Browse Subject Areas

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Non-Stomatal Limitation to Photosynthesis in Cinnamomum camphora Seedings Exposed to Elevated O3

  • Junfeng Niu,

    Affiliation Key Laboratory of Vegetation Restoration and Management of Degraded Ecosystems, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, Guangdong, China

  • Zhaozhong Feng,

    Affiliation State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China

  • Weiwei Zhang,

    Affiliation Key Laboratory of Black Soil Ecology, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Harbin, Heilongjiang, China

  • Ping Zhao,

    Affiliation Key Laboratory of Vegetation Restoration and Management of Degraded Ecosystems, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, Guangdong, China

  • Xiaoke Wang

    Affiliation State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China

Non-Stomatal Limitation to Photosynthesis in Cinnamomum camphora Seedings Exposed to Elevated O3

  • Junfeng Niu, 
  • Zhaozhong Feng, 
  • Weiwei Zhang, 
  • Ping Zhao, 
  • Xiaoke Wang


Ozone (O3) is the most phytotoxic air pollutant for global forests, with decreased photosynthesis widely regarded as one of its most common effects. However, controversy exists concerning the mechanism that underlies the depressing effects of O3 on CO2 assimilation. In the present study, seedlings of Cinnamomum camphora, a subtropical evergreen tree species that has rarely been studied, were exposed to ambient air (AA), ambient air plus 60 [ppb] O3 (AA+60), or ambient air plus 120 [ppb] O3 (AA+120) in open-top chambers (OTCs) for 2 years. Photosynthetic CO2 exchange and chlorophyll a fluorescence were investigated in the second growing season (2010). We aim to determine whether stomatal or non-stomatal limitation is responsible for the photosynthesis reduction and to explore the potential implications for forest ecosystem functions. Results indicate that elevated O3 (E-O3) reduced the net photosynthetic rates (PN) by 6.0-32.2%, with significant differences between AA+60 and AA+120 and across the four measurement campaigns (MCs). The actual photochemical efficiency of photosystem II (PSII) in saturated light (Fv/Fm) was also significantly decreased by E-O3, as was the effective quantum yield of PSII photochemistry (ΦPSII). Moreover, E-O3 significantly and negatively impacted the maximum rates of carboxylation (Vcmax) and electron transport (Jmax). Although neither the stomatal conductance (gs) nor the intercellular CO2 concentration (Ci) was decreased by E-O3, PN/gs was significantly reduced. Therefore, the observed reduction in PN in the present study should not be attributed to the unavailability of CO2 due to stomatal limitation, but rather to the O3-induced damage to Ribulose-1,5-bisphosphate carboxylase/oxygenase and the photochemical apparatus. This suggests that the down-regulation of stomatal conductance could fail to occur, and the biochemical processes in protoplasts would become more susceptible to injuries under long-term O3 exposure, which may have important consequences for forest carbon and water budget.


Of the known phytotoxic air pollutants, ozone (O3) has the greatest potential to detrimentally impact forests [1]. The negative effects of O3 on plants are commonly attributed to its highly oxidative properties that can damage cell membranes, denature critical enzymes, and give rise to various reactive oxygen species (ROS) [2], . One of the most consistent effects of O3 on trees is the inhibition of photosynthesis [5], [6], [7]. Based on a quantitative meta-analysis of over 100 studies of O3 effect on trees, it has been estimated that an 11% decrease in net photosynthetic rates (PN) has resulted from the increase in O3 levels that has occurred since the Industrial Revolution [8].

However, controversy exists concerning the mechanism that underlies the depressing effects of O3 on CO2 assimilation in plants [9]. While previous studies have frequently linked these effects to decreased stomatal conductance (gs), many others have related the O3-induced decline in photosynthesis to altered mesophyll activities, such as reduced maximum rates of carboxylation (Vcmax) and electron transport (Jmax) [10], [11], [12], [13]. Additionally, under high levels of O3, changes in chlorophyll a fluorescence, including reductions in photochemical quenching (qP), actual photochemical efficiency (Fv/Fm) and the effective quantum yield of photosystem II (PSII) in saturated light (ФPSII), have also been widely reported [14],[15]. In fact, due to its high-resolution, real-time, and non-invasive nature, chlorophyll a fluorescence measurement has become an important technique parallel to gas exchange analyses for confirming the primary site of photosynthetic limitation [16]. Combined measurements of chlorophyll a fluorescence and gas exchange can provide valuable information regarding plant photosynthetic performance [17].

