Effects of 60-day NO₂ fumigation on growth, oxidative stress and antioxidative response in Cinnamomum camphora seedlings

Effects of 60-day NO 2 fumigation on growth, oxidative stress and antioxidative response in Cinnamomum camphora seedlings 1 2 first-author †E-mail:zhuomeichen@163.comyxchen@zju.edu.cn †E-mail:zhuomeichen@163.comyxchen@zju.edu.cn zhuomeichen@163.com 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:zhuomeichen@163.comyxchen@zju.edu.cn †E-mail:zhuomeichen@163.comyxchen@zju.edu.cn zhuomeichen@163.com 2 3 2 College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310029, China College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310029, China Zhejiang Forestry Academy, Hangzhou 310023, China Zhejiang Forestry Academy, Hangzhou 310023, China Department of Geography, Minjiang University, Fuzhou 350108, China Department of Geography, Minjiang University, Fuzhou 350108, China Objective: 2 To study the oxidative stress and antioxidative response of Cinnamomum camphora seedlings exposed to nitrogen dioxide (NO 2 ) fumigation. Methods: 2 Measurements were made up of the growth, chlorophyll content, chlorophyll fluorescence, antioxidant system and lipid peroxidation of one-year-old C. camphora seedlings exposed to NO 2 (0.1, 0.5, and 4 μl/L) fumigation in open top chambers over a period of 60 d. Results: 2 After the first 30 d, 0.5 and 4.0 μl/L NO 2 showed insignificant effects on the growth of C. camphora seedlings. However, exposure to 0.5 and 4.0 μl/L NO 2 for 15 d significantly reduced their chlorophyll content ( P < 0.05), enhanced their malondialdehyde (MDA) content and superoxide dismutase (SOD) activity ( P < 0.05), and also significantly reduced the maximal quantum yield of PSII in the dark [the ratio of variable fluorescence to maximal fluorescence ( F v / F m )] ( P < 0.05). In the latter 30 d, 0.5 μl/L NO 2 showed a positive effect on the vitality of the seedlings, which was reflected by a recovery in the ratio of F v / F m and chlorophyll content, and obviously enhanced growth, SOD activity, ascorbate (AsA) content and glutathione reductase (GR) activity ( P < 0.05); 4.0 μl/L NO 2 then showed a negative effect, indicated by significant reductions in chlorophyll content and the ratio of F v / F m , and inhibited growth ( P < 0.05). Conclusion: 2 The results suggest adaptation of C. camphora seedlings to 60-d exposure to 0.1 and 0.5 μl/L NO 2 , but not to 60-d exposure to 4.0 μl/L NO 2 . C. camphora seedlings may protect themselves from injury by strengthening their antioxidant system in response to NO 2 -induced oxidative stress. Cinnamomum camphora Fumigation Growth Chlorophyll content Chlorophyll fluorescence Antioxidant Lipid peroxidation Introduction 1 Nitrogen dioxide (NO 2 ), one of the main traffic-related air pollutants, contributes to the forming of ozone via a photochemical reaction with hydroxyl radicals in the atmosphere (Takahashi et al., 2005 2005 ). Hourly concentrations of NO 2 in Japan, UK and USA are reported to reach levels of 0.02–0.19, 0.25–0.4 and 0.01–0.12 μl/L (Takahashi et al., 2005 2005 ). In China, atmospheric NO 2 concentration averagely in some large cities was recorded to be 0.017 μl/L in 2006 (Ministry of the Environmental Protection of the People’s Republic of China, 2007 2007 ). Plants have been shown to metabolize dissolved NO x through an NO 3 − assimilation pathway to form amino acids and proteins (Zeevaart, 1976 1976 ; Marie and Ormrod, 1984 1984 ). Stimulated growth has been reported in the presence of low concentrations of NO 2 (Sabaratnam et al., 1988 1988 ; Okano et al., 1985 1985 ; Marie and Ormrod, 1984 1984 ). However, negative effects on plant health were observed under either high