacity but low affinity enzyme for H2O2, whereas POD has a high affinity for H2O2 (Mittler, 2002). After NO2 exposure and shorter daily exposure to nitric acid mist, POD activity was significantly
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Inhibited growth and Chl content in the present study might be attributed to the damaged defense system and consequently unbalanced metabolism. In plant cells, ROS are unavoidable by-products of aerobic metabolism. Under normal growth conditions, amounts of ROS are modest and cells experience only mild oxidative stress, whereas many stresses enhance ROS production (Inzé and Van-Montagu, 1995; Mittler et al., 2004). The results of our present study clearly show that NO2 triggered a rapid increase in O−2 generation rate in Arabidopsis shoots. The enhanced production of ROS under stress can pose a threat to cells, but it is also thought that ROS serve as signal molecules to activate the stress responses and defense pathways (Apel and Hirt, 2004; Knight and Knight, 2001; Mittler, 2002). Thus, NO2-induced ROS can be viewed as cellular indicators of stress and as secondary messengers involved in the stress-response signal transduction pathway. Since membrane lipids and proteins are the preferred targets of ROS in plants under environmental stress (Prassad, 1996), they are considered to be reliable indicators of controlled modulation of ROS levels and oxidative stress (Halliwell and Gutteridge, 1993). Therefore, we further investigated the levels of lipid peroxidation and protein oxidation by measuring MDA and PCO contents. In Arabidopsis shoots, the MDA level and PCO content increased, and oxidative stress occurred after
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Fig. 3 – Effect of NO2 on levels of lipid peroxidation (a) and protein oxidation (b) in Arabidopsis shoots. Data represent the mean ± SE of three different experiments.*p < 0.05, **p < 0.01, ***p < 0.001 vs. control.
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Fig. 4 – Effect of NO2 on antioxidant enzyme activities (including: SOD (a), POD (b), CAT(c)) in Arabidopsis shoots. Data represent the mean ± SE of three different experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control. Please cite this article as: Liu, X., et al., Effects of nitrogen dioxide and its acid mist on reactive oxygen species production and antioxidant enzyme activity in Arabidopsis..., J. Environ. Sci. (2015), http://dx.doi.org/10.1016/j.jes.2015.03.011
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enhanced and eliminated part of the generated H2O2. As a result, the remaining H2O2 might not be enough to induce CAT activity. This was not the case for the longest daily exposure, which accumulated more H2O2 and induced CAT activity. Thus, POD, but not CAT, was the most efficient scavenging enzyme to decrease the cellular levels of H2O2 in plant cells under NO2 stress. Moreover, the increase of POD activity can contribute to the overall cell resistance to NO2 stress since POD participates in many other cell processes involved in the plant defense reaction (Valério et al., 2004). POD can initiate cell-wall toughening events such as phenolic cross-linking and lignification, which can strengthen leaf and stem tissues against potential damage (Polle et al., 1994). The roles that POD can play in cell wall toughening and in the production of secondary metabolites, and its simultaneous oxidant and antioxidant capabilities, make it an important factor in the integrated defense response of plants to NO2 stress. Additionally the AsA– GSH redox system in chloroplasts is also an effective scavenger of O−2 and H2O2 in plant cells (Foyer and Halliwell, 1976; Jiang and Chen, 2008). Thus as NO2 concentrations increased, GSH content increased, but AsA content decreased, suggesting that the antioxidants also played an important role in plant defenses against NO2 stress.
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Fig. 5 – Effect of nitric acid mist exposure on Chl content (a), oxidative stress (b) and antioxidant enzyme activities (c) in Arabidopsis shoots. Data represent the mean ± SE of three different experiments.*p < 0.05, **p < 0.01, ***p < 0.001 vs. control.
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NO2 exposure affected plant growth and chlorophyll level, and increased O−2 production rate in Arabidopsis shoots. Furthermore, NO2 elevated the levels of lipid peroxidation and protein oxidation, accompanied by the induction of SOD, POD and CAT activities and the change of antioxidant contents. Following this, we mimicked nitric acid mist under experimental conditions, and confirmed antioxidant mechanisms of the plant in response to the stress. Our results imply that NO2 and its acid mist caused pollution risk to plant systems, increased ROS acted as a signal to induce a defense response, and antioxidant status played an important role in plant protection against NO2/nitric acid mist-caused oxidative damages.
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Acknowledgments
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This study was supported by the National Natural Science 411 Foundation of China (Nos. 21477070, 21377076), the Specialized 412 Research Fund for the Doctoral Program of Higher Education 413
Please cite this article as: Liu, X., et al., Effects of nitrogen dioxide and its acid mist on reactive oxygen species production and antioxidant enzyme activity in Arabidopsis..., J. Environ. Sci. (2015), http://dx.doi.org/10.1016/j.jes.2015.03.011
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(SRFDP) (Nos. 20121401110003, 20131401110005), the Project for Science and Technology Development of Shanxi Province (No. 20120313009-2), the Research Project supported by the Shanxi Scholarship Council of China (No. 2012-009), and the Program for the Top Young and Middle aged Innovative Talents of Higher Learning Institutions of Shanxi (TYMIT, No. 20120201).
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Please cite this article as: Liu, X., et al., Effects of nitrogen dioxide and its acid mist on reactive oxygen species production and antioxidant enzyme activity in Arabidopsis..., J. Environ. Sci. (2015), http://dx.doi.org/10.1016/j.jes.2015.03.011
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