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Journal of Experimental Botany, Vol. 55, No. 397, pp. 771-781, March 1, 2004
© 2004 Oxford University Press

Plants and the Environment

Mechanisms underlying the amelioration of O3-induced damage by elevated atmospheric concentrations of CO2

Received 25 November 2003; Accepted 28 November 2003

João Cardoso-Vilhena1, Luis Balaguer2, Derek Eamus3, John Ollerenshaw4 and Jeremy Barnes4,*

1 Museu, Laboratório e Jardim Botânico, Universidade de Lisboa, Rua da Escola Politécnica 58, 1250-102 Lisboa, Portugal
2 Departamento de Biología Vegetal I, Facultad de Ciencias Biológicas, Universidad Complutense, 28040 Madrid, Spain
3 Institute for Water and Environmental Resource Management, Dunbar Building, University of Technology, Sydney, CNR. Westbourne Street and Pacific Highway, St. Leonards, NSW 2065, Australia
4 Environmental and Molecular Plant Physiology, Institute for Research on the Environment and Sustainability [IRES], School of Biology, Newcastle University, Newcastle Upon Tyne NE1 7RU, UK

* To whom correspondence should be addressed. Fax: +44 (0)191 222 5229. E-mail: J.D.Barnes{at}ncl.ac.uk
Abbreviations: {phi}app, apparent quantum yield of CO2 assimilation; A, rate of CO2 assimilation; A350, light-saturated rate of CO2 assimilation rate at an ambient CO2 concentration of 350 µl l–1; Amax, light- and CO2-saturated rate of CO2 assimilation; CFA, charcoal/Purafil®-filtered air; ci, intercellular CO2 concentration; DALE, days after leaf emergence; DAT, days after transfer; Fv/Fm, maximum quantum yield of PSII photochemistry; gH2O, stomatal conductance to water vapour; K, root/shoot allometric coefficient; RGR, plant relative growth rate; Vcmax, maximum in vivo rate of Rubisco carboxylation.


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
There is growing evidence that rising atmospheric CO2 concentrations will reduce or prevent reductions in the growth and productivity of C3 crops attributable to ozone (O3) pollution. In this study, the role of pollutant exclusion in mediating this response was investigated through growth chamber-based investigations on leaves 4 and 7 of spring wheat (Triticum aestivum cv. Hanno). In the core experiments, plants were raised at two atmospheric CO2 concentrations (ambient [350 µl l–1] or elevated CO2 [700 µl l–1] under two O3 regimes (charcoal/Purafil®-filtered air [<5 nl l–1 O3] or ozone-enriched air [75 nl l–1 7 h d–1]). A subsequent experiment used an additional O3 treatment where the goal was to achieve equivalent daily O3 uptake over the life-span of leaves 4 and 7 under ambient and CO2-enriched conditions, through daily adjustment of exposures based on measured shifts in stomatal conductance. Plant growth and net CO2 assimilation were stimulated by CO2-enrichment and reduced by exposure to O3. However, the impacts of O3 decreased with plant age (i.e. leaf 7 was more resistant to O3 injury than leaf 4); a finding consistent with ontogenic shifts in the tolerance of plant tissue and/or acclimation to O3-induced oxidative stress. In the combined treatment, elevated CO2 protected against the adverse effects of O3 and reduced cumulative O3 uptake (calculated from measurements of stomatal conductance) by c. 10% and 35% over the life-span of leaves 4 and 7, respectively. Analysis of the relationship between O3 uptake and the decline in the maximum in vivo rate of Rubisco carboxylation (Vcmax) revealed the protection afforded by CO2-enrichment to be due, to a large extent, to the exclusion of the pollutant from the leaf interior (as a consequence of the decline in stomatal conductance triggered by CO2-enrichment), but there was evidence (especially from flux–response relationships constructed for leaf 4) that CO2-enrichment resulted in additional effects that alleviated the impacts of ozone-induced oxidative stress on photosynthesis.

Key words: Air pollutant interactions, detoxification, ozone uptake, rising atmospheric CO2 concentrations, spring wheat.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Ground-level concentrations of CO2 and O3 continue to rise as a direct consequence of anthropogenic activities. Over the same time-scale that the tropospheric CO2 concentration has risen from ~280 µl l–1 to ~370 µl l–1, average background levels of O3 have roughly doubled (from 10–15 nl l–1 to 25–30 nl l–1), and UN-ECE critical levels (European exposure guidelines) for the protection of vegetation from O3 are now regularly exceeded during the summer months in many parts of Europe and North America (Stockwell et al., 1997). Even if CO2 emissions are restricted to the extent that the present rate of increase is sustained, atmospheric CO2 concentrations will have risen by a further ~80% by the end of the 21st century (Wigley et al., 1997). As ground-level O3 concentrations are also expected to continue to rise for the foreseeable future (Chameides et al., 1994), it is important to understand how these concomitant changes in atmospheric composition are likely to affect terrestrial vegetation (sensu Krupa, 2003; Fuhrer, 2003).

Elevated CO2 is expected to increase the productivity of C3 plants and enhance water use efficiency at the leaf level through a simultaneous increase in photosynthesis and a decline in stomatal conductance (Cure and Acock, 1986; Eamus, 1991; Drake et al., 1997). Some experiments have shown that the stimulation of photosynthesis induced by elevated CO2 may not be sustained in the long-term. When plants are grown under conditions affecting sink strength (e.g. restricted root volume and/or nutrient supply: Arp, 1991; Stitt and Krapp, 1999), photosynthetic adjustment may occur over a period of days or weeks in response to prolonged CO2-enrichment (Yelle et al., 1989; Thomas and Strain, 1991). This acclimation is generally associated with a decrease in the activity and/or amount of Rubisco (Sage et al., 1989; Rowland-Bamford et al., 1991; Jacob et al., 1995) and other Calvin cycle enzymes (Besford, 1990).

In contrast to elevated CO2, present-day O3 concentrations are known to be high enough to reduce plant growth and productivity through the suppression of photosynthesis, reduced stomatal conductance, enhanced rates of respiration (associated with detoxification and repair processes), shifts in carbon partitioning, and acceleration of the rate of leaf senescence (Reich, 1983; Reich and Amundson, 1985; Cooley and Manning, 1987; Amthor, 1988; Pell et al., 1999). Research has shown that the decline in photosynthetic capacity induced by O3 is primarily caused by a decrease in the maximum in vivo rate of Rubisco carboxylation (Vcmax) (Farage et al., 1991; Farage and Long, 1995, 1999) due to a reduction in the activity and/or quantity of Rubisco (Lenherr et al., 1988; Dann and Pell, 1989; Pell et al., 1992). The mechanisms responsible for triggering the decline in Rubisco content and/or activity remain to be elucidated. The effects of O3 are, however, consistent with the an enhanced rate of Rubisco degradation and subunit transcript abundance observed in leaves exposed to the pollutant (Reddy et al., 1993; Pell et al., 1997). Impacts of O3 on light-harvesting processes and photosynthetic electron transport are considered of secondary importance (Nie et al., 1993; Farage and Long, 1999).