concentration and short-term NO 2 exposure (Sabaratnam and Gupat, 1988 1988 ; Qiao and Murray, 1998 1998 ) or low concentration and long-term NO 2 exposure (Ashenden, 1970 1970 ; Ashenden et al., 1990 1990 ; Maggs and Ashmore, 1998 1998 ). Thus, the effect of NO 2 on plants depends on the experimental conditions such as the plant species studied, NO 2 concentration and stress duration. Uptake of NO 2 results in a reduction in photosynthesis, which can be explained by competition for nicotinamide adenine dinucleotide phosphate (NADPH) between the processes of nitrite reduction and carbon assimilation in the chloroplast, and thereby leads to the generation of reactive oxygen species (ROS) (Sabaratnam and Gupat, 1988 1988 ; Clyde Hill and Bennet, 1970 1970 ; Shimazaki et al., 1992 1992 ). When the production rate of ROS exceeds the elimination rate, lipid peroxidation and DNA injury may occur, and the organism may suffer from oxidative stress (Pan et al., 2006 2006 ). However, plants have their own defense mechanisms including antioxidants and antioxidant enzymes (Wu and Tiedemann, 2002 2002 ; Lai et al., 2007 2007 ). For instance, peroxidase (POD), catalase (CAT), superoxide dismutase (SOD) and the ascorbate-glutathione cycle (AGC) protect various vital physiological processes from injury by ROS produced under stress (Rai et al., 2004 2004 ; Wu and Tiedemann, 2002 2002 ). Studies have been carried out on the growth, chlorophyll content, photosynthesis, nitrogen content and enzymes related to nitrogen metabolism of plants exposed to NO 2 , including nitrite reductase, glutamine synthetase and glutamate synthase (Takahashi et al., 2005 2005 ; Yu et al., 1988 1988 ; Sabaratnam et al., 1988 1988 ; Qiao and Murray, 1998 1998 ; Okano et al., 1985 1985 ). However, little is known about the antioxidative response of plants and the regulation of photosynthesis. As motorized traffic is one of the main sources of atmospheric NO 2 , we should pay more attention to roadside species when studying the effects of NO 2 on plants. Cinnamomum camphora , the camphor tree, one of the most important trees in the subtropical evergreen broadleaf forest, is widely distributed in the south of China along the Yangtze River valley, and is also found in Korea, Japan and Vietnam (Zheng, 1983 1983 ). It has been widely planted in cities throughout southern China because of its high landscaping value and disease and pollution resistance (Tian et al., 2007 2007 ). Takahashi et al. ( 2005 2005 ) reported that C. camphora showed a high assimilation capacity at 0.1 μl/L NO 2 and a low assimilation capacity at 4.0 μl/L NO 2 , and was therefore classified in a group showing low resistance and high assimilation to NO 2 among 70 taxa of woody plants used as roadside trees. In this study, 60-d NO 2 fumigation was carried out to study oxidative stress and the antioxidative response of C. camphora . The results will be helpful in determining the resistance of C. camphora to NO 2 and in providing a base for future studies of environmental toxicology in plants. Materials and methods 1 Plant material and treatment 2 The study was conducted on one-year old C. camphora seedlings in a suburban area of Hangzhou, located in the southeast of China at 30°14′ N latitude, 120°09′ E longitude and 102 m above sea level. Seeds were germinated in a sand bed on Mar. 20, 2007 and seedlings were transplanted into 1-L pots (one plant per pot) after two leaves had grown. The culture medium was a mixture of turf (organic matter:crude ash=42:58, w/w), vermiculite and perlite in the proportions 4.5:4.5:1 (v/v/v). Seedlings were separated into four treatments: exposure to 0.1, 0.5 or 4.0 μl/L NO 2 or unfiltered air (the