Urban Forestry & Urban Greening 14 (2015) 1166–1173
Contents lists available at ScienceDirect
Urban Forestry & Urban Greening journal homepage: www.elsevier.com/locate/ufug
Differential sensitivity of four urban tree species to elevated O3 Sheng Xu, Xingyuan He ∗ , Wei Chen, Yanqing Huang, Yi Zhao, Bo Li State Key Laboratory of Forest and Soil Ecology, Institute of Applied Ecology, Chinese Academy of Sciences, Wenhua Road 72, 110016 Shenyang, China
a r t i c l e
i n f o
Article history: Received 21 March 2015 Received in revised form 22 October 2015 Accepted 27 October 2015 Available online 30 October 2015 Keywords: Air pollution Elevated O3 Open top chamber (OTC) Physiological Response Urban Trees Volatile organic compound (VOC)
a b s t r a c t Tropospheric ozone (O3 ) is regarded as one of the most wide-spread air pollutants, and it is likely to increase further in many urban areas of the world. Therefore, trees in urban areas are expected to be subjected to increasing elevated O3 stress. In this study, four 4-year-old urban tree species (Ginkgo biloba, Quercus mongolica, Pinus tabulaeformis and Pinus armandii) in the northeast of China were sampled to investigate their physiological responses and sensitivity to elevated O3 (80 ppb) simulated by using open top chambers (OTCs). The results showed that Q. mongolica was more sensitive to elevated O3 than G. biloba. P. armandii was most tolerant to O3 compared to other tree species. Among the four tree species, Q. mongolica showed a highest decrease in foliar weight, branch weight, stem weight and total above-ground biomass under elevated O3 , and decreasing by 51.0%, 48.5%, 38.2% and 44.9%, respectively. Elevated O3 decreased net photosynthetic rate (Pn) in leaves of Q. mongolica by 63.4% (P < 0.01) after 90 days of fumigation, and 45.5% (P < 0.01) in needles of P. tabulaeformis after 30 days of fumigation. Superoxide dismutase (SOD) activity in leaves of Q. mongolica is decreased by 31.9% (P < 0.01) after 90 days of fumigation. A signiﬁcant decrease of SOD activity was observed in P. tabulaeformis after 60 days of fumigation (P < 0.05). Compared to the ambient air control, elevated O3 signiﬁcantly increased the emission rates of volatile organic compounds (VOCs) from four tree species. VOCs emission rate increased to the highest level (327.2 g g−1 h−1 ) in Q. mongolica after 60 days of O3 fumigation. Our results provide a helpful recommendation in creating urban forest ecosystems considering ozone response and VOC emissions. © 2015 Elsevier GmbH. All rights reserved.
Introduction Atmospheric O3 has increased considerably since pre-industrial times, and is predicted to exceed 80 ppb by the end of this century (IPCC, 2013). In the middle latitudes of the northern hemisphere, the ground-level background O3 concentration is expected to persist throughout the 21st century to levels in the range of 42–84 ppb due to the increased emission of O3 precursors produced by the rapid industrialization and urbanization over the last three decades (Vingarzan, 2004; IPCC, 2013). Particularly in China, anthropogenic emission of O3 precursors have increased signiﬁcantly along with rapid urbanization and industrialization (Wang et al., 2007; Yan et al., 2010a; Xu et al., 2012), in contrast to a signiﬁcant decrease in Europe and little change in North America. Ground-level peak hourly O3 concentrations in some urban areas of China exceed up to 160 ppb, particularly during the May to September period in the recent years (Feng et al., 2014). As a phytotoxic secondary air pollutant, O3 is formed from photochemical reactions involving NOx and volatile organic
∗ Corresponding author. Tel.: +86 02483970349; fax: +86 02483970300. E-mail address: [email protected]
(X. He). http://dx.doi.org/10.1016/j.ufug.2015.10.015 1618-8667/© 2015 Elsevier GmbH. All rights reserved.