Several experiments have shown that higher atmospheric CO2 concentrations will protect C3 plants from the adverse effects of O3, although the combined effects of the gases on crop yield await clarification (reviewed by Turcsányi et al., 2000). The mechanisms underlying the protection against O3 afforded by rising atmospheric concentrations of CO2 are not fully understood (Rao et al., 1995; McKee et al., 2000). Ozone uptake at the leaf-level is predominantly controlled by stomatal aperture (Kerstiens and Lendzian, 1989). Consequently, the reduction in stomatal conductance under elevated CO2 may reduce O3 uptake in to foliage, on average by 32% across a range of C3 species (Barnes and Wellburn, 1998). However, in the vast majority of studies, it is not known to what extent this reduction in O3 uptake accounts for the protection afforded by elevated CO2. Some investigations reveal that CO2-enrichment can protect against O3 ‘injury’ without substantial reductions in O3 uptake (Heagle et al., 1993; Mulholland et al., 1997), while others imply that O3 exclusion is the chief factor responsible for the reduction in the impacts of O3 under CO2-enriched conditions (McKee et al., 1995, 1997; Olszyk and Wise, 1997; Broadmeadow and Jackson, 2000).

In this study, an ‘O3-sensitive’ cultivar of spring wheat (Triticum aestivum L. cv. Hanno) was used to elucidate the interactive effects of elevated CO2 and O3 on plant growth and leaf gas exchange at different stages of leaf and plant development, with the specific objective of establishing the relative importance of O3 exclusion versus detoxification in mediating the protection against O3 afforded by elevated CO2.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Plant culture and fumigation
Seed of spring wheat (Triticum aestivum L. cv. Hanno) was sown in seed trays containing vermiculite and germinated in a controlled environment chamber supplied with charcoal/Purafil®-filtered air (CFA). Six days after germination, seedlings at the same developmental stage (1st leaf) were transplanted individually to 3 dm3 pots containing a standard potting compost (John Innes No. 2), and transferred to eight controlled environment chambers which form part of a fumigation system described in detail elsewhere (Zheng et al., 1998). Chambers were illuminated by metal-halide lamps (Siemens HRH fittings plus 400 W HQI-T lamps supplied by OSRAM, St Helens, Merseyside, UK) providing a PPFD of {approx}250 µmol m–2 s–1 at plant canopy height as a 14 h photoperiod. Diurnal temperature fluctuations ranged from a night-time minimum of 15±0.2 °C to a day-time maximum of 23±0.3 °C, and air temperatures were logged continually (Delta-T Devices Ltd, Cambridge, UK). Relative humidity was maintained at 65±5%. A factorial experimental design was used to achieve four treatments duplicated in replicate chambers: ambient CO2 (346±3 µl l–1+CFA <5 nl l–1 O3); O3 (ambient CO2+74±3 nl l–1 O3 7 h d–1 (10.00–17.00 h)); elevated CO2 (702±7 µl l–1+CFA); and elevated CO2+O3 (elevated CO2+75±3 nl l–1 O3 7 h d–1). In a subsequent experiment, an additional O3 treatment was used with the aim of equalizing O3 uptake under ambient and elevated CO2. In this latter experiment, plants were grown in elevated CO2 and elevated CO2+O3 under the fumigation conditions described above and the O3 concentration in the elevated CO2+O3 treatment raised from 75±4 nl l–1 over the life-span of leaf 4 to 114±4 nl l–1 O3 or over the life-span of leaf 7 to 140±4 nl l–1 (level II ozone treatment). Plants were watered daily and fertilized once a week with a medium-strength commercial nutrient solution (Phostrogen, Corwen, Clwyd, UK).

Growth and dry matter partitioning
Twenty plants were harvested upon transfer to the respective treatments to provide baseline measurements for growth rate determinations. A subsequent harvest, comprising five independent plants per chamber, was made 77 d after transfer (DAT) to the respective treatments. Plants were separated into root and shoot, washed, and dried to constant weight in an oven at 70 °C. Plant relative growth rate (RGR), the RGR of component plant parts (shoot and root), and the allometric root:shoot coefficient (K=RGR of the root/RGR of the shoot) were calculated as described by Hunt (1990).

Gas exchange measurements
Leaves 4 and 7 on the main shoot were labelled upon emergence (15 and 33 DAT to the treatments, respectively) and measurements of gas exchange made in situ at regular intervals over the leaf life-span. Rates of CO2/H2O exchange were monitored using a standard Parkinson leaf cuvette (model PLC-B, PP Systems, Hitchin, UK) linked to a portable infra-red gas analyser (Ciras-II, PP Systems, Hitchin, UK). Measurements were made at the growth CO2 concentration under chamber conditions (PPFD=195±3 µmol m–2 s–1 at the position occupied by the leaf in the cuvette; leaf temperature=23.1±0.1 °C; leaf-air–vapour pressure deficit=0.75 ±0.02 kPa). The response of CO2 assimilation to changes in the intercellular CO2 concentration (ci) was determined at cuvette CO2 concentrations of 100, 150, 200, 250, 350, and 1000 µl l–1. These measurements were made using an automated Parkinson leaf cuvette (model auto-PLC-B, PP Systems; leaf temperature=22.9 ±0.1 °C; saturated vapour pressure deficit=0.67±0.03 kPa) under a PPFD shown by previous investigation to be high enough to achieve the light-saturated rate of CO2 assimilation (996±2 µmol m–2 s–1). Stomatal conductance to water vapour (gH2O) and the rate of net CO2 assimilation (A) under growth conditions, the light-saturated rate of CO2 assimilation measured at 350 µl CO2 l–1 (A350), and the light- and CO2-saturated rate of CO2 assimilation measured at 1000 µl CO2 l–1 (Amax) were calculated according to von Caemmerer and Farquhar (1981). Vcmax was calculated from the linear portion of the measured A/ci response as described by Harley et al. (1992). Values for the Rubisco Michaelis–Menten constant for CO2 (Kc), Rubisco Michaelis–Menten constant for O2 (Ko), and Rubisco specificity factor ({tau}) measured by Jordan and Ogren (1984) were used in calculations, correcting for temperature dependencies according to Harley et al. (1992).

Leaf boundary layer resistance under chamber conditions was measured using the wet-blotting paper method described by Unsworth et al. (1984). Ozone uptake at the leaf-level was calculated by analogy with Fick’s first law for gas diffusion from measurements of leaf boundary layer conductance and gH2O, multiplying by 0.612 (Nobel, 1983) to account for the slower rate of molecular diffusion of O3 in air compared with H2O. The intercellular O3 concentration was assumed to be zero based on the measurements of Laisk et al. (1989).

Photosynthetic light response
Steady-state rates of leaf gas exchange were determined over a range of PPFDs on leaves 4 and 7, at a stage when the leaves had attained full expansion. Rates of CO2/H2O exchange were determined for individual leaves in a temperature-controlled spherical leaf cuvette, similar to that described by Long and Drake (1991), incorporated into an open-gas exchange system (Balaguer et al., 1995). Leaf temperature was maintained at 23±0.5 °C. Three measurements were made on pooled leaves from plants in each treatment at the growth CO2 concentration, by enclosing the leaves of two independent plants in the cuvette. The PPFD was then reduced to zero and the rate of gas exchange recorded, PPFD was increased in seven steps to approximately 1000 µmol m–2 s–1. Following the completion of A/ci and A/PPFD response curves, the projected leaf area enclosed in the cuvette was determined with the aid of a leaf area meter (Delta-T Devices, UK). The apparent quantum yield of CO2 assimilation ({phi}app) was derived from asymptotic curves (Delgado et al., 1993) fitted to the A/PPFD data (mean r2 >0.90) using INPLOT 4.0 (GraphPad Software, CA, USA).