Field and experimental evidence suggests that broadleaf evergreen trees in Mediterranean areas are comparatively more O3 tolerant than deciduous species in temperate and boreal regions [18], [19]. This can be largely attributed to the sclerophyllous traits of the former, as well as the uniquely Mediterranean climate that concentrates high levels of O3 during seasons of drought, which triggers stomatal closure [20], [21], [22], [23]. In subtropical regions, trees also develop glossy and leathery leaves, however, the climate is generally characterized by four clearly demarcated seasons with rain and heat co-occurring in summers, and so far very little information is available concerning the impact of O3 on the regional evergreen tree species.

In the present study, seedlings of C. camphora, a subtropical evergreen broadleaf tree species native to the Yangtze River Delta in eastern China, were exposed to ambient air (AA), ambient air plus 60 [ppb] O3 (AA+60), or ambient air plus 120 [ppb] O3 (AA+120) in open-top chambers (OTCs) for 2 years. During the second growing season (2010), gas exchange and chlorophyll a fluorescence were measured and analyzed. The aims of this experiment were to: (1) determine the extent to which photosynthesis is reduced in the experimental seedlings exposed to elevated O3 (E-O3, AA+60 or AA+120), (2) clarify whether stomatal or non-stomatal limitation is responsible for this reduction, and (3) explore the ecological meaning of our findings to broad-scale studies.

Materials and Methods

Experimental site and plant material

Permits and approvals for the work were obtained from East China Normal University and Tiantong Forest Farm, which are responsible for the protection of the Tiantong National Forest Park. The experiment was carried out within the park, at the Tiantong National Field Observation and Research Station for Forest Ecosystems (29°48′N, 121°47′E), Ningbo, Zhejiang province, China. One-year-old C. camphora seedlings, a typical subtropical evergreen broadleaf tree species widely distributed throughout eastern China and Japan, were planted in 5-[L] plastic pots and fumigated with E-O3 for 2 years. These seedlings were purchased from a nearby commercial nursery and selected for phenotypic homogeneity. The potting soil consisted of a mixture of native yellowish-brown lateritic soil and litter collected from a fir forest at a 1∶1 ratio. All seedlings were acclimated to OTC conditions for two weeks before O3 fumigation. More information about the climate conditions of the experimental site and the plant cultivation prior to O3 treatment were described in Niu et al.[24].

OTCs and treatments

Six OTCs (octagonal base, 5.5 [m2] of basal area, and 2.6 [m] in height) were set up at the experimental field in early 2009. The rate of light transmittance of the OTCs was 98.3% and the average air velocity corresponded to a turnover rate of two complete air changes per minute. O3 was generated from pure oxygen using an electrical discharge O3 generator (HY003, Chuangcheng Technology Co., Ltd., Jinan, China) and piped into four of the six OTCs in mixture with ambient air. O3 flow was modulated using mass flow meters (SY-9311, Beijing Shengye Science and Technology Development Co., Ltd., Beijing, China) in order to obtain the designated O3 concentration within each OTC. The seedlings were fumigated from 9:00–17:00, 7 days per week, 25 May to 10 September 2009 and 1 May to 7 October 2010, except for rainy and mostly cloudy days.

Replicate AA, AA+60, and AA+120 chambers were randomly arranged in the experimental field. Seedling positions within each OTC were changed every 3-5 days. Every 10-15 days, all chambers were emptied and randomly reassigned a new O3 level, and the seedlings were replaced according to their specified treatment levels. This allowed us to eliminate position and chamber effects, treating each plant as an independent experimental unit. Five seedlings within each OTC and a total of 30 plants (5 plants×3 groups×2 OTCs) were investigated.


Temperature and relative air humidity were recorded at 30-minute intervals using thermo-hygrographs (DSR-TH, ZOGLAB Microsystem Inc., Hangzhou, China) inside and outside the OTCs. O3 concentrations at approximately 10 [cm] above the plant canopy were monitored using a UV-absorption O3 analyzer (Model 49i, Thermo Scientific Inc., Connecticut, USA).