compounds (VOCs) (West et al., 2006). Ozone usually induces visible injury, decreases the growth, damages the photosynthetic apparatus, and disturbs the physiological and biochemical processes of trees (He et al., 2007; Feng et al., 2008; Contran et al., 2009; Xu et al., 2014), which often results in reduced chlorophyll content, decreased net photosynthetic rate and stomatal conductance (Karnosky et al., 2007; He et al., 2007; Kitao et al., 2009), decline in the activities of photosynthetic enzymes, and inhibition of photosynthetic electron transport rate and photochemical reactions in PSII (Calatayud et al., 2006; Contran et al., 2009; Wang et al., 2009; Thwe et al., 2014). In China, Ginkgo biloba and Quercus mongolica are the two economically and ecologically important temperate deciduous tree species. Pinus tabulaeformis and Pinus armandii are the two endemic and dominant species of coniferous forests for timber and soil conservation. The four tree species also are widely used as landscape ornamentals and in forestation of urban areas in northeast China (e.g., Shenyang city). According to our observations, the highest O3 concentration at ground level is often over 40 ppb or even up to more than 80 ppb during the summer in the urban area of Shenyang city (Yan et al., 2010a; Xu et al., 2014). In recent years, we have reported physiological responses of urban plants exposed to elevated O3 (He et al., 2006, 2007; Lu et al., 2009; He et al., 2009; Yan
S. Xu et al. / Urban Forestry & Urban Greening 14 (2015) 1166–1173
et al., 2010a,b; Li et al., 2011; Xu et al., 2012, 2014), but little information is known or reported for the differences in the response of urban tree species to elevated O3 . The O3 -tolerance is usually attributed to the ability of leaves or needles to perform photosynthesis, which associates with stomatal conductance indirectly, as well as to the capacity to activate detoxifying systems (e.g., antioxidant enzyme activity). To our knowledge, this is the ﬁrst study to compare physiological differences and O3 -sensitivity comprehensively in terms of visible injury, growth, net photosynthetic rate, antioxidant enzyme activity, and VOCs emission rate of four tree species under elevated O3 concentration, which will provide a scientiﬁc basis for the bio-monitoring of urban forest to O3 pollution and the selection of urban tree species for sustainable development of urban forest ecosystem under climate change. Materials and methods Study site The study was conducted at Shenyang Arboretum with an area of 5 hm2 , Chinese Academy Science, located in the populated central area of Shenyang city (41◦ 46 31.29 N, 123◦ 26 27.51 E), the largest typical-forest city in the north-eastern China with temperate continental monsoon climate, average annual precipitation 755.4 mm, and average annual temperature 7.41 ◦ C. Experimental treatments and plant materials The experiment was carried out using the open top chambers (OTCs) according to the design of Heagle et al. (1973). Chambers were 4 m in diameter, 3 m in height, with a 45◦ sloping frustum, and distributed randomly without mutual shading in an open area of the Arboretum. Ozone was generated by electric discharge (GP5J, China) from pure compressed oxygen, and then added to the OTCs. The diurnal pattern of ozone exposure was set at a constant concentration (about 80 ppb). The generated O3 was directly dispensed to the open top chamber through a PVC pipe. The duration of ozone fumigation was 9 h per day. The O3 concentrations were controlled by computers, using a professional program for O3 dispensing and monitoring. The O3 concentration in the chambers was monitored by an O3 analyzer (S-900 Aeroqual, New Zealand). The average daytime ambient O3 concentration was about 40 ppb, and it was higher in summer and spring and lower in winter and autumn in the study area (Yan et al., 2010a,b). Four tree species (4-year-old G. biloba, P. tabulaeformis, Q. mongolica and P. armandii) were obtained from a local nursery (Shenyang city). Nine plants of each tree species were planted in ground (loamy soil, no extra fertilizer) in an OTC, and cultured and acclimated for 60 days with uniform management. Three OTCs (replicates) were used for one treatment. There were two treatments in this study, including ambient air and elevated O3 (80 ppb). The trees were randomly distributed among the chambers and fumigated by elevated O3 for 9 h daily in the daytime (08:00–17:00) from June 13 to September 12 in 2008. Mature leaves
or needles were already present on the plants when the experiment began. All the samples and data in this study were obtained during 9:00–11:00am after 0, 30, 60, and 90 days of gas fumigation, respectively. During the experiment, data related to O3 concentrations and microclimatic conditions in OTCs are shown in Table 1. During the experiment, the average and maximum daily concentration of O3 is 84.6 and 111.2 ppb, respectively. Measurements of visible injury and physiological parameters During the experiment, the symptoms of injured leaves were recorded for each tree species, and the percentage of injured leaves was calculated in relation to the total leaf number. Injury index used to evaluate the extent of foliar damage induced by elevated O3 was according to the method of Chaudhary and Agrawal (2013). The range of injury index is from 0 to 5 with the categories of injury deﬁned as no injury (0), very slight (1), slight (2), moderate (3), severe (4), and very severe (5) with the percentages of injured leaves for each category being 0%, 1–15%, 16–25%, 26–50%, 51–75%, and 76–100%, respectively. Growth parameters in this study including foliar, branch, stem and total weights of above-ground plant were measured at the end of the experiment. Biomass samples were oven dried (65 ◦ C) till constant weight was achieved (72 h). The biomass of each part was expressed in terms of grams per plant. Gas exchange was measured on the fully expanded leaves or healthy one-year-old needles on sunny days. Measurements were taken on eight intact, sun-exposed leaves, or needles in the middle of lateral branches selected from middle canopy. All photosynthetic parameters were measured by a portable photosynthesis system (Li-6400, Li-Cor Inc., Lincoln NE, USA) with a red/blue LED light source (Li-6400-02B) mounted onto a 6 cm2 clamp-on