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Published online 30 July 2007
Published in Crop Sci 47:1475-1487 (2007)
© 2007 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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CROP PHYSIOLOGY & METABOLISM

Elevated Carbon Dioxide and Ozone Effects on Peanut: I. Gas-Exchange, Biomass, and Leaf Chemistry

Fitzgerald L. Booker*, Kent O. Burkey, Walter A. Pursley and Allen S. Heagle

USDA-ARS, Plant Science Research Unit, and Dep. of Crop Science, North Carolina State Univ., 3127 Ligon St., Raleigh, NC 27607. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA or the North Carolina Agric. Res. Serv

* Corresponding author (fitz.booker{at}ars.usda.gov).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The effects of elevated CO2 and ozone (O3) on net photosynthetic rate (A) and growth are generally antagonistic although plant responses are highly dependent on crop sensitivity to the individual gases and their concentrations. In this experiment, we evaluated the effects of various CO2 and O3 mixtures on leaf gas-exchange, harvest biomass, and leaf chemistry in peanut (Arachis hypogaea L.), an O3–sensitive species, using open-top field chambers. Treatments included ambient CO2 (about 375 µmol mol–1) and CO2 enrichment of approximately 173 and 355 µmol mol–1 in combination with charcoal-filtered air (22 nmol O3 mol–1), nonfiltered air (46 nmol O3 mol–1), and nonfiltered air plus O3 (75 nmol O3 mol–1). Twice-ambient CO2 in charcoal-filtered air increased A by 23% while decreasing seasonal stomatal conductance (gs) by 42%. Harvest biomass was increased 12 to 15% by elevated CO2. In ambient CO2, nonfiltered air and added O3 lowered A by 21% and 48%, respectively, while added O3 reduced gs by 18%. Biomass was not significantly affected by nonfiltered air, but was 40% lower in the added O3 treatment. Elevated CO2 generally suppressed inhibitory effects of O3 on A and harvest biomass. Leaf starch concentration was increased by elevated CO2 and decreased by O3. Treatment effects on foliar N and total phenolic concentrations were minor. Increasing atmospheric CO2 concentrations should attenuate detrimental effects of ambient O3 and promote growth in peanut but its effectiveness declines with increasing O3 concentrations.

Abbreviations: A, net photosynthetic rate • AA, ambient air • CF, charcoal-filtered • gs, stomatal conductance • LMPA, leaf mass per unit leaf area • NF, nonfiltered • OZ, 1.56 x ambient O3 • PPFD, photosynthetic photon flux density • WAP, weeks after planting • WUE, water use efficiency


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Atmospheric concentrations of CO2 have risen from preindustrial levels of approximately 280 µmol mol–1 in 1750 to about 375 µmol mol–1 currently (Keeling and Whorf, 2005). The expected continued rise in atmospheric CO2 concentration, apart from possible influences of increasing temperature and other changes in environmental conditions, is anticipated to stimulate biomass production and perhaps yield of many C3 crops (Ainsworth and Long, 2005; Jablonski et al., 2002; Kimball et al., 1993; Prasad et al., 2005). Elevated CO2 typically improves net photosynthetic rates (A), plant–water relations, and photosynthetic water use efficiency (Ainsworth and Long, 2005; Polley, 2002; Prasad et al., 2005). It also lessens O3–induced stress in some crop species (Fiscus et al., 2002; Olszyk et al., 2000; Poorter and Pérez-Soba, 2001). Conversely, stimulation of plant growth at elevated CO2 can be curtailed by O3 (Barnes and Wellburn, 1998; Olszyk et al., 2000; Poorter and Pérez-Soba, 2001).

Tropospheric O3 levels since the 1960s have been sufficiently high to suppress growth and yield of many C3 crops in a number of industrialized countries worldwide (Treshow and Bell, 2002; Mauzerall and Wang, 2001). In addition, emissions of O3 precursors and areas affected by O3 pollution continue to grow (Dentener et al., 2005; Prather et al., 2003). Ozone impairs growth primarily by inhibiting A and perhaps translocation processes, which limit availability of photosynthate needed for biomass production (Fiscus et al., 2005; Heath and Taylor, 1997; Long and Naidu, 2002). Root production appears particularly susceptible to O3 exposure (Cooley and Manning, 1987; Grantz et al., 2006). Allocation of C and energy resources to detoxification and repair processes in O3–stressed plants likely detracts from growth as well (Heath and Taylor, 1997). Experiments with Arabidopsis thaliana mutants and pairs of O3–sensitive and –tolerant plant lines suggest that many detrimental effects of O3 are initiated and mediated by increased reactive oxygen species formation along with changes in plant hormone levels, antioxidant metabolism, cellular ion fluxes, and gene expression (Heath and Taylor, 1997; Kangasjarvi et al., 2005).