Gas exchange and chlorophyll a fluorescence under light conditions were measured using an infrared gas analyzer (IRGA) fitted with a 6400-40 leaf chamber fluorometer (LI-6400, LI-COR Inc., Lincoln, NE, USA). All measurements were made from 9:00–12:00 and recorded when the coefficient of variance (CV) was less than 3%. The photosynthetic photon flux density (PPFD) was fixed at a saturating intensity of 1200 [µmol m−2 s−1]. CO2 was supplied with pure CO2 cylinders and maintained at 380 [µmol mol−1]. Block temperature of the cuvette was set to 30±0.5°C, and relative humidity 60±5%. Maximum, minimum and steady state fluorescence under light conditions (Fm, Fo and Fs) were measured, and ФPSII, qP and Fv/Fm were calculated as (Fm-Fs)/Fm, (Fm-Fs)/(Fm-Fo) and (Fm- Fo)/Fm, respectively. Six plants per treatment were analyzed, and only fully expanded upper leaves were screened. Tracking analyses of leaves in the same leaf position were carried out monthly (2 July, 7 August, 7 September, and 8 October 2010).

In order to determine the maximum photochemical efficiency of PSII (Fv/Fm), a field-portable chlorophyll fluorometer (FMS 2, Hansatech Instruments Ltd., Norfolk, UK) was employed. The same leaves used for gas exchange analyses were screened. Leaves were adapted to dark for 30 minutes and the minimum fluorescence (Fo) was measured by switching on the modulating light (0.6 [kHz]). Then, the application of a saturating light pulse (8000 [m−2 s−1] for 1 [s]) led to the rapid closure of PSII reaction centers, yielding the maximum fluorescence (Fm). Fv/Fm was calculated as (Fm-Fo)/Fm.

The response of carbon assimilation rates to changing CO2 concentrations (A-Ci response curves) was determined by sequentially measuring the rates of photosynthesis at CO2 concentrations of 380, 200, 150, 100, 50, 400, 600, 900, 1200 and 1500 [µmol mol−1]. Light intensity, block temperature and relative humidity were set equal to those used in gas exchange analyses. Vcmax and Jmax were determined and adjusted to 25°C according to Long and Bernacchi [25]. Four seedlings per treatment were screened, and two measurement campaigns (MCs) were carried out for the A-Ci response curves, on 22 July and 24 September 2010, respectively.

Data analysis

Data were analyzed using SAS software (Version 9.1.3, SAS Institute, Cary, NC, USA). Repeated measures analyses of variance (RANOVAs) were performed in order to analyze the overall effect of O3 on the examined parameters throughout the growing season. Variances across MCs, as well as the interaction between O3 and MCs were also investigated. For each MC, ANOVA model was applied to test the O3 effect and Bonferroni methods were adopted for post-hoc multiple comparisons. Normality of distribution and homogeneity of variance were tested before all analyses. Differences between treatments were considered significant if p≤0.05.


OTC microclimate and O3 monitoring

Table 1 shows AOT40s (accumulated O3 exposure over a threshold of 40 [ppb]) and SUM60s (sum of hourly O3 concentration when the concentration is equal to or greater than 60 [ppb]) during each MC of gas exchange and chlorophyll a fluorescence during the 2010 growing season. Because of persistent rain (19 days) from 7 August to 7 September, the accumulation of AOT40 and SUM60 was lower during this period, as shown in Table 1. Under AA, the total dose of O3 was 6.7 and 8.7 [ppm h] in the forms of AOT40 and SUM60, respectively. AOT40s were generally lower than values expressed as SUM60s in all O3 regimes. Detailed descriptions of the average O3 concentrations, as well as the OTC microclimate conditions throughout the 2 years of this experiment can be found elsewhere [24], [26].

Table 1. AOT40s and SUM60s under different O3 exposure regimes during the 2010 growing season of Cinnamomum camphora seedlings.

Gas exchange

Throughout the 2010 growing season, PN was reduced, on average, by 13.0% and 25.3% under AA+60 and AA+120, respectively. Differences between these two treatment regimes were statistically significant, except on 2 July (Table 2 and Figure 1a). PN/gs was also significantly reduced, while Ci was significantly increased by AA+120 on 8 October. A negative O3 effect on PN/gs was also observed on 7 September (Figure 1d). Variations of PN, gs, Ci, and PN/gs across the four MCs were statistically significant, and O3 interacted significantly with MCs for PN and Ci (Table 2).

Figure 1. Effects of elevated O3 on gas exchange parameters.