leaf chamber. Net photosynthetic rate (Pn ) and stomatal conductance (gs ) were recorded every 30-day period from the beginning of experiment under saturated light (1000 mol photons m–2 s–1 of PPFD) provided by the red/blue LED light source from 9:00am to 11:00am on leaves or one-year-old needles on the middle of lateral branches. The temperature, CO2 concentration and air relative humidity in the leaf cuvette were not artiﬁcially modiﬁed but depended on the corresponding OTC conditions. All measurements were done at a constant air ﬂow rate of 500 mol s–1 . Lipid peroxidation was estimated by malondialdehyde (MDA) content according to the method of Buege and Aust (1987). The content was determined by thiobarbituric acid reaction. One g of leaf tissue was homogenized in 5 mL of 0.6% (v/v) TBA solution in 10% (v/v) trichloroacetic acid. The homogenate was centrifuged at 12,000g for 15 min. and the supernatant was heated in a boiling water bath for 15 min and then cooled quickly in an ice bath. The resulting mixture was centrifuged at 12,000g for 15 min, and the absorbance of the supernatant was measured at 532 nm. Measurements were corrected for unspeciﬁc turbidity by subtracting the absorbance at 600 nm. MDA concentrations were calculated by means of an extinction coefﬁcient of 155 mmol L−1 cm−1 (Zhangyuan and Bramlage, 1992). The assay of superoxide dismutase (SOD) activity was based on the method described by Beyer and Fridovich (1987) with a
Table 1 Concentrations of O3 , AOT40, and microclimatic conditions in OTCs during the experiment. OTCs
Ambient air Elevated O3
[O3 ]mean , average daily (08:00–17:00) concentrations of O3 (ppb); [O3 ]max, average maximum daily concentrations of O3 (ppb); AOT40, cumulative the sum of the differences between the hourly mean ozone concentration in ppb and 40 ppb for each hour of gas exposure; AOT40(x) , indicates the accumulated values of AOT40 from the beginning gas fumigation until each sampling day (ppb h); DPPFD, average daily photosynthetic photo ﬂux density at the canopy level (mol m−2 day−1 ); [CO2 ]mean , average air CO2 concentration in OTC (mol mol−1 ); RHmean , average daily air relative humidity (%); Tmean , average daily air temperature (◦ C).
S. Xu et al. / Urban Forestry & Urban Greening 14 (2015) 1166–1173
slight modiﬁcation. One unit of enzymatic activity was deﬁned as the amount of enzyme required to bring about a 50% inhibition of the rate of nitro blue tetrazolium (NBT) reduction measured at 560 nm. The 3 ml reaction mixture included 50 l diluted enzyme extract, 2.05 ml 50 mM potassium phosphate buffer (pH 7.8), 0.3 ml 13 mM methionine (Met), 0.3 ml 63 M NBT, and 0.3 ml 1.3 M riboﬂavin. After 0, 30, 60, and 90 days of gas fumigation, VOCs samples were collected in the transparent polyethylene bags covering welllighted plant branches. The samples were collected from inside of the bags on glass adsorbent tubes (11.5 cm long and 0.4 cm internal diameter) ﬁlled with Tenax-TA, Carboxen 1000 and Carbosieve SIII (Supelco Inc., Bellefonte, PA, USA) using a constant-ﬂow type pump. The ﬂow rates were 100 mL min−1 and the sampling time was 5 min. The air samples were kept at 4 ◦ C until analysis. VOCs were separated and detected by a gas chromatograph with ﬂame ionization detection (FID). Analysis of VOCs was carried out using a thermal desorption sample injection system (ACEM 9300, CDS, USA) connected by a thermal transfer line maintained at 250 ◦ C to a gas chromatograph (14B, Shimadzu, Japan) with FID. The details of the experimental methods can be found in our previous studies (Li et al., 2009; Xu et al., 2012). Statistical analysis Chambers corresponding to each treatment were respectively considered as statistical replicates (n = 3). Three independent measurements were performed for every parameter in each chamber, and these independent measurements were averaged to obtain a chamber mean. One-way ANOVA was carried out using SPSS computer package (SPSS Inc. 1999, Chicago, IL, USA) for all sets of data at each sampling day. The values presented are the means of all measurements at every 30-day sampling point (from the beginning of June 13 to the end of September 12 for O3 exposure), and comparisons of means were determined using the least signiﬁcant difference test. Differences were considered statistically signiﬁcant when P < 0.05 or highly signiﬁcant at P < 0.01.
Table 2 Percentage of visible foliar injury on urban tree species after 90 days of elevated O3 (80 ppb) exposure. Tree species
Injured leaves (%)
Rating of injury
G. biloba P. tabulaeformis Q. mongolica P. armandii
13.6 25.0 36.5 20.4
1 2 3 2
Very slight Slight Moderate Slight
was the highest for Q. Mongolica among these tree species under elevated O3 (Table 2). Growth and photosynthesis Elevated O3 signiﬁcantly decreased growth of four tree species after long-term fumigation and decreased the total above-ground biomass of G. biloba, P. tabulaeformis, Q. mongolica and P. armandii by 14.3%, 34.5%, 44.9%, and 29.8%, respectively (Table 3). Among the four tree species, Q. mongolica showed the greatest loss of foliar weight, branch weight and stem weight under elevated O3 with decreases of 51.0%, 48.5%, and 38.2%, respectively. Elevated O3 decreased net photosynthetic rate (Pn ) in leaves of G. biloba and Q. mongolica, with declines of 35.6% (P < 0.05) and 53.6% (P < 0.01) after 60 days of fumigation, respectively (Fig. 1A and C). A decrease of 63.4% (P < 0.01) in Pn of Q. mongolica induced by elevated O3 was found after 90 days of fumigation compared to ambient air (Fig. 1C). Elevated O3 induced a 45.5% decrease (P < 0.01) of Pn in needles of P. tabulaeformis after 30 days of fumigation (Fig. 1B), and a 57.7% decrease (P < 0.01) of Pn in needles of P. armandii after 60 days of fumigation (Fig. 1D). After 90 days of O3 exposure, elevated O3 decreased Pn in needles of P. tabulaeformis and P. armandii by 39.1% (P < 0.05) and 28.0% (P > 0.05), respectively (Fig. 1B and D)). Stomatal conductance (gs ) showed a signiﬁcant decrease during O3 exposure (Fig. 2), particularly in leaves of Q. mongolica after 30 days (P < 0.01). The decrease rate of gs varied with the growing season. By the end of experiment (after 90 days), gs decreased by 15.5%, 85.1%, 84.5%, and 47.5% in G. biloba, P. tabulaeformis, Q. mongolica, and P. armandii, respectively (Fig. 2A–D).