Chlorophyll fluorescence
Measurements were made on leaves 4 and 7, 15 d after leaf emergence (DALE), on 3–4 independent plants per chamber (6–8 plants per treatment) using a PAM-2000 fluorometer (Waltz, Effeltrich, Germany). The minimum level of fluorescence (Fo) was obtained under modulated red light (2 µmol m–2 s–1; frequency 20 kHz) and maximal fluorescence yields (Fm) recorded following exposure to a saturating light pulse (0.8 s) of 4000 µmol m–2 s–1, provided by an 8 V/20 W halogen lamp (Bellaphot, Osram). The ratio of variable to maximal fluorescence (Fv/Fm; the maximum quantum yield of PSII photochemistry) was measured after 1 h dark adaptation.

Ex vivo Rubisco activity
Rubisco activity was measured in parallel with leaf gas exchange over the life-span of leaves 4 and 7. Assays were conducted using the method of Sharkey et al. (1991). Crude extracts were prepared by rapidly grinding fresh leaf tissue (c. 200 mg) in ice-cold extraction buffer; 100 mM HEPES-KOH (pH 7.5) containing 15 mM MgCl2, 5 mM EGTA, 15% (w/w) PEG (20 000), and 14 mM mercaptoethanol; adding 30% (w/w) insoluble polyvinylpolypyrrolidone (PVPP; 25 000) immediately after grinding. The extracts were centrifuged at 12 000 g for 1 min at 4 °C, then the supernatant was immediately decanted in to fresh 1.5 ml Eppendorf tubes and a subsample stored at –20 °C for the subsequent determination of soluble protein concentration using a commercially available kit (Coomassie Plus, Pierce & Warriner Ltd., Chester, UK).

Initial Rubisco activity (i.e. the maximum activity measured as near as possible to the in vivo state of enzyme activation) was assayed at 25 °C immediately following the preparation of crude extracts in a reaction mixture containing 150 mM Bicine (pH 8.0), 25 mM NaHCO3, 20 mM MgCl2, 3.5 mM ATP, 0.25 mM NADH, 5 mM phosphocreatine, 80 nkat glyceraldehyde-3-phosphate dehydrogenase, 80 nkat 3-phosphoglyceric phosphokinase, 80 nkat creatine phosphokinase, plus 50 µl of extract. NADH oxidation was initiated in the spectrophotometer by the addition of RuBP (to provide a final RuBP concentration of 0.5 mM). Total Rubisco activity (i.e. the maximum in vivo activity achievable following activation of the enzyme with Mg2+ and CO2) was assayed following a 15 min incubation of the reaction mixture minus the three enzymes and RuBP at 25 °C. Absorbance changes were recorded at 340 nm using an automated Pye-Unicam SP8700 UV/Vis spectrophotometer (Pye-Unicam Ltd., Cambridge, UK). Each assay was performed in duplicate and was corrected for independent controls run for every sample to which RuBP was not added. Rubisco activity was calculated from the change in absorbance over the first minute, based on a reaction stoichiometry of 2:1 (NADH:CO2). The activation state of Rubisco was estimated by expressing the initial activity as a percentage of the total activity.

Statistical analyses
Statistical analyses were performed using SPSS (SPSS Inc., Chicago, Illinois, USA). Data were first checked for normal distribution and homogeneity of variance, then log-transformed as required prior to analysis. Treatment means were compared using the least significant difference calculated at the 5% level. Because of elevated CO2xO3 interactions, the main effects of elevated CO2 and O3 were tested using an independent t-test to compare individual means. Interactive effects of elevated CO2, O3, leaf age, and, where appropriate, plant age, were tested by MANOVA. Data were analysed under the assumption that plants in replicate chambers were as likely to be as similar, or as different from, plants within an individual chamber.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Plant growth and dry matter partitioning
Table 1 shows the effects of elevated CO2 and/or O3 on accumulated plant biomass, RGR and K, 77 DAT to the treatments. Elevated CO2 significantly (P ≤0.05) increased RGR and this was reflected in a 66% increase in biomass compared with plants raised at ambient CO2. By contrast, O3 significantly (P ≤0.05) reduced RGR, causing a 32% decline in plant biomass at ambient CO2. Root growth was more affected by O3 than shoot growth (i.e. K was significantly (P ≤0.05) reduced by O3). In the combined treatment, elevated CO2 offered significant (CO2xO3, P ≤0.01) protection against the growth suppression caused by O3; the RGR of plants grown in elevated CO2 was not significantly different from those exposed to elevated CO2+O3. Elevated CO2 did not, however, protect against the shift in root:shoot partitioning induced by O3 (i.e. O3-induced decline in K).


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  		Table 1. Effects of elevated CO2 and O3 on total plant dry weight (DW), relative growth rate (RGR) and allometric root:shoot growth (K=RGRR/RGRS) in 11-week-old wheat plants (Triticum aestivum cv. Hanno) Values represent the mean ±SE, n=10; values followed by a different letter in the same column are significantly different at the 5% level.
 
Stomatal conductance and O3 uptake
Figure 1 shows the impact of CO2 and/or O3 on gH2O of leaves 4 and 7. Independently, both elevated CO2 and O3 resulted in a decline (P ≤0.001) in gH2O over the life-span of leaves 4 and 7. In the combined treatment, effects on gH2O were significantly less than additive (CO2xO3 P ≤0.05) and not dissimilar to plants grown in elevated CO2 alone.



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  		Fig. 1. Impacts of exposure to elevated CO2 and/or O3 on stomatal conductance to water vapour (gH2O) over the life-span of wheat (Triticum aestivum L. cv. Hanno) leaves 4 and 7. Gas exchange measurements were made under growth conditions at 5, 8, 10, 13, 15, 18, 20, and 23 d after leaf emergence. Plants were grown in duplicate controlled environment chambers and exposed to either ambient CO2, (open squares) (346±3 µl l–1 CO2+CFA <5 nl l–1 O3); O3, (filled squares) (ambient CO2+74±3 nl l–1 O3 7 h d–1); elevated CO2, (open circles) (702±7 µl l–1 CO2+CFA); elevated CO2+O3 level I, (filled circles) (elevated CO2+75±4 nl l–1 O3 7 h d–1), elevated CO2+O3 level II, (filled triangles) (elevated CO2+75±4 nl l–1 O3 7 h d–1 increased to 114±4 (over the life-span of leaf 4) or 140±4 (over the life span of leaf 7) nl l–1 O3 7 h d–1. Values represent mean ±SE (n=6–10).