Vertical bars represent average levels and distinct letters indicate significant differences among O3 regimes (n = 6) (AA: ambient air; AA+60: ambient air plus 60 [ppb] O3; AA+120: ambient air plus 120 [ppb] O3).

Table 2. Repeated measures ANOVAs (RANOVAs) of the gas exchange and chlorophyll a fluorescence parameters of Cinnamomum camphora seedlings during the 2010 growing season (P values are shown, n = 4 for Vcmax, Jmax and Jmax/Vcmax, n = 6 for other parameters).

Vcmax was significantly decreased by AA+60 and AA+120 on 24 September, but only by AA+120 on 22 July (Figure 2b). Both AA+60 and AA+120 exerted significantly negative effects on Jmax (Figure 2a) across the two MCs. The difference in Jmax between AA+60 and AA+120 was statistically significant on 22 July, but not on 24 September (Figure 2a). Jmax varied significantly, while Vcmax maintained the same levels across the two MCs (Table 2). Jmax/Vcmax was not significantly affected by E-O3 in the present study (Figure 2c).

Figure 2. Effects of elevated O3 on the maximum rates of carboxylation (Vcmax) and electron transport (Jmax).

Vertical bars represent average levels and distinct letters indicate significant differences among O3 regimes (n = 4) (AA: ambient air; AA+60: ambient air plus 60 [ppb] O3; AA+120: ambient air plus 120 [ppb] O3).

Chlorophyll a fluorescence

Fv/Fm and ФPSII were significantly decreased by E-O3 (Table 2). ФPSII was significantly decreased by AA+120 across all four MCs, and also by AA+60 on 2 July and 8 October. For Fv/Fm, only AA+120 exerted significant impact, on 2 July and 8 October (Figure 3a, c). Additionally, qP was significantly depressed by AA+120 across all four MCs, and also by AA+60 on 7 August and 8 October (Figure 3b). However, Fv/Fm was not significantly influenced by E-O3 (Figure 3d). Differences across MCs were statistically significant for all fluorescence parameters. However, no interactions were found between O3 and MCs (Table 2).

Figure 3. Effects of elevated O3 on the chlorophyll a fluorescence parameters.

Vertical bars represent average levels and distinct letters indicate significant differences among O3 regimes (n = 6) (AA: ambient air; AA+60: ambient air plus 60 [ppb] O3; AA+120: ambient air plus 120 [ppb] O3).


E-O3 significantly reduced PN in C. camphora over the course of the present study. At the end of the 2010 growing season, AA+60, which corresponded to an AOT40 of 26.1 [ppm h], reduced PN by 11.7%. A similar 11.4% decrease in PN occurred with an AOT40 of 36.2 [ppm h] in the Mediterranean evergreen Satsuma mandarin (Cirtus unshiu [Mak.] Marc.) [15]. However, in deciduous Quercus pyrenaica, Quercus robur and Quercus faginea, an AOT40 of 26.2-28.8 [ppm h] decreased PN by 64%, 38% and 33%, respectively [27]. Based on the comparisons with these results, our findings suggest that C. camphora is less resistant to O3 than Mediterranean evergreen broadleaves, but more tolerant than deciduous species [7], [19]. Reduction in PN under E-O3 has also been reported in other species, deciduous (Quercus serrata, Populus tremuloides Michx., Betula pendula Roth.) as well as evergreen (Pinus taeda L.) [28], [29], [30], [31], and therefore this may represent a common response behavior to high levels of O3 in woody plants [32].

Similar to observations in European beech (Fagus sylvatica) and black aspen (Populus nigra) [33], gs was not affected by E-O3 in the present study (Figure 1b), indicating that the significant reduction in PN of C. camphora cannot be attributed to stomatal behavior. Additionally, Ci was not reduced, but in fact significantly increased by AA+120 on 8 October, confirming that CO2 supply was not the limiting factor in reducing PN. Moreover, significant decrease in PN/gs was detected under AA+120 on 7 September and 8 October, further suggesting that factors other than gs should be considered when attempting to clarify the mechanism responsible for the reduction in PN that results from O3 stress. Increased Ci, as well as the negative relationship between PN and Ci under elevated O3, has also been documented in previous literature [34], [35].