Results Lipid peroxidation and antioxidant enzyme activity Visible injury Visible foliar injury in G. biloba, appearing as light-yellow ﬂecks, was ﬁrst observed after 70 days of exposure to elevated O3 , and 13.6% of the leaves were affected after 90 days of fumigation (Table 2). For Q. Mongolica, light-yellow ﬂecks were ﬁrst observed after 42 days of gas exposure, and 36.5% of the leaves were injured after 90 days of fumigation. Slight tip-burn symptom on needles was ﬁrst observed after 45 days for P. tabulaeformis and 25.0% of the needles were affected after 90 days of gas exposure. Slight tipburn symptom on needles of P. armandii was ﬁrst displayed after 49 days of fumigation, and the percentage of visible injury symptom was up to 20.4% after 90 days of O3 exposure. The injury index
Among four tree species, inter-speciﬁc differences in MDA content were found (Fig. 3). No signiﬁcant increase in MDA content in leaves of G. biloba was found under elevated O3 in any sampling point, compared to ambient air (Fig. 3A). Elevated O3 signiﬁcantly increased MDA content in leaves of Q. mongolica after 60 (P < 0.05) and 90 (P < 0.01) days of fumigation (Fig. 3C). A signiﬁcant increase in MDA content in needles of P. tabulaeformis was observed after 60 and 90 days of fumigation by elevated O3 , respectively (Fig. 3B). MDA content in needles of P. armandii was increased by 38.1% (P < 0.05) after 60 days of fumigation (Fig. 3D). Elevated O3 signiﬁcantly decreased SOD activity in leaves of G. Biloba by 47.7%, 32.5%, and 34.4% after 30, 60, and 90 days of
Table 3 Changes in growth parameters of urban tree species exposure to elevated O3 by the end of the experiment (n = 9). Tree species
G. biloba P. tabulaeformis Q. mongolica P. armandii
35.6 (4.7) 40.1 (7.9) 42.9 (2.0) 37.5 (7.6)
68.9 (11.8) 48.1 (14.5) 53.2 (11.9) 50.4 (16.7)
280.4 (82.9) 112.5 (28.5) 95.6 (8.0) 86.6 (13.4)
384.9 (99.4) 200.7 (50.9) 191.8 (21.9) 174.5 (37.7)
22.3 (2.5) 20.3 (3.5) 21.0 (2.9) 23.1 (3.4)
51.9 (8.0) 31.8 (9.2) 27.4 (13.8) 28.7 (5.8)
255.8 (44.9) 79.4 (33.4) 57.3 (22.4) 70.7 (16.5)
330.1 (55.4) 131.5 (46.1) 105.8 (39.1) 122.5 (25.7)
Each value represents the mean of nine measurements. The value in the parenthesis is standard error (SD). FW (fresh weight), BW (branch dry weight), SW (stem dry weight), and TW (total dry weight; gram per plant).
S. Xu et al. / Urban Forestry & Urban Greening 14 (2015) 1166–1173
Fig. 1. Effect of elevated O3 on net photosynthetic rates (Pn ) of four urban tree species: G. biloba (A), P. tabulaeformis (B), Q. mongolica (C), P. armandii (D). Data in the ﬁgure are means of three independent OTCs (±SD). Signiﬁcant differences between ambient air and elevated O3 at each time point are indicated by asterisks: * P < 0.05 and ** P < 0.01.
fumigation, respectively (Fig. 4A). For Q. mongolica, SOD activity decreased by 31.9% (P < 0.01) after 90 days of fumigation (Fig. 4C). A signiﬁcant decrease of SOD activity was observed in P. tabulaeformis after 60 days of fumigation and in P. armandii after 90 days of fumigation (P < 0.05) (Fig. 4B, 4D). Q. mongolica among four tree species showed the highest values in SOD activity regardless of any treatment. The inter-speciﬁc differences in SOD activity are much greater than the ozone effect in many cases (Fig. 4).