 
Instantaneous O3 uptake was calculated from measurements of gH2O and leaf boundary layer conductance. Figure 2 shows cumulative O3 uptake modelled over the life-span of leaves 4 and 7 for plants grown at ambient and/or elevated CO2. Maximum rates of instantaneous O3 uptake coincided with the attainment of full leaf expansion (10–13 DALE). At ambient CO2, cumulative O3 uptake was consistently greater for leaf 7 than leaf 4. Elevated CO2 reduced cumulative O3 uptake by c. 10% and 35% over the life-span of leaves 4 and 7, respectively, compared with plants grown in ambient CO2. In the additional (Level II) elevated CO2+O3 treatment, the experimental objective to equalize daily O3 uptake in plants raised under ambient and elevated CO2 proved difficult to achieve (since ozone concentrations had to be manipulated on the basis of measurements of gH2O made the previous day) and there was a degree of over-compensation resulting in greater cumulative O3 uptake in plants exposed to the combined treatment compared with those exposed to ambient CO2.



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  		Fig. 2. Cumulative O3 uptake modelled from measurements of stomatal conductance to water vapour (gH2O) and leaf boundary layer over the life-span of wheat (Triticum aestivum L. cv. Hanno) leaves 4 and 7. Gas exchange measurements were made in situ under growth conditions at 5, 8, 10, 13, 15, 18, 20, and 23 d after leaf emergence. Ozone uptake modelled on the assumption that there was no major change in gH2O over the course of the day under the growth conditions (an assumption supported by previous experimental measurements reported by Balaguer et al., 1995). Plants were grown in duplicate controlled environment chambers and exposed to O3, (filled squares) (346±3 µl l–1 CO2+74±3 nl l–1 O3 7 h d–1); elevated CO2+O3, (filled circles) (702±7 µl l–1 CO2+75±4 nl l–1 O3 7 h d–1) or elevated CO2+O3 level II, (filled triangles) (elevated CO2+75±4 nl l–1 O3 7 h d–1 increased to 114±4 (over the life-span of leaf 4) or 140±4 (over the life-span of leaf 7) nl l–1 O3 7 h d–1.

 
In situ CO2 assimilation
Figures 3 and 4 show the impact of elevated CO2 and/or O3 on A and Amax, respectively, over the life-span of leaves 4 and 7. Elevated CO2 stimulated A by a maximum of 30% and 15% in leaves 4 and 7, respectively. However, the extent of the increase in A declined as the leaves aged (leaf life-spanxCO2, P ≤0.05 (leaf 4); P ≤0.001 (leaf 7)), and leaf 7 exhibited a greater decline in A following the attainment of full expansion compared with leaf 4 (plant agexCO2, P ≤0.001). CO2-enrichment significantly (P ≤0.05) increased A350 and Amax in leaf 4, whereas in leaf 7 A350 and Amax declined (P ≤0.05) once leaves attained full expansion. Ozone exposure resulted in a significant decline in A, A350 and Amax in leaves 4 and 7, although the effects of O3 on leaf 7 were not as extensive as observed for leaf 4 (plant agexO3, P ≤0.001). The impact of O3 increased as leaves aged, particularly following the attainment of full expansion (leaf life-spanxO3, P ≤0.001). There was no significant difference in A, A350 and Amax between plants grown in elevated CO2+O3 and those grown in elevated CO2 alone.



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  		Fig. 3. Impacts of exposure to elevated CO2 and/or O3 on the rate of CO2 assimilation (A) over the life-span of wheat (Triticum aestivum L. cv. Hanno) leaves 4 and 7. Gas exchange measurements were made in situ under growth conditions at 5, 8, 10, 13, 15, 18, 20, and 23 d after leaf emergence. Plants were grown in duplicate controlled environment chambers and exposed to either ambient CO2, (open squares) (346±3 µl l–1 CO2+CFA <5 nl l–1 O3); O3, (filled squares) (ambient CO2+74±3 nl l–1 O3 7 h d–1); elevated CO2, (open circles) (702±7 µl l–1 CO2+CFA); elevated CO2+O3 level I, (filled circles) (elevated CO2+75±4 nl l–1 O3 7 h d–1) or elevated CO2+O3 level II, (filled triangles) (elevated CO2+75±4 nl l–1 O3 7 h d–1 changed to 114±4 (over the life-span of leaf 4) or 140±4 (over the life-span of leaf 7) nl l–1 O3 7 h d–1. Values represent mean ±SE (n=6–10).

 


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  		Fig. 4. Impacts of exposure to elevated CO2 and/or O3 on the light- and CO2-saturated rate of CO2 assimilation (Amax) over the life-span of wheat (Triticum aestivum L. cv. Hanno) leaves 4 and 7. Gas exchange measurements were made at 6, 11, 16, and 21 d after leaf emergence. Plants were grown in duplicate controlled environment chambers and exposed to ambient CO2, (open squares) (346±3 µl l–1 CO2+CFA <5 nl l–1 O3); O3, (filled squares) (ambient CO2+74±3 nl l–1 O3 7 h d–1 (10.00–17.00 h)); elevated CO2, (open circles) (702±7 µl l–1 CO2+CFA) or elevated CO2+O3, (filled circles) (elevated CO2+75±4 nl l–1 O3 7 h d–1). Values represent mean ±SE (n=6).

 
Apparent quantum yield of CO2 assimilation and maximum quantum yield of PSII photochemistry
Table 2 shows the impact of elevated CO2 and/or O3 on {phi}app and Fv/Fm. Elevated CO2 increased {phi}app (although the effect did not attain statistical significance in leaf 7). Fv/Fm was not affected by elevated CO2. Ozone, on the other hand, significantly (P ≤0.05) reduced {phi}app in leaf 4, but resulted in no change in {phi}app in leaf 7. Ozone had a marginal impact on Fv/Fm (P ≤0.01; equivalent to only a 4% reduction in Fv/Fm). In the combined treatment, elevated CO2 protected against the decline in {phi}app induced by O3, but the decline in Fv/Fm persisted.


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  		Table 2. Effects of elevated CO2 and O3 on the impact of exposure to elevated CO2 and/or O3 on {phi}app and Fv/Fm in wheat (Triticum aestivum L. cv. Hanno) leaves 4 and 7 Measurements were made 15 d after leaf emergence. Values represent the mean ±SE, n=6–8; values followed by a different letter in the same column are significantly different at the 5% level.
 
Rubisco activity
The impact of elevated CO2 and/or O3 on Vcmax (measured in situ) and ex vivo Rubisco activity is shown in Figs 5 and 6, respectively. Estimates of Vcmax were linearly related to ex vivo measurements of initial Rubisco activity (y=0.75x+37.45; r2=0.48, n=23, P ≤0.001), but relationships were weakened by the fact that measurements were made on different plants. Ex vivo measurements of Rubisco activity were, on average, 40% lower than in vivo estimates, although the extent and pattern of the changes induced by exposure to elevated CO2 and/or O3 were similar.



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  		Fig. 5. Impact of exposure to elevated CO2 and/or O3 on the maximum in vivo rate of Rubisco carboxylation (Vcmax) over the life-span of wheat (Triticum aestivum L. cv. Hanno) leaves 4 and 7. Gas exchange measurements were made at 6, 11, 16, and 21 d after leaf emergence. Plants were grown in duplicate controlled environment chambers and exposed to either ambient CO2, (open squares) (346±3 µl l–1 CO2+CFA <5 nl l–1 O3); O3, (filled squares) (ambient CO2+74±3 nl l–1 O3 7 h d–1); elevated CO2, (open circles) (702±7 µl l–1 CO2+CFA); elevated CO2+O3 level I, (filled circles) (elevated CO2+75±4 nl l–1 O3 7 h d–1) or elevated CO2+O3 level II, (filled triangles) (elevated CO2+75±4 nl l–1 O3 7 h d–1 was increased to 114±4 (over the life-span of leaf 4) or 140±4 (over the life-span of leaf 7) nl l–1 O3 7 h d–1. Values represent mean ±SE (n=6).