control, CK). One hundred seedlings per treatment were transferred to open-top chambers (OTC) on July 12, 2007. Seedlings were treated for a period of 60 d during daytime hours (10 h/d, 7 d/week). Measurements of their growth, chlorophyll fluorescence, chlorophyll content, antioxidant system and lipid peroxidation were made every 15 d. OTC and the experimental environment 2 Potted seedlings were placed in eight OTCs. Each OTC was a hexagonal prism of 1.85 m height and 1.16 m diameter. A board with 1 200 holes (each 12 mm in diameter) was inserted in the chamber at a height of 30 cm from the bottom. Air was pumped continuously through the chamber from the bottom. NO 2 from a compressed source was ventilated into the bottom of the chamber through a Teflon tube connected by a solenoid valve. The concentration of the gas in the chamber was monitored using a system consisting of a solenoid valve, an online-sensor, a single chip micyoco (SCM) and a computer. The concentration was sensed by an electrochemical sensor, converted to digital signal, and then sent to a computer by the SCM. The computer database was compared against the target concentration and used to send a command to the SCM to control the solenoid valve to open or close each second. The solenoid valve was in mic-flux and low-frequency pulse width modulation (PWM). The fluctuation of the gas concentration in different parts of the chamber was measured and found to remain within 5% of the target concentration. Natural sunlight was used as the light source. The measured data of temperature, humidity and light density inside the chamber during the experiment are shown in Table 1 1 . 2 Date 2 Mean temperature (°C) 2 Relative humidity (%) 2 PFD of the sunshine (μmol/(m 2 ·s)) Max Min Max Min Max Min  July, 2007 34.0 28.7 42.9 28.2 180.6 6.45  August, 2007 34.1 29.4 39.8 22.8 182.4 7.23  September, 2007 26.8 24.9 54.0 41.5 135.3 4.01 Growth measurement 2 Every 15 d, eight seedlings from each treatment were collected. Ground diameter and height were measured and the ratio of height/ground diameter was calculated. The seedlings were washed and heated to 80 °C and the above-ground dry weight and below-ground dry weight were recorded until they reached a constant level. Chlorophyll content determination 2 Fully expanded leaves from area around the middle of the trunk of each of eight seedlings per treatment were randomly selected for measurement every 15 d. The petiole and the primary veins were removed from the leaves and discarded. Three independent chlorophyll measurements were made for each leaf sample. Portions of the samples were also used for measurements of the antioxidant system and lipid peroxidation determinations. Frozen leaf tissue was homogenized in 80% (v/v) ice-cold acetone in the dark. The supernatant was separated by centrifugation at 2 000× g for 10 min. Chlorophyll content was determined by measuring the absorbance of the supernatant at 646 and 663 nm, as described by Lichtenthaler ( 1987 1987 ). Chlorophyll fluorescence 2 Measurements of chlorophyll fluorescence were made every 15 d from the upper face of designated seedling leaves using an LI-6400 photosynthesis system (LI-COR Inc., Lincoln, NE, USA) fitted with an integral fluorescence chamber. Before each measurement, eight fully developed seedlings were taken and placed in the dark for 15 min at (22±1) °C. After recording the dark signal level, the minimum fluorescence F 0 was obtained by excitation of a probing light beam. The intensity of this light was sufficiently low so as to not produce any significant variable fluorescence [1.6 kHz, photon flux density (PFD)=0.02 μmol photons/(m 2 ·s)]. A single saturating flash [1 s, PFD=4 500 μmol