VOCs emission rate Compared to ambient air, elevated O3 signiﬁcantly increased VOCs emission rates of four tree species after 30 days of fumigation (Fig. 5). VOCs emission rate has increased by a factor of 7.3 (P < 0.01) in G. biloba and a factor of 2.0 (P < 0.01) in Q. mongolica after 60 days of fumigation by elevated O3 (Fig. 5A and C). After 30 days of gas exposure, elevated O3 increased VOCs emission rate by factors of
Fig. 2. Effect of elevated O3 on stomatal conductance (gs ) of four urban tree species: G. biloba (A), P. tabulaeformis (B), Q. mongolica (C), and P. armandii (D). Data in the ﬁgure are means of three independent OTCs (±SD). Signiﬁcant differences between ambient air and elevated O3 at each time point are indicated by asterisks: * P < 0.05 and ** P < 0.01.
S. Xu et al. / Urban Forestry & Urban Greening 14 (2015) 1166–1173
Fig. 3. Effect of elevated O3 on malondialdehyde (MDA) content of four urban tree species: G. biloba (A), P. tabulaeformis (B), Q. mongolica (C), and P. armandii (D). Data in the ﬁgure are means of three independent OTCs (±SD). Signiﬁcant differences between ambient air and elevated O3 at each time point are indicated by asterisks: * P < 0.05 and ** P < 0.01.
3.0 and 25.4 (P < 0.01) in needles of P. tabulaeformis (Fig. 5B) and P. armandii (Fig. 5D), respectively. Under elevated O3 , the highest VOCs emission rate for each tree species was 327.2 g g−1 h−1 in leaves of Q. mongolica after 60 days of fumigation, 2.2 g g−1 h−1 in G. biloba after 90 days of fumigation, 6.5 g g−1 h−1 in P. tabulaeformis and 9.5 g g−1 h−1 in P. armandii after 30 days of fumigation. Discussion Plants exposed to elevated O3 stress often show visible injury, especially, in short-term and acute fumigation of deciduous tree
species (Novak et al., 2005; Bagard et al., 2008). In this study, both G. biloba and Q. mongolica exhibited foliar symptoms of light yellow ﬂecks consistent with those observed during previous studies within the OTC facility for many trees (Wei et al., 2004; He et al., 2007; Calatayud et al., 2011). The timing of injury onset was approximately one month earlier in Q. mongolica than in G. biloba, which indicated that Q. mongolica was more sensitive than G. biloba to the elevated O3 treatments imposed in this study (80 ppb). The O3 -induced symptoms in coniferous trees generally appeared as chlorotic mottling, necrotic banding or tip necrosis in needles. Visible injury symptoms can vary among coniferous tree species.
Fig. 4. Effect of elevated O3 on superoxide dismutase (SOD) activity of four urban tree species: G. biloba (A), P. tabulaeformis (B), Q. mongolica (C), and P. armandii (D). Data in the ﬁgure are means of three independent OTCs (±SD). Signiﬁcant differences between ambient air and elevated O3 at each time point are indicated by asterisks: * P < 0.05 and ** P < 0.01.
S. Xu et al. / Urban Forestry & Urban Greening 14 (2015) 1166–1173
Fig. 5. Effect of elevated O3 on volatile organic compounds (VOCs) emission rates of four urban tree species: G. biloba (A), P. tabulaeformis (B), Q. mongolica (C), and P. armandii (D). Data in the ﬁgure are means of three independent OTCs (±SD). Signiﬁcant differences between ambient air and elevated O3 at each time point are indicated by asterisks: * P < 0.05 and ** P < 0.01.
Chlorotic mottling is the most common symptom on sensitive genotypes of southern pines at low to moderate (< 60 ppb) ozone concentrations (Flagler and Chappelka, 1995). In this study, tip necrosis was observed in one-year-old needles of both P. tabulaeformis and P. armandii exposed to 80 ppb. The tip-burn symptoms were very similar to a study on eastern white pine exposed to 100 ppb O3 for 6 h (Karnosky et al., 2007). The tip-burn symptoms were most common on recently expanded needles of southern pines, although they can occur on older, more mature tissue. In addition, no signiﬁcant relationship was found between symptoms occurrence and O3 levels. Differences between trees could be a variety of reasons, including the non-linear relationship between exposure and O3 uptake as well as the inter- and intra-speciﬁc variability in sensitivity (Ferretti et al., 2007; Chaudhary and Agrawal, 2013). In fact, we hypothesize that the observed differences in the occurrence of visible injury among G. biloba and Q. mongolica, or P. tabulaeformis and P. armandii were due to differences in the extent of oxidative stress and antioxidant capacity under elevated O3 conditions. Sensitivity in response to O3 exposure ranged from very sensitive to tolerant based on visible injury and growth. In this study, Q. mongolica total biomass declined more than G. biloba, and P. tabulaeformis declined more than P. armandii after long-term O3 fumigation, relative to ambient air controls. These results implied that Q. mongolica and P. tabulaeformis were more sensitive to elevated O3 than other urban tree species, resulting in reduced overall carbon gain. Photosynthesis is the most important physiological process affected in sensitive plants by rising levels of O3 in the troposphere. The reduction of Pn and stomatal conductance, following exposure to high levels of O3 , has been described in a number of scientiﬁc studies (Bortier et al., 2000; Novak et al., 2005; Bagard et al., 2008). Stomatal conductance was related to a larger potential of O3 uptake. In general, photosynthesis and stomatal conductance of plants decline in response to elevated O3 . Our results here showed that elevated O3 reduced stomatal conductance in leaves of all urban tree species tested. Pn and stomatal conductance decrease in Q. mongolica were greater under elevated O3 than G. biloba, and P. tabulaeformis more than P. armandii. Declines in stomatal conductance under elevated O3 may indicate that O3 has an impact