RuBisCO is commonly regarded as one of the primary targets of O3-induced damage [36], [37]. On both 22 July and 24 September, AA+120 significantly reduced Vcmax, which was also notably decreased by AA+60 on 24 September (Figure 2b). These findings concur broadly with those reported in aspen (P. tremuloides) and birch (B. pendula Roth.) [28], [38], [39]. Decreases in RuBisCO quantity and activity may be responsible for the decline of Vcmax under E-O3 [40]. Jmax was also significantly decreased by E-O3 in the present study, while Jmax/Vcmax was not affected (Figure 2c). The constant Jmax/Vcmax ratio indicates the close coupling between RuBP carboxylation and light-driven electron transport. Activities of these two processes are commonly related, and vary in parallel with environmental conditions [41]; thus the observed decrease in Jmax might have occurred in response to the declining Vcmax [42]. Therefore, O3-induced degradation and deactivation of RuBisCO, as well as its feedback inhibitory effect on the electron transport system, might be a primary cause of the reduction in PN.

Confirming the previous findings in evergreen Mediterranean species [43], [44], Fv/Fm in C. camphora was not influenced by E-O3 (Figure 3d). However, Fv′/Fm′ was significantly reduced by AA+120 on 2 July and 8 October, implying enhanced energy decay via non-radiative processes at the PSII reaction centers [45]. Meanwhile, in the present study, significant reductions of qP (notably under AA+120 at all four MCs and under AA+60 on 7 August and 8 October) and ФPSII (notably under AA+120 at all four MCs and under AA+60 on 2 July and 8 October) were also observed under E-O3 (Figure 3a, b). Similar results have been reported in Scots pine (Pinus sylvestris L.) and Satsuma mandarin (C. unshiu [Mak.] Marc.) [15], [17]. Reduction in qP and ФPSII may correlate with a decrease in the proportion of available excitation energy used in the photochemistry [11]. Decreased photochemistry but enhanced non-radiative decay suggests that more energy absorbed by PSII is dissipated as heat, and this could then lead to the overheating of PSII reaction centers. Therefore, direct oxidative damage, as well as indirect heat-related injuries to the photochemical apparatus, may also play an important role in mediating the down-regulation of PN in C. camphora exposed to E-O3.

Previous studies have frequently linked the O3-induced decline in plant photosynthesis to its inhibitory effect on foliar stomatal conductance. Torsethaugen et al. found that acute O3 exposure inhibited the guard cell K+ channels, which mediate K+ uptake that drives stomatal opening, and thus led to decreased photosynthesis in Vicia faba [46]. Zhang et al. reported that the reduction in photosynthesis (-27%) of Liriodendron chinense (Hemsl.) Sarg seedlings was accompanied by a significant decrease of stomatal conductance (-34.7%) after O3 exposure for 40 days at a concentration of [150 ppb] [47]. During the first growing season (2009) of the present study, we also observed concurrent reduction in photosynthesis (-24.6%) and stomatal conductance (-34.2%) under [AA+60] [26], which was however not observed during the second growing season (2010). This suggests that the coupling between photosynthesis and stomatal conductance in plants could fail and the biochemical processes in protoplasts would become more susceptible to injuries under long-term O3 exposure.

Decoupling between photosynthesis and stomatal conductance under elevated O3 may have important implications for water use and carbon cycling of forest ecosystems [48]. One the one hand, failure or sluggishness of stomatal closure could give rise to excessive plant transpiration [49], resulting in unnecessary water loss, leading to regional water shortage, or even causing tree wilt and dieback if soil water supply is particularly tight, especially in arid and semi-arid areas. At the same time, increased exposure of mesophyll cells to O3 through open stoma, on the other hand, could decrease the efficiency of light use by photosystem II for CO2 assimilation [25], resulting in lower forest carbon sequestration and leading to a warmer atmosphere. Therefore, the potential impact of O3 under both current and future enriched conditions should be considered adequately in carbon budget calculations, forest hydrology simulations and climate change predictions at regional and global scales.

It should be noted that the present study was conducted on just one subtropical evergreen species of 2 to 3 years age. The unique physiological characteristics of seedlings and the optimal water status under OTC conditions as well as the restriction of root growth in pots may bias tree performance [4], [24]. To attain a comprehensive understanding of the effect of O3 on forests, as well as forest responses and feedbacks to global changes, further investigations based on mature trees of a wider range of other species are critically needed.