 


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  		Fig. 6. Impacts of exposure to elevated CO2 and/or O3 on ex vivo Rubisco activity (bars) and soluble protein content (symbols) over the life-span of wheat (Triticum aestivum L. cv. Hanno) leaves 4 and 7. Measurements were made at 11, 16, and 21 d after leaf emergence Plants were grown in duplicate controlled environment chambers and exposed to either ambient CO2, (open squares) (346±3 µl l–1 CO2+CFA <5 nl l–1 O3); O3, (filled squares) (ambient CO2+74±3 nl l–1 O3 7 h d–1); elevated CO2, (open circles) (702±7 µl l–1 CO2+CFA); elevated CO2+O3, (filled circles) (elevated CO2+75±4 nl l–1 O3 7 h d–1). Bars represent ambient CO2 (open bars); O3 (filled bars); elevated CO2 (diagonally-striped bars); elevated CO2+O3 (cross-hatched bars). Bars are divided to show initial (bottom) and final (top) Rubisco activity. Values represent mean ±SE (n=6).

 
Overall, CO2-enrichment resulted in no significant change in Vcmax in leaves 4 and 7. However, at 21 DALE, a 25% reduction in Vcmax was observed in leaf 7 compared with plants raised in ambient CO2. This decline was accompanied by a reduction in leaf soluble protein content (Fig. 6), with no change in the activation state of the enzyme. Ozone exposure depressed Vcmax and ex vivo Rubisco activity following the attainment of full leaf expansion, but resulted in no change in the activation state of Rubisco, despite a significant (P ≤0.01) reduction in the soluble protein content of leaf 4. In the combined treatment, elevated CO2 protected against the reduction in Vcmax induced by O3 in both leaves 4 and 7. When plants in elevated CO2+O3 were exposed to higher O3 concentrations (additional elevated CO2+O3 treatment), elevated CO2 significantly (CO2xO3, P ≤0.01) reduced the extent of O3 damage in leaf 4, but not in leaf 7, and there was a significant plant agexCO2xO3 interaction (P ≤0.01).

The reduction in Vcmax caused by O3 was not linearly related to cumulative O3 uptake (Fig. 7), suggesting evidence of an uptake threshold prior to injury. Once this cumulative threshold was exceeded, Vcmax declined linearly in response to cumulative O3 uptake. In ambient CO2, tolerance to the pollutant increased with plant age, since equivalent O3 uptake resulted in a greater impact on leaf 4 than leaf 7 (plant agexO3, P ≤0.05). Although cumulative O3 uptake at ambient and elevated CO2 was similar for leaf 4, CO2-enrichment still reduced the extent of O3 damage. This indicates that elevated CO2 enhanced the tolerance of leaf 4 to O3. In leaf 7, the decline in gH2O induced by CO2-enrichment was sufficient to reduce cumulative O3 uptake below the threshold level required to damage this leaf at ambient CO2. By considering the data for plants exposed to the additional elevated CO2+O3 treatment, it proved possible to show that elevated CO2 did not increase the O3 tolerance of leaf 7, since equivalent O3 uptake resulted in a similar decline in Vcmax under ambient and elevated CO2.



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  		Fig. 7. Relationship between cumulative O3 uptake and change in the maximum in vivo rate of Rubisco carboxylation (Vcmax) for wheat (Triticum aestivum L. cv. Hanno) leaves 4 and 7. Data expressed as % decline in Vcmax caused by O3 at ambient or elevated atmospheric CO2 concentrations Cumulative O3 uptake modelled from measurements of gH2O made at regular intervals over the leaf life-span, based on the assumption that there was no major change in gH2O over the course of the day under the growth conditions (an assumption supported by previous experimental measurements reported by Balaguer et al., 1995). Plants were grown in duplicate controlled environment chambers and exposed to O3, (filled squares) (346±3 µl l–1 CO2+74±3 nl l–1 O3 7 h d–1); elevated CO2+O3 level I, (filled circles) (702±7 µl l–1 CO2+75±4 nl l–1 O3 7 h d–1), elevated CO2+O3 level II, (filled triangles), (elevated CO2+75±4 nl l–1 O3 7 h d–1 increased to 114±4 (over the life-span of leaf 4) or 140±4 (over the life-span of leaf 7) nl l–1 O3 7 h d–1.

 
Visible injury
Shortly after leaves attained full expansion, typical visible symptoms of O3 damage (chlorotic mottling and chlorosis developing from the leaf tip) became apparent on both leaves 4 and 7 (i.e. 16 DALE). Injury was not quantified, but exposure to elevated CO2 was observed to prevent the development of visible O3 symptoms. In addition, symptoms of O3 damage appeared to develop more rapidly in leaf 4 compared with leaf 7.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Growth and biomass accumulation was substantially increased when wheat plants were grown at an atmospheric CO2 concentration of 700 µl l–1 (i.e. a doubling of the present day ambient CO2 concentration). The stimulation in plant growth was accompanied by an increase in A and a sustained decrease in gH2O over the life-span of both leaves 4 and 7. These are common responses of C3 plants to CO2-enrichment (Cure and Acock, 1986; Drake et al., 1997). Consistent with studies on field-grown wheat (Miglietta et al., 1996; Mulholland et al., 1997; Garcia et al., 1998), there was little evidence of photosynthetic adjustment in this study’s chamber-grown plants. However, the stimulation in photosynthesis induced by CO2-enrichment declined after leaves attained full expansion, for example, Vcmax was reduced by 25% in leaf 7 after 23 DALE. The decline in Rubisco activity at this stage may reflect a reduction in Rubisco content, since elevated CO2 did not affect the activation state of the enzyme, but reduced leaf soluble protein content (of which Rubisco is a major determinant). Some studies have suggested that the decline in Rubisco content in the later stages of leaf development may be due to accelerated rates of leaf senescence under CO2-enrichment (Nie et al., 1995; Garcia et al., 1998; Sicher and Bunce, 1998).

Exposure to O3 resulted in a marked reduction in RGR and a decrease in K. This finding supports previous observations that root growth is often more affected by O3 than shoot growth (Cooley and Manning, 1987; Davison and Barnes, 1998). Consistent with the majority of studies on the impacts of O3 on leaf gas exchange (Reich and Amundson, 1985; Darrall, 1989; Ojanperä et al., 1998), the pollutant resulted in a marked decline in A and gH2O in both leaves 4 and 7. Studies on different taxa (Lenherr et al., 1988; Farage et al., 1991; Pell et al., 1992; Farage and Long, 1995) suggest that the decline in gH2O induced by O3 results in the main from the impacts of the pollutant on carboxylation efficiency. However, measurements made on leaf 7 between 10 and 15 DALE, revealed that O3 caused a decline in gH2O without affecting A. These observations lend additional support to the growing opinion that shifts in stomatal conductance induced by O3 do not solely reflect effects on photosynthetic capacity, but involve direct effects of the pollutant on the stomatal complex (Torsethaugen et al., 1999; Zheng et al., 2002).