on photosynthetic gas exchange (Noormets et al., 2001; Yan et al., 2010a). In fact, our recent work (Yan et al., 2010a) supports the present ﬁndings for the greater sensitivity of Q. mongolica compared to G. biloba in terms of O3 impacts on photosynthesis. The accurate evaluation of plant sensitivity to elevated O3 relies on the assessment of the changes, e.g., in chlorophyll content, antioxidant systems, chlorophyll ﬂuorescence, and growth (Yan et al., 2010a; Pellegrini et al., 2013). In our previous study, chlorophyll bleaching, or leaf yellowing indicated that leaf senescence was accelerated by chronic O3 fumigation (Yan et al., 2010a; Wang et al., 2009). During the growing season, greater decrease in chlorophyll content was observed in Q. mongolica compared to other tree species (Yan et al., 2010a), supporting the conclusion that Q. mongolica was more sensitive to elevated O3 . The extent of lipid peroxidation as represented by MDA content reﬂects the state and integrity of membranes in plant cells, and has been correlated with the level of O3 exposure (Calatayud et al., 2002). In our study, the increase in MDA content observed in the leaves of Q. mongolica and in needles of P. tabulaeformis under elevated O3 exposure indicated an occurrence of oxidative stress. No signiﬁcant increase in the MDA contents of G. biloba and P. armandii implied that urban tree species with high O3 -tolerance might had the greater antioxidant capacity to O3 pollution (He et al., 2006; Yan et al., 2013). Enhanced antioxidant capacity in O3 -exposed plants, as a common response, has been observed in several other tree species under elevated O3 exposure (Nali et al., 2004; Lu et al., 2009). SOD is the only enzyme found to date in either plants or other organisms for the reduction of O− 2 to H2 O2 and the oxidation of O− 2 to O2 (Asada, 2000). In this study, the inter-speciﬁc differences in SOD activity are much greater than the O3 effect in many cases. Species differences in SOD response may reﬂect differences in acclimatory physiological adjustment in response to elevated O3 . Indeed, the increase of antioxidant ability was also associated with rising of other antioxidant enzyme activities or contents in antioxidants of urban tree species under different developmental stages, as had been reported by our recent studies (Lu et al., 2009; Yan et al., 2010a,b). Loreto and Velikova (2001) suggested that some VOCs (mainly isoprene) have antioxidant properties and can protect plants from O3 damage. Many studies have shown that elevated O3 exposure
S. Xu et al. / Urban Forestry & Urban Greening 14 (2015) 1166–1173
induced the VOCs emission from plants. In our study, elevated O3 signiﬁcantly increased VOCs emission rates of four urban tree species, particularly in Q. mongolica. Our result is similar to the report of Velikova et al. (2005), which indicates that exposure to elevated O3 caused a signiﬁcant increase of VOCs from Phragmites australis. At the same time, in laboratory experiments, O3 fumigation had increased the emission rate of VOCs in Q. ilex (Loreto et al., 2004). In addition, VOCs emission varied with urban tree species (Paoletti, 2009; Shi et al., 2011). Therefore, the absolute emission rates of VOCs may be much more dependent on species than O3 exposure. Conclusion Our study provides a helpful recommend in creating urban forest ecosystems considering O3 response and VOC emissions. The results for visible injury, foliar injury index, biomass changes, photosynthetic gas exchange, physiological, and biochemical indices support the conclusion that the four urban tree species express different levels of O3 -sensitivity. Q. mongolica showed comparatively higher sensitivity to elevated O3 and as such could be used for bio-monitoring of O3 in O3 -polluted urban areas. Our data conﬁrmed that the O3 impacts were different depending on the urban tree species. However, the inter-speciﬁc differences of urban tree species in physiological and biochemical changes are much greater than the ozone effect in many cases. The comparison of O3 -relevant characteristics between young and old trees with different developmental stages remains an important issue. Further research for the long-term O3 exposure is also necessary to determine the injury level and adaptation of urban plants under climate change. Acknowledgments This work would not have been possible but the tireless efforts of Prof. Dali Tao helped much in physiological measurements and critical reading and language polishing of the manuscript. This work was supported by the National Natural Science Foundation of China (31170573 and 31270518), the National Youth Scientiﬁc Funds (31000225) and the National Key Project (90411019). References Asada, K., 2000. The water-water cycle as an alternative photon and electron sinks. Philosoph. Trans. Royal Soc. B-Biol. Sci. 355, 1419–1431. Bagard, M.L., Thiec, D., Delacote, E., Hasenfratz-Sauder, M.P., Banvoy, J., Gerard, J., Dizengremel, P., Jolivet, Y., 2008. Ozone-induced changes in photosynthesis and photorespiration of hybrid poplar in relation to the developmental stage of the leaves. Physiol. Plant. 134, 559–574. Beyer, W.F., Fridovich, I., 1987. Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions. Anal. Biochem. 161, 559–566. Bortier, K., Ceulemans, R., De, T.L., 2000. Effects of tropospheric ozone on woody plants. In: Agrawal, M. (Ed.), Environmental Pollution and Plant Responses. Lewis Publishers, Boca Raton, FL, pp. 153–182. Buege, J.A., Aust, S.D., 1987. Microsomal lipid peroxidation. Methods Enzymol. 52, 302–310. Calatayud, A., Iglesias, D.J., Talon, M., Barreno, E., 2006. Effects of long-term ozone exposure on citrus: Chlorophyll a ﬂuorescence and gas exchange. Photosynthetica 44, 548–554. Calatayud, A., Ramirez, J.W., Iglesias, D.J., Barreno, E., 2002. Effects of ozone on photosynthetic CO2 exchange, chlorophyll a ﬂuorescence and antioxidant systems in lettuce leaves. Physiol. Plant. 116, 308–316. Calatayud, V., Cervero, J., Calvo, E., Garcia-Breijo, F.J., Reig-Arminana, J., Sanz, M.J., 2011. Responses of evergreen and deciduous Quercus species to enhanced ozone levels. Environ. Pollut. 159, 55–63. Chaudhary, N., Agrawal, S.B., 2013. Intraspeciﬁc responses of six Indian clover cultivars under ambient and elevated levels ozone. Environ. Sci. Pollut. Res. 20, 5318–5329. Contran, N., Paoletti, E., Manning, W.J., Tagliaferro, F., 2009. Ozone sensitivity and ethylenediurea protection in ash trees assessed by JIP chlorophyll a ﬂuorescence transient analysis. Photosynthetica 47, 68–78.