E-O3 (AA+60 or AA+120) significantly reduced PN in C. camphora. Comparisons of this reduction with those observed in other species suggest that C. camphora is less tolerant to O3 than Mediterranean evergreen trees, but more resistant than deciduous species. Reduction of stomatal conductance is not a reasonable explanation for the decline of PN in the present study, as manifested by the increased Ci and decreased PN/gs. As with PN, decreases in Vcmax, Jmax, ФPSII and qP were detected, indicating that direct oxidative damage and indirect heat-related injuries to RuBisCO and photochemical apparatus were responsible for the reduction in PN that was observed in C. camphora under E-O3. This suggests that the biochemical processes in protoplasts will become more susceptible to injuries under long-term O3 exposure, which may bear important implications for forest water use and carbon cycling.


For fieldwork assistance, we wish to thank Mr Wenzhi Sun. Great appreciation is also given to Professor Zongwei Feng for his constructive suggestions on the experimental design.

Author Contributions

Conceived and designed the experiments: JFN ZZF. Performed the experiments: JFN WWZ. Analyzed the data: JFN. Contributed reagents/materials/analysis tools: JFN. Wrote the paper: JFN. Helpful suggestions in data analyses and manuscript preparation: XKW PZ.


  1. 1. Bussotti F (2008) Functional leaf traits, plant communities and acclimation processes in relation to oxidative stress in trees: a critical overview. Global Change Biology 14: 2727–2739.
  2. 2. Karnosky DF, Pregitzer KS, Zak DR, Kubiske ME, Hendrey GR, et al. (2005) Scaling ozone responses of forest trees to the ecosystem level in a changing climate. Plant Cell and Environment 28: 965–981.
  3. 3. Matyssek R, Sandermann H, Wieser G, Booker F, Cieslik S, et al. (2008) The challenge of making ozone risk assessment for forest trees more mechanistic. Environmental Pollution 156: 567–582.
  4. 4. Samuelson L, Kelly JM (2001) Scaling ozone effects from seedlings to forest trees. New Phytologist 149: 21–41.
  5. 5. Bortier K, Ceulemans R, De Temmerman L (2000) Effects of ozone exposure on growth and photosynthesis of beach seedlings (Fagus sylvatica). New Phytologist 146: 271–280.
  6. 6. Karnosky DF, Skelly JM, Percy KE, Chappelka AH (2007) Perspectives regarding 50 years of research on effects of tropospheric ozone air pollution on US forests. Environmental Pollution 147: 489–506.
  7. 7. Paoletti E (2006) Impact of ozone on Mediterranean forests: A review. Environmental Pollution 144: 463–474.
  8. 8. Wittig VE, Ainsworth EA, Long SP (2007) To what extent do current and projected increases in surface ozone affect photosynthesis and stomatal conductance of trees? A meta-analytic review of the last 3 decades of experiments. Plant Cell and Environment 30: 1150–1162.
  9. 9. Degl'Innocenti E, Vacca C, Guidi L, Soldatini GF (2003) CO2 photoassimilation and chlorophyll fluorescence in two clover species showing different response to O3. Plant Physiology and Biochemistry 41: 485–493.
  10. 10. Darbah JNT, Kubiske ME, Nelson N, Kets K, Riikonen J, et al. (2010) Will photosynthetic capacity of aspen trees acclimate after long-term exposure to elevated CO2 and O3? Environmental Pollution 158: 983–991.
  11. 11. Feng Z, Pang J, Kobayashi K, Zhu J, Ort DR (2011) Differential responses in two varieties of winter wheat to elevated ozone concentration under fully open-air field conditions. Global Change Biology 17: 580–591.
  12. 12. He XY, Fu SL, Chen W, Zhao TH, Xu S, et al. (2007) Changes in effects of ozone exposure on growth, photosynthesis, and respiration of Ginkgo biloba in Shenyang urban area. Photosynthetica 45: 555–561.
  13. 13. Matyssek R, Gunthardtgoerg MS, Keller T, Scheidegger C (1991) Impairment of gas-exchange and structure in birch leaves (betula-pendula) caused by low ozone concentrations. Trees-Structure and Function 5: 5–13.