Ozone depressed Vcmax and ex vivo Rubisco activity in leaves 4 and 7 without significant changes in the activation state of the enzyme. Loss of Rubisco triggered by exposure to O3 has been reported previously in several species (Dann and Pell, 1989; Pell et al., 1992; Nie et al., 1993) and is considered to constitute the primary cause of the O3-induced decline in CO2 assimilation (Farage et al., 1991; Farage and Long, 1995). This conclusion is supported by the finding that the capacity to regenerate RuBP and the potential for electron transport were not impaired by O3 (ozone reduced Vcmax by 17% by 16 DALE without effects on Amax, {phi}app, and Fv/Fm) in leaf 7. In leaf 4 (which appeared more sensitive to O3 than leaf 7) O3 depressed both Amax and {phi}app, a finding that could not be explained by a marked decline in the potential for PSII photochemistry. Similar findings have been reported previously (Nie et al., 1993; Farage and Long, 1995) and lend support to the view that one of the primary effects of O3 is to promote premature leaf senescence, because similar effects have been observed in senescing leaves (Lu and Zhang, 1998).

The impact of O3 on photosynthesis was negligible until leaves attained full expansion. This is consistent with the findings of Nie et al. (1993) who reported that the O3-induced decline in Rubisco content in mature wheat leaves was restricted to the oldest parts of the leaf. Previous studies have indicated that the maximal effect of O3 on Rubisco coincides with the period when Rubisco concentration reaches its peak (Dann and Pell, 1989; Pell et al., 1992). These observations show that this period (11–14 DALE) also coincided with highest gH2O. This implies that variations in the effects of O3 with leaf development stage may be related, at least in part, to shifts in O3 uptake with leaf ontogeny.

The greater resistance of older leaves to O3 did not correlate with changes in stomatal conductance. Equivalent O3 uptake resulted in a greater decline in Vcmax in leaf 4 than leaf 7; cumulative O3 uptake of 4.2 mmol m–2 reducing Vcmax by 71% and 17% in leaves 4 and 7, respectively. This is consistent with previous findings that suggest increasing resistance to O3 with plant age is related, at least in part, to changes in the tolerance of plant tissue (Walmsley et al., 1980; Mulholland et al., 1997; Lyons and Barnes, 1998).

Elevated CO2 protected against the adverse effects of O3 on plant growth and photosynthesis. This amelioration of O3-injury by elevated CO2 has been observed in a variety of species (reviewed by Barnes and Wellburn, 1998; Turcsányi et al., 2000; Fangmeier and Bender, 2002). In the present study, elevated CO2 reduced cumulative O3 uptake by c. 10% and 35% in leaves 4 and 7, respectively. Exclusion of the pollutant (via stomatal closure) under CO2-enrichment is thought to be one of the chief mechanisms underlying the protective action of elevated CO2 (Fiscus et al., 1997; McKee et al., 1997; Vandermeiren et al., 2002). This conclusion is supported by the finding that a ‘wilty’ mutant of tomato (Lycopersicon esculentum Mill. flacca), which has non-functional stomata, is not protected against O3 by elevated CO2 (Olszyk and Wise, 1997). However, previous studies on wheat (Rao et al., 1995; Mulholland et al., 1997; McKee et al., 2000) and clover (Trifolium repens L.) (Heagle et al., 1993) suggest the protection against O3 afforded by elevated CO2 is not mediated solely by exclusion. To investigate if additional factors were involved in mediating the alleviation of O3 ‘injury’ by elevated CO2, an extra O3-treatment was used in the present study. The treatment aimed specifically at achieving comparable cumulative O3 uptake over the life-span of leaves 4 or 7 in plants raised at ambient and elevated CO2. Ozone flux-response data revealed that in addition to the exclusion of the pollutant, CO2-enrichment enhanced the tolerance of leaf tissue (especially leaf 4) to O3-induced oxidative stress, presumably via shifts in leaf metabolism and/or anatomy (Rao et al., 1995).

The present study revealed a non-linear relationship between cumulative O3 uptake and impacts on photosynthetic capacity lending strong support to the view that a flux threshold exists for O3 injury. This contention is supported by (i) empirical data derived from flux–response relationships for wheat and other species (UNECE, 2003) and (ii) simulations of ozone flux to the plasmalemma based on measured elements of the diffusion–reaction network following uptake of ozone in to foliage (Pleijel et al., 2003). The tolerance of leaves to O3 was also shown to increase with plant age, highlighting the need to ‘weight’ impacts in relation to phenology (Soja et al., 2000). Data revealed that rising atmosperic CO2 concentrations are likely to afford protection against the adverse effects of O3 on plant growth and photosynthesis, with the effect due, at least in part, to the decline in stomatal conductance triggered by increases in atmospheric CO2. However, the present study suggested that rising atmosperic CO2 concentrations may also enhance the tolerance of leaf tissue to O3-induced oxidative stress. This finding is consistent with reported shifts in the antioxidant status of leaves under the combined influence of elevated CO2+O3 (Rao et al., 1995), but there is a need for further investigation, in particular, to dissect the impacts of rising CO2 on the fate of ozone following uptake into the leaf interior (i.e. to partition changes in the O3 diffusion–reaction network in the leaves of plants raised at elevated CO2) as well as the fuelling of oxidative repair processes. The findings illustrate the uncertainties in predicting plant responses to the combination of rising CO2+O3 based on predictions from the effects of the individual gases.


   Acknowledgements
 
The authors thank Alan White, Phil Green, and Keith Taylor, Tom Lyons (Newcastle University), Kate Maxwell (Cambridge University), and Ian McKee (Essex University) for assistance during the course of this study. The work was performed during JDBs tenure as a Royal Society Research Fellow and was financed by grants from The Portuguese Ministry of Science and Technology–PRAXIS XXI Scheme (BD/9155/96) and The Spanish Ministry of Education and Science, DGICYT (FPU-EX92). Derek Eamus was on sabbatical leave from NTU, Darwin.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Amthor JS. 1988. Growth and maintenance respiration in leaves of bean (Phaseolus vulgaris L.) exposed to ozone in open-top chambers in the field. New Phytologist 110, 319–325.[CrossRef][Web of Science]

Arp WJ. 1991. Effects of source–sink relations on photosynthetic acclimation to elevated CO2. Plant, Cell and Environment 14, 869–875.[CrossRef]

Balaguer L, Barnes JD, Panicucci A, Borland AM. 1995. Production and utilization of assimilates in wheat (Triticum aestivum L.) leaves exposed to elevated O3 and/or CO2. New Phytologist 129, 557–568.[CrossRef][Web of Science]

Barnes JD, Wellburn AR. 1998. Air pollutant combinations. In: De Kok LJ, Stulen I, eds. Responses of plant metabolism to air pollution and global change. Leiden: Backhuys, 147–164.

Besford RT. 1990. The greenhouse effect: acclimation of tomato plants growing in high CO2, relative changes in Calvin cycle enzymes. Journal of Plant Physiology 136, 458–463.