photons/(m 2 ·s)] was then applied to reach maximal fluorescence ( F m ). The yield of variable fluorescence ( F v ) was calculated as F m − F 0 . Photosynthetic capacity (also termed the maximal quantum yield) was estimated by the ratio of ( F m − F 0 )/ F m (also termed F v / F m ) for dark-adapted leaves (Genty et al., 1990 1990 ; Frankart et al., 2002 2002 ). Antioxidant system analysis 2 SOD activity was assayed by its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) (Dhindsa et al., 1981 1981 ). The absorbance was recorded at 560 nm. It was expressed as U/g fresh weight, and 1 U was defined as the amount of enzyme causing 50% inhibition of NBT reduction under the assay conditions. Ascorbic acid (AsA) content was determined following the procedure of Ella et al. ( 2003 2003 ). Frozen leaf tissue was homogenized with 3 ml of 5% (w/v) metaphosphoric acid and then centrifuged at 20 000× g . The mixture of 0.25 ml of 3 mmol/L 2,6-dichloroindophenol (DCIP) and 0.5 ml supernatant was kept at room temperature for 20 min, incubated at 50 °C for 1 h after adding 0.5 ml of 1% (w/v) thiourea in 5% metaphosphoric acid and 0.5 ml of 10 mmol/L dinitrophenylhydrazone (DNPH), and then cooled in an ice bath for 15 min while adding 1.25 ml of ice-cold 85% (v/v) sulfuric acid (H 2 SO 4 ). An aliquot of 0.5 ml of 20% (v/v) H 2 SO 4 was added and absorbance at 520 nm was measured. Corrections were made for the oxidation of ascorbate by replacing DCIP with distilled water. Glutathione reductase (GR) activity was assayed as described by Foyer and Halliwell ( 1976 1976 ) and Liu et al. ( 2007 2007 ). The reaction mixture consisted of 50 mmol/L phosphate buffered saline (pH 7.8), 0.15 mmol/L NADPH, 0.5 mmol/L oxidized glutathione (GSSG) and enzyme extract. The decrease in absorption at 340 nm [ ε =6.2 (mmol/L·cm) −1 ] due to NADPH oxidation was recorded over 2.5 min and was expressed as U/g fresh weight (1 U=1 mmol substrate activated during 1 min at 25 °C). Lipid peroxidation determination 2 The degree of lipid peroxidation was estimated using malondialdehyde (MDA) as an indicator. The MDA content was determined using the thiobarbituric acid (TBA) method (Shalata and Tal, 1998 1998 ). Samples were mixed with 1 ml of 10% (w/v) trichloroacetic acid (TCA) and 1 ml of 0.67% (w/v) TBA, and were then heated in a boiling water bath for 15 min. The amount of TBA reactive substance (TBARS) formed was determined by measuring absorbance at 535 nm and correcting for nonspecific absorbance at 600 nm. The MDA content was expressed as nmol/L MDA/g fresh weight. Statistical analysis 2 Statistical data analysis was performed using SPSS version 10.0 software. Treatments were compared using one-way analysis of variance (ANOVA). Prior to the analysis, data were checked for normality and homogeneity of variance, and were square root transformed to equalize variances. Differences between treatments were considered significant at P < 0.05. Results 1 Growth 2 In the first 30 d of the experiment, all levels of NO 2 fumigation yielded insignificant differences in growth, including the height, ground diameter, above-ground dry weight, below-ground dry weight and ratio of height/diameter. By the end of the experiment, NO 2 fumigation still showed no significant effects on the height. However, by then 0.5 μl/L NO 2 had increased the ground diameter, above-ground dry weight and below-ground dry weight to the highest in the treatments, significantly higher than the corresponding control values. Treatment with 0.1 μl/L NO 2 also significantly promoted the growth of ground diameter, while 4.0 μl/L NO 2 significantly reduced the above-ground dry weight and below-ground dry weight (Table 2 2 ). The values