  14. 14. Bussotti F, Desotgiu R, Cascio C, Pollastrini M, Gravano E, et al. (2011) Ozone stress in woody plants assessed with chlorophyll a fluorescence. A critical reassessment of existing data. Environmental and Experimental Botany 73: 19–30.
  15. 15. Calatayud A, Iglesias DJ, Talon M, Barreno E (2006) Effects of long-term ozone exposure on citrus: Chlorophyll a fluorescence and gas exchange. Photosynthetica 44: 548–554.
  16. 16. Soja G, Pfeifer U, Soja AM (1998) Photosynthetic parameters as early indicators of ozone injury in apple leaves. Physiologia Plantarum 104: 639–645.
  17. 17. Wang KY, Kellomaki S (1997) Effects of elevated CO2 and soil-nitrogen supply on chlorophyll fluorescence and gas exchange in Scots pine, based on a branch-in-bag experiment. New Phytologist 136: 277–286.
  18. 18. Bussotti F, Gerosa G (2007) Are the Mediterranean forests in Southern Europe threatened from ozone? Journal of Mediterranean Ecology 3: 23–34.
  19. 19. Nali C, Paoletti E, Marabottini R, Della Rocca G, Lorenzini G, et al. (2004) Ecophysiological and biochemical, strategies of response to ozone in Mediterranean evergreen broadleaf species. Atmospheric Environment 38: 2247–2257.
  20. 20. Gerosa G, Finco A, Mereu S, Vitale M, Manes F, et al. (2009) Comparison of seasonal variations of ozone exposure and fluxes in a Mediterranean Holm oak forest between the exceptionally dry 2003 and the following year. Environmental Pollution 157: 1737–1744.
  21. 21. Grulke NE, Paoletti E (2005) A field system to deliver desired O3 concentrations in leaf-level gas exchange measurements: results for Holm Oak near a CO2 spring. Phyton-Annales Rei Botanicae 45: 21–31.
  22. 22. Manes F, Vitale M, Fabi AM, De Santis F, Zona D (2007) Estimates of potential ozone stomatal uptake in mature trees of Quercus ilex in a Mediterranean climate. Environmental and Experimental Botany 59: 235–241.
  23. 23. Ribas A, Penuelas J, Elvira S, Gimeno BS (2005) Ozone exposure induces the activation of leaf senescence-related processes and morphological and growth changes in seedlings of Mediterranean tree species. Environmental Pollution 134: 291–300.
  24. 24. Niu J, Zhang W, Feng Z, Wang X, Tian Y (2011) Impact of elevated O3 on visible foliar symptom, growth and biomass of Cinnamomum camphora seedlings under different nitrogen loads. Journal of Environmental Monitoring 13: 2873–2879.
  25. 25. Long SP, Bernacchi CJ (2003) Gas exchange measurements, what can they tell us about the underlying limitations to photosynthesis? Procedures and sources of error. Journal of Experimental Botany 54: 2393–2401.
  26. 26. Feng Z, Niu J, Zhang W, Wang X, Yao F, et al. (2011) Effects of ozone exposure on sub-tropical evergreen Cinnamomum camphora seedlings grown in different nitrogen loads. Trees-Structure and Function 25: 617–625.
  27. 27. Calatayud V, Cervero J, Calvo E, Garcia-Breijo F-J, Reig-Arminana J, et al. (2011) Responses of evergreen and deciduous Quercus species to enhanced ozone levels. Environmental Pollution 159: 55–63.
  28. 28. Noormets A, Kull O, Sober A, Kubiske ME, Karnosky DF (2010) Elevated CO2 response of photosynthesis depends on ozone concentration in aspen. Environmental Pollution 158: 992–999.
  29. 29. Oksanen E (2003) Responses of selected birch (Betula pendula Roth) clones to ozone change over time. Plant Cell and Environment 26: 875–886.
  30. 30. Richardson CJ, Sasek TW, Fendick EA, Kress LW (1992) Ozone exposure-response relationships for photosynthesis in genetic strains of loblolly pine seedlings. Forest Ecology and Management 51: 163–178.
  31. 31. Watanabe M, Yamaguchi M, Tabe C, Iwasaki M, Yamashita R, et al. (2007) Influences of nitrogen load on the growth and photosynthetic responses of Quercus serrata seedlings to O3. Trees-Structure and Function 21: 421–432.