Broadmeadow MSJ, Jackson SB. 2000. Growth responses of Quercus petraea, Fraxinus excelsior and Pinus sylvestris to elevated carbon dioxide, ozone and water supply. New Phytologist 146, 437–451.[CrossRef][Web of Science]

Chameides WL, Kasibhatla PS, Yienger J, Levy H. 1994. Growth of continental-scale metro-agro-plexes, regional ozone pollution, and world food production. Science 264, 74–77.[Abstract/Free Full Text]

Cooley DR, Manning WJ. 1987. The impact of ozone on assimilate partitioning in plants: a review. Environmental Pollution 47, 95–113.[CrossRef][Medline]

Cure JD, Acock B. 1986. Crop responses to carbon dioxide doubling: a literature survey. Agricultural and Forest Meteorology 38, 127–145.[CrossRef][Web of Science]

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.[Abstract/Free Full Text]

Darrall NM. 1989. The effect of air pollutants on physiological processes in plants. Plant, Cell and Environment 12, 1–30.

Davison AW, Barnes JD. 1998. Effects of ozone on wild plants. New Phytologist 139, 135–151.[CrossRef]

Delgado E, Parry MAJ, Lawlor DW, Keys AJ, Medrano H. 1993. Photosynthesis, ribulose-1,5-bisphosphate carboxylase and leaf characteristics of Nicotiana tabacum L. genotypes selected by survival at low CO2 concentrations. Journal of Experimental Botany 44, 1–7.[Abstract/Free Full Text]

Drake BD, Gonzalez-Meler MA, Long SP. 1997. More efficient plants: a consequence of rising atmospheric CO2. Annual Review of Plant Physiology and Plant Molecular Biology 48, 608–639.

Eamus D. 1991. The interaction of rising CO2 and temperatures with water use efficiency. Plant, Cell and Environment 14, 843–852.[CrossRef]

Fangmeier A, Bender J. 2002. Air pollutant combinations—significance for future impact assessments on vegetation. Phyton 42, 63–51.

Farage PK, Long SP. 1995. An in vivo analysis of photosynthesis during short-term O3 exposure in three contrasting species. Photosynthesis Research 43, 11–18.[CrossRef]

Farage PK, Long SP. 1999. The effects of O3 fumigation during leaf development on photosynthesis of wheat and pea: an in vivo analysis. Photosynthesis Research 59, 1–7.[CrossRef]

Farage PK, Long SP, Lechner EG, Baker NR. 1991. The sequence of change within the photosynthetic apparatus of wheat following short-term exposure to ozone. Plant Physiology 95, 529–535.[Abstract/Free Full Text]

Fiscus EL, Reid CD, Miller JE, Heagle AS. 1997. Elevated CO2 reduces O3 flux and O3-induced yield losses in soybeans: possible implications for elevated CO2 studies. Journal of Experimental Botany 48, 307–313.[Abstract/Free Full Text]

Fuhrer J. 2003. Agroecosystem responses to combinations of elevated CO2, ozone and global climate change. Agriculture, Ecosystems and Environment 97, 1–20.

Garcia RL, Long SP, Wall GW, Osborne CP, Kimball BA, Nie GY, Pinter PJ, Wechsung F. 1998. Photosynthesis and conductance of spring-wheat leaves: field response to continuous free-air atmospheric CO2 enrichment. Plant, Cell and Environment 21, 659–669.[CrossRef]

Harley PC, Thomas RB, Reynolds JF, Strain BR. 1992. Modelling photosynthesis of cotton grown in elevated CO2. Plant, Cell and Environment 15, 271–282.[CrossRef]

Heagle AS, Miller JE, Sherrill DE, Rawlings JO. 1993. Effects of ozone and carbon dioxide mixtures on two clones of white clover. New Phytologist 123, 751–762.[CrossRef][Web of Science]

Hunt R. 1990. Basic growth analysis. London: Unwin Hyman.

Jacob J, Greitner C, Drake BG. 1995. Acclimation of photosynthesis in relation to Rubisco and non-structural carbohydrate contents and in situ carboxylase activity in Scirpus olneyi grown at elevated CO2 in the field. Plant, Cell and Environment 18, 875–884.[CrossRef]

Jordan DB, Ogren WL. 1984. The CO2/O2 specificity of ribulose 1,5-bisphosphate carboxylase/oxygenase: dependence on ribulose-bisphosphate concentration, pH and temperature. Planta 161, 308–313.[CrossRef][Web of Science]

Kerstiens G, Lendzian K. 1989. Interactions between ozone and plant cuticles. I. Ozone deposition and permeability. New Phytologist 112, 13–19.[CrossRef]

Krupa S. 2003. Atmosphere and agriculture in the new millenium. Environmental Pollution 126, 293–300.[CrossRef][Medline]

Laisk A, Kull O, Moldau H. 1989. Ozone concentration in leaf intercellular air spaces is close to zero. Plant Physiology 90, 1163–1167.[Abstract/Free Full Text]

Lenherr B, Machler F, Grandjean A, Fuhrer J. 1988. The regulation of photosynthesis in leaves of field-grown spring wheat (Triticum aestivum L. cv. Albis) at different levels of ozone in ambient air. Plant Physiology 88, 1115–1119.[Abstract/Free Full Text]

Long SP, Drake BG. 1991. Effect of the long-term elevation of CO2 concentration in the field on the quantum yield of photosynthesis of the C3 sedge, Scirpus olneyi. Plant Physiology 96, 221–226.[Abstract/Free Full Text]

Lu C, Zhang J. 1998. Changes in photosystem II function during senescence of wheat leaves. Physiologia Plantarum 104, 239–247.[CrossRef]

Lyons TM, Barnes JD. 1998. Influence of plant age on ozone resistance in Plantago major. New Phytologist 138, 83–89.[CrossRef]

McKee IF, Eiblmeier M, Polle A. 1997. Enhanced ozone-tolerance in wheat grown at an elevated CO2 concentration: ozone exclusion and detoxification. New Phytologist 137, 275–284.[CrossRef][Web of Science]

McKee IF, Farage PK, Long SP. 1995. The interactive effects of elevated CO2 and O3 concentration on photosynthesis in spring wheat. Photosynthesis Research 45, 111–119.

McKee IF, Mulholland BJ, Craigon J, Black CR, Long SP. 2000. Elevated concentrations of atmospheric CO2 protect against and compensate for O3 damage to photosynthetic tissues of field-grown wheat. New Phytologist 146, 427–435.[CrossRef][Web of Science]

Miglietta E, Giuntoli A, Bindi M. 1996. The effect of free air carbon dioxide enrichment (FACE) and soil nitrogen availability on the photosynthetic capacity of wheat. Photosynthesis Research 47, 281–290.[CrossRef]

Mulholland BJ, Craigon J, Black CR, Colls JJ, Atherton J, Landon G. 1997. Impact of elevated atmospheric CO2 and O3 on gas exchange and chlorophyll content in spring wheat (Triticum aestivum L.). Journal of Experimental Botany 48, 1853–1863.[Abstract/Free Full Text]

Nie GY, Long SP, Garcia RL, Kimball BA, Lamorte RL, Pinter PJ, Wall GW, Webber AN. 1995. Effects of free-air CO2 enrichment on the development of the photosynthetic apparatus in wheat, as indicated by changes in leaf proteins. Plant, Cell and Environment 18, 855–864.[CrossRef]

Nie G-Y, Tomasevic M, Baker NR. 1993. Effects of ozone on the photosynthetic apparatus and leaf proteins during leaf development in wheat. Plant, Cell and Environment 16, 643–651.[CrossRef]

Nobel PS. 1983. Biochemical plant physiology and ecology. New York: Freeman.