represent mean±SD ( n =8). Different lower case letters following the values show significant differences ( P < 0.05) between treatments according to one-way ANOVA NO 2 (μl/L) Duration (d) Ground diameter (cm) Height (cm) Above-ground dry weight (g) Below-ground dry weight (g) Ratio of height/ground diameter CK 0 1.92±0.124 15.18±1.22 0.2768±0.0357 0.2416±0.0250 79.036±9.78 15 1.96±0.10a 17.60±1.23a 0.3792±0.0341a 0.2697±0.0247a 89.802±6.72a 30 2.18±0.11a 21.21±0.93a 0.4389±0.0255a 0.2875±0.0309a 97.298±4.71a 45 2.52±0.09a 22.18±1.87a 0.5134±0.0378a 0.3247±0.0351ab 88.027±4.07a 60 2.66±0.10a 22.37±1.67a 0.5980±0.0486a 0.4531±0.0278a 84.104±9.23a 0.1 0 − − − − − 15 2.01±0.16a 17.82±1.56a 0.3809±0.0350a 0.2875±0.0267a 88.662±9.07a 30 2.34±0.16a 21.35±1.81a 0.4359±0.0323a 0.3525±0.0254a 91.242±5.16a 45 2.74±0.17ab 22.69±2.11a 0.5695±0.0440a 0.3987±0.0381a 82.812±6.53a 60 2.93±0.13b 23.68±1.01a 0.6563±0.0442a 0.4840±0.0452a 80.821±8.86a 0.5 0 − − − − − 15 1.98±0.13a 18.21±0.56a 0.3154±0.0464a 0.2733±0.0298a 91.970±6.45a 30 2.25±0.15a 21.72±1.25a 0.4251±0.0422a 0.3588±0.0398a 96.533±9.13a 45 2.87±0.16b 23.05±2.03a 0.6391±0.0650b 0.4839±0.0365c 80.314±5.75b 60 3.10±0.17b 24.06±0.86a 0.7493±0.0558b 0.6184±0.0372b 77.613±5.51b 4.0 0 − − − − − 15 1.99±0.12a 17.93±1.39a 0.3322±0.0286a 0.2516±0.0369a 90.101±4.89a 30 2.13±0.15a 21.06±1.71a 0.3769±0.0398a 0.2675±0.0449a 98.874±5.32a 45 2.35±0.16a 21.66±1.08a 0.4682±0.0380a 0.2871±0.0465b 92.171±8.77a 60 2.45±0.15a 21.73±1.59a 0.5028±0.0315c 0.3430±0.0350c 88.698±9.04a Chrolophyll fluorescence 2 Exposure to 0.5 and 4.0 μl/L NO 2 for 15 d brought about an enhancement in F 0 ( P < 0.05); however, in the subsequent treatment period, no statistical difference from the control was found. Exposure to 0.5 μl/L NO 2 had little impact on F m but 0.1 μl/L NO 2 reduced F m to 977.7 after 15 d ( P < 0.05). Exposure to 4.0 μl/L NO 2 reduced F m significantly to 988.4 and 977.3 after 15 and 60 d, respectively. After 15 d of fumigation, NO 2 caused a significant reduction in the ratio of F v / F m at NO 2 concentrations of 0.5 and 4.0 μl/L. In subsequent periods, NO 2 showed insignificant effects on the ratio of F v / F m with the exception of 30 d×4.0 μl/L NO 2 and 60 d×4.0 μl/L NO 2 (Table 3 3 ). The values represent mean±SD ( n =8). Different lower case letters following the values show significant differences ( P < 0.05) between treatments according to one-way ANOVA NO 2 (μl/L) Duration (d) F 0 F m F v / F m CK 0 165.7±5.1 922.8±34.94 0.820±0.004 15 170.0±7.1a 1058.6±47.7a 0.839±0.014a 30 175.7±7.2a 958.8±31.3a 0.817±0.004ab 45 178.4±9.1a 974.7±32.2ab 0.817±0.005ab 60 180.5±7.1a 1005.2±8.6a 0.820±0.009a 0.1 0 − − − 15 163.7±6.5a 977.7±22.6c 0.832±0.011ab 30 169.5±11.4a 892.4±47.8a 0.810±0.004a 45 176.1±11.4a 1001.3±40.3a 0.824±0.009a 60 178.3±13.5a 1028.2±14.5a 0.827±0.013a 0.5 0 − − − 15 190.7±7.0b 1034.5±12.8ab 0.815±0.006b 30 173.7±8.3a 968.5±32.0a 0.821±0.007b 45 171.4±9.4a 958.8±43.8ab 0.821±0.004ab 60 165.1±10.2a 964.9±38.9a 0.829±0.007a 4.0 0 − − − 15 201.2±9.4b 988.4±12.6bc 0.797±0.007c 30 182.0±9.3a 903.0±6.5a 0.799±0.005c 45 183.5±9.7a 915.2±33.3b 0.799±0.009b 60 174.9±7.7a 877.3±3.5b 0.801±0.009b Chlorophyll content 2 NO 2 fumigation at each concentration reduced the Chl a, Chl b and Chl (a+b) contents in the first 30 d ( P < 0.05), except that 0.5 μl/L NO 2 showed no statistical difference from the control in Chl a×30 d and Chl (a+b)×30 d (Fig. 1 1 ). In the second 30 d period, 0.1 and 0.5 μl/L NO 2 had little effects, while 4.0 μl/L NO 2 significantly reduced the chlorophyll contents. In the control samples, overall decreases were found of 18.36%, 15.32% and 17.53% in Chl a, Chl b and Chl (a+b) contents, respectively, compared