  32. 32. Bussotti F, Desotgiu R, Cascio C, Strasser RJ, Gerosa G, et al. (2007) Photosynthesis responses to ozone in young trees of three species with different sensitivities, in a 2-year open-top chamber experiment (Curno, Italy). Physiologia Plantarum 130: 122–135.
  33. 33. Bussotti F, Strasser RJ, Schaub M (2007) Photosynthetic behavior of woody species under high ozone exposure probed with the JIP-test: A review. Environmental Pollution 147: 430–437.
  34. 34. Novak K, Cherubini P, Saurer M, Fuhrer J, Skelly JM, et al. (2007) Ozone air pollution effects on tree-ring growth, delta C-13, visible foliar injury and leaf gas exchange in three ozone-sensitive woody plant species. Tree Physiology 27: 941–949.
  35. 35. Yan K, Chen W, He X, Zhang G, Xu S, et al. (2010) Responses of photosynthesis, lipid peroxidation and antioxidant system in leaves of Quercus mongolica to elevated O3. Environmental and Experimental Botany 69: 198–204.
  36. 36. Fontaine V, Pelloux J, Podor M, Afif D, Gerant D, et al. (1999) Carbon fixation in Pinus halepensis submitted to ozone. Opposite response of ribulose-1,5-bisphosphate carboxylase/oxygenase and phosphoenolpyruvate carboxylase. Physiologia Plantarum 105: 187–192.
  37. 37. Noormets A, Sober A, Pell EJ, Dickson RE, Podila GK, et al. (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 and Environment 24: 327–336.
  38. 38. Eichelmann H, Oja V, Rasulov B, Padu E, Bichele I, et al. (2004) Photosynthetic parameters of birch (Betula pendula Roth) leaves growing in normal and in CO2- and O3-enriched atmospheres. Plant, Cell & Environment 27: 479–495.
  39. 39. Kets K, Darbah JNT, Sober A, Riikonen J, Sober J, et al. (2010) Diurnal changes in photosynthetic parameters of Populus tremuloides, modulated by elevated concentrations of CO2 and/or O3 and daily climatic variation. Environmental Pollution 158: 1000–1007.
  40. 40. Dann MS, Pell EJ (1989) Decline of activity and quantity of ribulose bisphosphate carboxylase oxygenase and net photosynthesis in ozone-treated potato foliage. Plant Physiology 91: 427–432.
  41. 41. Warren CR, Dreyer E, Adams MA (2003) Photosynthesis-Rubisco relationships in foliage of Pinus sylvestris in response to nitrogen supply and the proposed role of Rubisco and amino acids as nitrogen stores. Trees-Structure and Function 17: 359–366.
  42. 42. Rogers A, Humphries SW (2000) A mechanistic evaluation of photosynthetic acclimation at elevated CO2. Global Change Biology 6: 1005–1011.
  43. 43. Ribas A, Penuelas J, Elvira S, Gimeno BS (2005) Contrasting effects of ozone under different water supplies in two Mediterranean tree species. Atmospheric Environment 39: 685–693.
  44. 44. Vitale M, Salvatori E, Loreto F, Fares S, Manes F (2008) Physiological responses of Quercus ilex leaves to water stress and acute ozone exposure under controlled conditions. Water Air and Soil Pollution 189: 113–125.
  45. 45. Strand M, Oquist G (1985) Inhibition of photosynthesis by freezing temperatures and high light levels in cold-acclimated seedlings of scots pine (pinus-sylvestris) .2. effects on chlorophyll fluorescence at room-temperature and 77-k. Physiologia Plantarum 65: 117–123.
  46. 46. Torsethaugen G, Pell EJ, Assmann SM (1999) Ozone inhibits guard cell K+ channels implicated in stomatal opening. Proceedings of the National Academy of Sciences of the United States of America 96: 13577–13582.
  47. 47. Zhang WW, Niu JF, Wang XK, Tian Y, Yao FF, et al. (2011) Effects of ozone exposure on growth and photosynthesis of the seedlings of Liriodendron chinense (Hemsl.) Sarg, a native tree species of subtropical China. Photosynthetica 49: 29–36.
  48. 48. Lombardozzi D, Levis S, Bonan GB, Sparks JP (2012) Predicting photosynthesis and transpiration responses to ozone: Decoupling modeled photosynthesis and stomatal conductance. Biogeosciences 9: 3113–3130.
  49. 49. Sun GE, McLaughlin SB, Porter JH, Uddling J, Mulholland PJ, et al. (2012) Interactive influences of ozone and climate on streamflow of forested watersheds. Global Change Biology 18: 3395–3409.