Ojanperä K, Pätsikkä E, Yläranta T. 1998. Effects of low ozone exposure of spring wheat on net CO2 uptake, Rubisco, leaf senescence and grain filling. New Phytologist 138, 451–460.[CrossRef]

Olszyk DM, Wise C. 1997. Interactive effects of elevated CO2 and O3 on rice and flacca tomato. Agriculture, Ecosystems and Environment 66, 1–10.[CrossRef]

Pell EJ, Eckardt N, Enyedi AJ. 1992. Timing of ozone stress and resulting status of ribulose bisphosphate carboxylase/oxygenase and associated net photosynthesis. New Phytologist 120, 397–405.[CrossRef]

Pell EJ, Schlagnhaufer CD, Arteca RC. 1997. Ozone-induced oxidative stress: mechanisms of action and reaction. Physiologia Plantarum 100, 264–273.[CrossRef]

Pell EJ, Sinn JP, Brendley BW, Samuelson L, Vinten-Johansen C, Tien M, Skillman J. 1999. Differential response of four tree species to ozone-induced acceleration of foliar senescence. Plant, Cell and Environment 22, 779–790.[CrossRef]

Pleijel H, Barnes JD, Gerosa G. 2003. Critical flux thresholds for ozone. Proceedings of the UNECE-EMEP Mapping and Modelling Workshop, Manchester, 20–24.

Rao MV, Hale BA, Ormrod DP. 1995. Amelioration of ozone-induced oxidative damage in wheat plants grown under high carbon dioxide. Plant Physiology 109, 421–432.[Abstract]

Reddy GN, Arteca RN, Dai Y-R, Flores HE, Negm FB, Pell EJ. 1993. Changes in ethylene and polyamines in relation to mRNA levels of the large and small subunits of ribulose bisphosphate carboxylase/oxygenase in ozone stresses potato foliage. Plant, Cell and Environment 16, 819–826.[CrossRef]

Reich PB. 1983. Effects of low concentrations of O3 on net photosynthesis, dark respiration, and chlorophyll contents in ageing hybrid poplar leaves. Plant Physiology 73, 291–296.[Abstract/Free Full Text]

Reich PB, Amundson RG. 1985. Ambient levels of ozone reduce net photosynthesis in tree and crop species. Science 230, 566–570.[Abstract/Free Full Text]

Rowland-Bamford AJ, Baker JT, Allen LH, Bowes G. 1991. Acclimation of rice to changing atmospheric carbon dioxide concentration. Plant, Cell and Environment 14, 577–583.[CrossRef]

Sage RF, Sharkey TD, Seemann JR. 1989. Acclimation of photosynthesis to elevated CO2 in five C3 species. Plant Physiology 89, 590–596.[Abstract/Free Full Text]

Sharkey TD, Savitch LV, Butz ND. 1991. Photometric method for routine determination of kcat and carbamylation of Rubisco. Photosynthesis Research 28, 41–48.

Sicher RC, Bunce JA. 1998. Evidence that premature senescence affects photosynthetic decline of wheat flag leaves during growth in elevated carbon dioxide. International Journal of Plant Science 159, 798–804.[CrossRef]

Soja G, Barnes JD, Posch M, Vandermeiren K, Pleijel H, Mills G. 2000. Phenological weighting of ozone exposures in the calculation of critical levels for wheat, bean and plantain. Environmental Pollution 109, 517–524.[CrossRef][Medline]

Stitt M, Krapp A. 1999. The interaction between elevated carbon dioxide and nitrogen nutrition: the physiological and molecular background. Plant, Cell and Environment 22, 583–621.[CrossRef]

Stockwell WR, Kramm G, Scheel H-E, Mohen VA, Seiler W. 1997. Ozone formation, destruction and exposure in Europe and the United States. In: Sandermann H, Wellburn AR, Heath RL, eds. Forest decline and ozone. A comparison of controlled chamber and field experiments. Berlin: Springer-Verlag, 1–38.

Thomas RB, Strain BR. 1991. Root restriction as a factor in photosynthetic acclimation of cotton seedlings grown in elevated carbon dioxide. Plant Physiology 96, 627–634.[Abstract/Free Full Text]

Torsethaugen G, Pell EJ, Assmann SM. 1999. Ozone inhibits guard cell K+ channels implicated in stomatal opening. Proceedings of the National Academy of Sciences, USA 96, 13577–13582.[Abstract/Free Full Text]

Turcsányi E, Cardoso-Vilhena J, Daymond J, Gillespie C, Balaguer L, Ollerenshaw J, Barnes J. 2000. Impacts of tropospheric ozone: past, present and likely future. In: Singh S, ed. Trace gas emissions. Berlin: Springer-Verlag, 249–272.

Unsworth MH, Heagle AS, Heck WW. 1984. Gas exchange in open-top field chambers-I. Measurement and analysis of atmospheric resistances to gas exchange. Atmospheric Environment 18, 373–380.[CrossRef]

UNECE. 2003. Establishing ozone critical levels. II. Karlsson PE, Selldén G, Pleijel H, eds. UNECE Workshop Report, IVL Publications-service, Box 21060, Stockholm.

Vandermeiren K, Black C, Lawson T, Casanova MA, Ojanpera K. 2002. Photosynthetic and stomatal responses of potatoes grown under elevated CO2 and/or O3—results from the European CHIP programme. European Journal of Agronomy 17, 337–352.[CrossRef]

von Caemmerer S, Farquhar GD. 1981. Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. Planta 153, 376–387.[CrossRef][Web of Science]

Walmsley L, Ashmore MR, Bell JNB. 1980. Adaptation of radish Raphanus sativus L. in response to continous exposure to ozone. Environmental Pollution 23, 165–177.[CrossRef]

Wigley TML, Jain AK, Joos F, Nyenzi BS, Shukla PR. 1997. Implications of proposed CO2 emissions limitations. In: Houghton JT, Meira-Filho JG, Griggs DJ, Noguer M, eds. Implications of proposed CO2 emissions limitations. Cambridge: Cambridge University Press.

Yelle S, Beeson RC, Trudel MJ, Gosselin A. 1989. Acclimation of two tomato species to high atmospheric CO2. Plant Physiology 90, 1465–1472.[Abstract/Free Full Text]

Zheng Y, Shimizu H, Barnes JD. 2002. Limitations to CO2 assimilation in ozone-exposed leaves of Plantago major L. New Phytologist 155, 67–78.[CrossRef]

Zheng Y, Stevenson KJ, Barrowcliffe R, Chen S, Wang H, Barnes JD. 1998. Ozone levels in Chongqing: a potential threat to crop plants commonly grown in the region? Environmental Pollution 99, 299–308.[CrossRef][Medline]


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