with values obtained at the beginning of the experiment. Lipid peroxidation 2 In every phase of the experiment, the MDA contents in NO 2 treatments were significantly higher than those of the control (Fig. 2 2 ). By the end of the experiment, the MDA contents in the 4.0, 0.5 and 0.1 μl/L NO 2 treatments were higher by 87.42%, 30.62% and 26.96%, respectively, than that of the control. Antioxidant system 2 As the experiment proceeded, the SOD activities first rose and then, in the 0.5 and 4.0 μl/L NO 2 treatments, stabilised at a level significantly higher than that of the control (Fig. 2 2 ). In the 0.1 μl/L NO 2 treatment, after the initial rise, levels fluctuated slightly. At the end of the fumigation period, SOD activities had risen in proportion to the concentration of NO 2 . In the 4.0 and 0.5 μl/L NO 2 treatments, SOD activities were higher than that of the control by 88.81% and 54.80% ( P < 0.05), respectively. Compared with the control, in the presence of 0.1 and 0.5 μl/L NO 2 , the AsA contents decreased significantly in the first 30 d with the exception of 0.1 μl/L NO 2 ×15 d and increased significantly in the following 30 d with the exception of 0.1 μl/L NO 2 ×45 d (Fig. 2 2 ). Treatment with 4.0 μl/L NO 2 increased the AsA content after 30 d to a much higher level than the other NO 2 treatments and 56.11% higher than the control ( P < 0.05), but reduced it sharply to a level significantly lower than the control after 45 d, and after 60 d showed an insignificant effect. The AsA content of control samples decreased by 46.16% from the start to the end of the experiment. During the fumigation, GR activity showed a double-peak tendency in the 4.0 μl/L NO 2 treatment and a single-peak tendency in the 0.1 and 0.5 μl/L NO 2 treatments (Fig. 2 2 ). In the 4.0 μl/L NO 2 treatment, GR activity increased to a significantly higher level than in the other treatments after 15 d, but weakened steeply to a level significantly lower than in the control after 30 d, and remained statistically insignificant from the control in the last period of the experiment. In the 0.1 and 0.5 μl/L NO 2 treatments, compared with the control, GR activities were insignificantly different after 15 d, significantly lower after 30 d, then significantly higher in the last phase with the exception of 0.1 μl/L NO 2 ×60 d. To sum up, the effects of NO 2 fumigation on GR activity differed in the changing phases of the experiment. The GR activity of control samples fell by 51.32% from the start to the end of the experiment. Discussion 1 Growth 2 In this study, 30-d NO 2 fumigation caused insignificant differences in the growth of C. camphora seedlings. 60-d treatment with 4.0 μl/L NO 2 inhibited growth, but 60-d treatment with 0.5 or 0.1 μl/L NO 2 promoted growth (Table 2 2 ). This suggests that C. camphora lacks adaptation to NO 2 at the level of 4.0 μl/L but is adapted to levels of 0.1 and 0.5 μl/L. Pandey and Agrawal ( 1994 1994 ) reported that the relative growth rate and net assimilation rate of tomato plants increased initially but then declined after exposure for longer periods to 0.2 μl/L NO 2 . A lack of adaptation to long-term and low-concentration NO 2 was also reported for P. pratensis and Pakistan rice (Ashenden, 1970 1970 ; Maggs and Ashmore, 1998 1998 ). Differences in adaptability to NO 2 among plant species may be caused by their different metabolic and antioxidant systems (Liu et al., 2007 2007 ). Similar adaptation responses of plants to long-term ozone have been reported (Walmsley et al., 1980 1980 ; Mehlhorn et al., 1991 1991 ). Oxidative stress 2 The view is commonly held that uptake of NO 2 results in the generation of ROS in plants because of competition for