Feng, Z.Z., Zeng, H.Q., Wang, X.K., Zheng, Q.W., Feng, Z.W., 2008. Sensitivity of Metasequoia glyptostroboides to ozone stress. Photosynthetica 46, 463–465. Feng, Z., Sun, J., Wan, W., Hu, E., Calatayud, V., 2014. Evidence of widespread ozone-induced visible injury on plants in Beijing. Chin. Environ. Pollut. 193, 296–301. Ferretti, M., Calderisi, M., Bussotti, F., 2007. Ozone exposure, defoliation of beech (Fagus sylvatica) and visible foliar symptoms on native plants in selected plots of South-Western Europe. Environ. Pollut. 145, 644–651. Flagler, R.B., Chappelka, A.H., 1995. Growth responses of Southern pines to acidic precipitation and ozone. In: Fox, S., Mickler, R.A. (Eds.), Impacts of Air Pollutants on Southern Pine Forests. Springer-Verlag, New York, pp. 388–424. He, X., Huang, W., Chen, W., Dong, T., Liu, C., Chen, Z., Xu, S., Ruan, Y.N., 2009. Changes of main secondary metabolites in leaves of Ginkgo biloba in response to ozone fumigation. J. Environ. Sci. 21, 199–203. He, X., Ruan, Y., Chen, W., Lu, T., 2006. Responses of the anti-oxidative system in leaves of Ginkgo biloba to elevated ozone concentration in an urban area. Bot. Stud. 47, 409–416. He, X.Y., Fu, S.L., Chen, W., Zhao, T., Xu, S., Tuba, Z., 2007. Changes in effects of ozone exposure on growth, photosynthesis, and respiration of Ginkgo biloba in Shenyang urban area. Photosynthetica 45, 555–561. Heagle, A.S., Body, D.E., Heck, W.W., 1973. An open-top chamber to assess the impact of air pollution on plants. J. Environ. Qual. 2, 2031–2039. IPCC. 2013. Climate Change 2013. Fifth Assessment Report http://www.ipcc.ch/ report/ar5. Karnosky, D.F., Skelly, J.M., Percy, K.E., Chappelka, A.H., 2007. Perspectives regarding 50 years of research on effects of tropospheric ozone air pollution on US forests. Environ. Pollut. 147, 489–506. Kitao, M., Löw, M., Heerdt, C., Grams, T.E.E., Häberle, K.H., Matyssek, R., 2009. Effects of chronic elevated ozone exposure on gas exchange responses of adult beech trees (Fagus sylvatica) as related to the within-canopy light gradient. Environ. Pollut. 157, 537–544. Li, D., Chen, Y., Shi, Y., He, X., Chen, X., 2009. Impact of Elevated CO2 and O3 concentrations on biogenic volatile organic compounds emissions from Ginkgo biloba. Bull. Environ. Contam. Toxicol. 82, 473–477. Li, X.M., Zhang, L.H., Li, Y.Y., Ma, L.J., Chen, Q., Wang, L.L., He, X.Y., 2011. Effects of elevated carbon dioxide and/or ozone on endogenous plant hormones in the leaves of Ginkgo biloba. Acta Physiol. Plant. 33, 129–136. Loreto, F., Velikova, V., 2001. Isoprene produced by leaves protects the photosynthetic apparatus against ozone damage, quenches ozone products, and reduces lipid peroxidation of cellular membranes. Plant Physiol. 127, 1781–1787. Loreto, F., Pinelli, P., Manes, F., Kollist, H., 2004. Impact of ozone on monoterpene emissions and evidence for an isoprene-like antioxidant action of monoterpenes emitted by Quercus ilex leaves. Tree Physiol. 24, 361–367. Lu, T., He, X., Chen, W., Yan, K., Zhao, T., 2009. Effects of elevated O3 and/or elevated CO2 on lipid peroxidation and antioxidant systems in Ginkgo biloba leaves. Bull. Environ. Contam. Toxicol. 8, 92–96. Nali, C., Paoletti, E., Marabottini, R., Della, R.G., Lorenzini, G., Paolacci, A.R., Ciafﬁ, M., Badiani, M., 2004. Ecophysiological and biochemical, strategies of response to ozone in Mediterranean evergreen broadleaf species. Atmospher. Environ. 