NADPH between the processes of nitrite reduction and carbon assimilation in the chloroplast, and the strong radical nature of NO 2 (Sabaratnam and Gupat, 1988 1988 ; Clyde Hill and Bennet, 1970 1970 ; Shimazaki et al., 1992 1992 ; Ramge et al., 1993 1993 ). Although growth was not significantly affected by NO 2 in the first 30 d, variation occurred in lipid peroxidation, chlorophyll fluorescence and chlorophyll contents, each of which might be an indicator of air pollution injury (Tables 2 2 and 3 3 , Fig. 1 1 ). MDA, the decomposition product of polyunsaturated fatty acids (PUFA) of biomembranes, is a good indicator of the severity of cell injury during oxidative stress (Price et al., 1990 1990 ). In this study, three concentrations of NO 2 increased the MDA contents in every treatment period, indicating the existence of oxidative injury induced by NO 2 during the experiment (Fig. 2 2 ). In this study, F 0 was significantly increased after 15 d at 0.5 and 4.0 μl/L NO 2 , indicating that modifications were induced at the antenna pigment level (Calatayud and Barreno, 2001 2001 ) (Table 3 3 ). After the same duration, a significant decrease in the ratio of F v / F m in these two treatments indicated that the photochemistry of PSII and its functional efficiency were affected (Maxwell and Johnson, 2000 2000 ) (Table 3 3 ). Similar results were observed by Barnes et al. ( 1988 1988 ), Guidi et al. ( 1997 1997 ) and Carrasco-Rodriguez and Valle-Tascon ( 2001 2001 ) in higher plants exposed to ozone. In the subsequent time period, the photosynthetic efficiency recovered at 0.5 μl/L NO 2 but remained lower at 4.0 μl/L NO 2 than at the control, which was reflected by the F m and the ratio of F v / F m (Table 3 3 ). Although it was not possible to conclude that the decline in photosynthesis was the result or cause of ROS accumulation, the inverse correlation between the decreased ratio of F v / F m and increased MDA contents in the 15 d×0.5 μl/L NO 2 , 15 d×4.0 μl/L NO 2 and 60 d×4.0 μl/L NO 2 indicated a possible association between them (Table 3 3 , Fig. 2 2 ). Chlorophyll pigments are essential for photosynthesis and a decrease in chlorophyll content has been used as an indicator of air pollution injury (Darrall and Jäger, 1984 1984 ). In this study, NO 2 reduced the Chl a, Chl b and Chl (a+b) contents in the first 30 d (Fig. 1 1 ). Similar phenomena have been observed in plants such as Hedera helix (Della-Torre et al., 1998 1998 ) and Avena sativa L. (Pleijel et al., 1994 1994 ), and may be caused by the disintegration of pigments by ROS (Sakaki et al., 1983 1983 ). In the subsequent 30 d, 4.0 μl/L NO 2 significantly reduced pigmentation, but 0.1 and 0.5 μl/L NO 2 had an insignificant effect (Fig. 1 1 ). Ra et al. ( 2005 2005 ) found that moderate levels of fertilizer air pollutants may permit higher protein synthesis rates, improve the ratio of intact chlorophyll to degraded forms, and increase overall concentrations of pigments and CO 2 uptake of lichen. The availability of nitrogen from 0.1 and 0.5 μl/L NO 2 may promote the synthesis of pigments and counteract the destruction by ROS, leading to a recovery in chlorophyll content. The continuously declining chlorophyll contents in the 4.0 μl/L treatment indicated that oxidative stress was still inhibiting the plant (Fig. 1 1 ). The tendency of chlorophyll contents to decrease in the experiment may have resulted from the increasing senescence of the seedlings as growth was taking place (Makino and Osmond, 1991 1991 ). Antioxidative response 2 SOD is essential for the elimination of ROS in plants because its enzymatic action dismutates superoxide (O 2 − ) to H 2 O 2 and O 2 (Meloni et al., 2003 2003 ). A correlation between increased SOD activity and tolerance to air pollutants has been observed in many plants, and is considered to be an adjustment response to stress (Lee and Bennett, 1982 1982 ). In this study, SOD activities increased with increasing NO 2 concentrations, suggesting a dose-dependent effect of NO 2 on SOD activity (Fig. 2 2 ). AGC contributes greatly to antioxidant protection against H 2 O 2 (Potters et al., 2002 2002 ). Several enzymes and antioxidants are involved in these reactions including AsA and GR (Foyer and Halliwell, 1976 1976 ; Smirnoff, 1996 1996 ). In general, the biosynthesis of AsA and reduced glutathione (GSH, catalyzed from oxidized glutathione by GR) is stimulated when the cell encounters stress conditions (Foyer and Halliwell, 1976 1976 ; Horemans et al., 2000 2000 ; Potters et al., 2002 2002 ). In the present study, 0.5 μl/L NO 2 reduced AsA contents and GR activities in the first 30 d, but induced them in the subsequent 30 d, indicating that AGC was efficiently activated to detoxify ROS in the second 30 d period (Fig. 2 2 ). Activated AGC, together with enhanced SOD, formed a stronger antioxidant system in the final 30 d. A new balance between the production and elimination of ROS was established to control the oxidative stress within an acceptable level, as shown by the recovered chlorophyll contents and growth promotion in the 0.5 μl/L NO 2 treatment (Table 2 2 , Figs. 1 1 and 2 2 ). Alternatively, C. camphora has a substantial reserve of phenolics, which can act as reducing agents, hydrogen donators and singlet oxygen quenchers (Rice-Evans et al., 1996 1996 ; Zhang and Lin, 2008 2008 ). The production of phenolics is considered to be a response to injuries (Dixon and Paiva, 1995 1995 ) and may have contributed to the recovery from injury at 0.5 μl/L NO 2 , and thus the continued growth and early leaf senescence in C. camphora (Table 2 2 , Figs. 1 1 and 2 2 ). Further studies on phenolic compounds should be carried out to test this possibility. In the 4.0 μl/L NO 2 treatment, the AsA content and GR activity fluctuated widely and asynchronously (Fig. 2 2 ), indicating a low efficiency AGC and thus an unbalanced antioxidant system less able to contribute to the scavenging of ROS. A similar asynchronous tendency between AsA content and GR activity was reported for Populus euramericana (Edjolo et al., 2001 2001 ). As AsA and GSH not only act as antioxidants, but also have other functions in physiological processes (Horemans et al., 2000 2000 ; Potters et al., 2002 2002 ), it was difficult to identify the mechanisms by which they were affected by NO 2 treatment. This needs further study at the cellular level. The general duration-dependent fall in AsA content and GR activity in the control suggests a physiological process of senescence in leaves, which is similar to time-dependent changes in GSH content found in chloroplasts of tomato (Kuźniak and Skłodowska, 2001 2001 ). Conclusion 1 In conclusion, according to the antioxidative responses and the parameters, there was an adaptation response of C. camphora seedlings after 60-d of 0.1 and 0.5 μl/L NO 2 , but not after 60-d of 4.0 μl/L NO 2 , suggesting that the critical toxic value of atmospheric NO 2 in inducing influential oxidative injury to C. camphora seedlings is between 0.5 and 4.0 μl/L. 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Chinese Tree Records 10.1631/jzus.B0910350 Project supported by Zhejiang Keystone Projects (No. 2005C22056), and the Zhejiang Provincial Natural Science Foundation of China (No. Y5080011) 2009-06-13 2010-02-05 2009-08-31 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2010 2010 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 03 11