38, 2247–2257. Noormets, A., Sober, A., Pell, E.J., Dickson, R.E., Podila, G.K., Sober, J., Isebrands, J.G., Karnosky, D.F., 2001. Stomatal and non-stomatal limitation to photosynthesis in two trembling aspen (Populus tremuloides Michx.) clones exposed to elevated CO2 and/or O3 . Plant. Cell Environ. 24, 327–336. Novak, K., Schaub, M., Fuhrer, J., Skelly, J.M., Hug, C., Landolt, W., Bleuler, P., Krauchi, N., 2005. Seasonal trends in reduced leaf gas exchange and ozone-induced foliar injury in three ozone sensitive woody plants species. Environ. Pollut. 136, 33–45. Paoletti, E., 2009. Ozone and urban forests in Italy. Environ. Pollut. 157, 1506–1512. Pellegrini, E., Nali, C., Lorenzini, G., 2013. Ecophysiology of Tilia Americana under ozone fumigation. Atmospher. Pollut. Res. 4, 142–146. Shi, Y., Li, D., Chen, Y., He, X., 2011. Emissions of isoprenoid from major planting tree species in Shenyang. Adv. Mat. Res. 183–185, 1041–1045. Thwe, A.A., Vercambre, G., Gautier, H., Gay, F., Phattaralerphong, J., Kasemsap, P., 2014. Response of photosynthesis and chlorophyll ﬂuorescence to acute ozone stress in tomato (Solanum lycopersicum Mill.). Photosynthetica 52 (1), 105–116. Velikova, V., Pinelli, P., Pasqualini, S., Reale, L., Ferranti, F., Loreto, F., 2005. Isoprene decreases the concentration of nitric oxide in leaves exposed to elevated ozone. New Phytol. 166, 419–426. Vingarzan, R., 2004. A review of surface O3 background levels and trends. Atmospher. Environ. 38, 3431–3442. Wang, L., He, X., Chen, W., 2009. Effects of elevated ozone on photosynthetic CO2 exchange and chlorophyll a ﬂuorescence in leaves of Quercus mongolica grown in urban area. Bull. Environ. Contam. Toxicol. 82, 478–481. Wang, X.K., Manning, W., Feng, Z.W., Zhu, Y.G., 2007. Ground-level ozone in China: Distribution and effects on crop yields. Environ. Pollut. 147, 394–400. Wei, C., Skelly, J.M., Pennypacker, S.P., Ferdinand, J.A., Savage, J.E., Stevenson, R.E., Davis, D.D., 2004. Inﬂuence of light ﬂeck and low light on foliar injury and physiological responses of two hybrid poplar clones to ozone. Environ. Pollut. 130, 215–227. West, J.J., Fiore, A.M., Horowitz, L.W., Mauzerall, D.L., 2006. Global health beneﬁts of mitigating ozone pollution with methane emission controls. Proc. Natl. Am. Sci. USA 103, 3988–3993.
S. Xu et al. / Urban Forestry & Urban Greening 14 (2015) 1166–1173 Xu, S., Chen, W., Huang, Y., He, X., 2012. Responses of growth, photosynthesis and VOCs emissions Pinus tabulaeformis Carr. exposure to elevated CO2 and/or elevated O3 in an urban area. Bull. Environ. Contam. Toxicol. 88, 443–448. Xu, S., He, X., Chen, W., Su, D., Huang, Y., 2014. Elevated CO2 ameliorated the adverse effect of elevated O3 in previous-year and current-year needles of Pinus tabulaeformis in Urban area. Bull. Environ. Contam. Toxicol. 92, 733–737. Yan, K., Chen, W., He, X., Zhang, G., Xu, S., Wang, L., 2010a. Responses of photosynthesis, lipid peroxidation and antioxidant system in leaves of Quercus mongolica to elevated O3 . Environ. Exp. Bot. 69, 198–204.
Yan, K., Chen, W., Zhang, G., Xu, S., Liu, Z., He, X., Wang, L., 2010b. Elevated CO2 ameliorated oxidative stress induced by elevated O3 in Quercus mongolica. Acta Physiol. Plant. 32, 375–385. Yan, K., He, X., Chen, W., Lu, T., Xu, S., Shao, H., Zhang, G., 2013. Variation of antioxidant system in Pinus armandii under elevated O3 in an entire growth season. Clean-Soil Air Water 41, 5–10. Zhangyuan, D., Bramlage, W.J., 1992. Modiﬁed thiobarbituric acid assay for measuring lipid oxidation in sugar-rich plant tissue extracts. J. Agric. Food Chem. 40, 1566–1570.