Ozone toxicity is likely reduced at elevated CO2 by lowered O3 uptake due to CO2–induced partial stomatal closure (Allen, 1990; Barnes and Wellburn, 1998; Booker and Fiscus, 2005; Fiscus et al., 1997; McKee et al., 1997; Olszyk et al., 2000; Polle and Pell, 1999; Poorter and Pérez-Soba, 2001). It has also been suggested that increased availability of photosynthate and energy equivalents for growth and detoxification processes at elevated CO2 aid in ameliorating O3 damage (Allen, 1990; Barnes and Wellburn, 1998; Booker and Fiscus, 2005; McKee et al., 1997; Polle and Pell, 1999; Poorter and Pérez-Soba, 2001). Nevertheless, plant responses to elevated CO2 and O3 depend in part on the gas concentrations, crop sensitivity, developmental stage, cumulative exposure, and other experimental conditions. For example, multiple studies with wheat (Triticum aestivum L.) found few statistically significant interactions between elevated CO2 and O3 on biomass production because O3 levels or crop cultivar sensitivity were too low to result in significant O3 effects (Bender et al., 1999). Other studies with highly O3–susceptible lines of clover (Trifolium repens L.), potato (Solanum tuberosum L.), and snap bean (Phaseolus vulgaris L.) found that growth inhibition by O3 was altered little by CO2 enrichment (Heagle et al., 1993, 2002, 2003). In the main, however, most studies determined that elevated CO2 partially or completely ameliorated the damaging effects of O3 on A and biomass production. This was observed in cotton (Gossypium hirsutum L.), soybean [Glycine max (L.) Merr.], wheat, and other crop species (Barnes and Pfirrman, 1992; Booker and Fiscus, 2005; Booker et al., 2005; Cardoso-Vilhena et al., 2004; Heagle et al., 1999, 2000; Miller et al., 1998; Olszyk et al., 2000; Plessl et al., 2005; Poorter and Pérez-Soba, 2001; Reid and Fiscus, 1998). Cumulative exposure and ontogeny were significant factors in several experiments in which amelioration of O3 effects on A by elevated CO2 declined as development progressed and O3 injury accumulated (Barnes and Pfirrman, 1992; Mulchi et al., 1992; Reid and Fiscus, 1998). It is unclear how peanut (Arachis hypogaea L.), an agronomically important and O3–sensitive species, would respond to increasing concentrations of atmospheric CO2 and O3.

Previous controlled environment and field experiments indicated that ambient O3 concentrations in the southeastern United States caused foliar injury and suppressed growth in peanut (Ensing et al., 1985; Heagle et al., 1983). Previous studies also found that peanut was quite responsive to increasing levels of atmospheric CO2 (Prasad et al., 2005). The objective of our study was to compare the effects of season-long exposures to various concentrations of CO2 and O3, administered singly and in mixtures, on foliar injury, leaf gas-exchange, harvest biomass production, and leaf chemistry in peanut. It was hypothesized that elevated CO2 and O3 would have concentration-dependent, counteracting effects on A and biomass production, which would be reflected by foliar injury assessments and some leaf chemistry components, such as chlorophyll and nonstructural carbohydrate concentrations. In addition, plant responses to nonfiltered (NF) air in open-top chambers were compared with plant responses to ambient air to assess chamber effects at ambient levels of CO2 and O3.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Culture Conditions and Gas Treatments
The experiment was conducted with peanut cuiltivar NC-V11, during 2002 and 2003 at a site 5 km south of Raleigh, NC (35°44' N, 78°41' W). The original topsoil in the field was excavated in 1984 and replaced with about 30 cm of Norfolk sandy loam (fine-loamy, kaolinitic, thermic Typic Kandiudult) to improve soil uniformity (Miller et al., 1988). The Norfolk sandy loam overlies an Appling sandy loam (fine, kaolinitic, thermic Typic Kanhapludult). Since 1985, the field has been cultivated periodically with cotton and soybean using conventional tillage and soil fertilization practices.

In preparation for the peanut experiment, the field was chisel-plowed and disked. The field was then limed and fertilized according to soil test recommendations with 94 kg K ha–1 on 15 Apr. 2002 and 13 Mar. 2003. Granular gypsum (86% CaSO4) was applied to the field at a rate of 897 and 1344 kg ha–1 on 1 July 2002 and 3 July 2003. Before planting, seeds were treated with a commercial Bradyrhizobium preparation (Rhizo-Flo, Becker Underwood Inc., Ames, IA) and then planted on 15 May 2002 and 13 May 2003. Plants were sown in rows with 1-m spacing and with plant spacing of 9 cm (11 plants m–2). Plants were irrigated with soaker hoses in 2002 and with emitters (Spot Spitters, Roberts Irrigation, San Marcos, CA) in 2003 installed parallel to each row at a distance of approximately 10 cm. Total irrigation in 2002 and 2003 was 35 cm and 17 cm, respectively. Plots were sprayed to control insects with acephate (O,S-dimethyl acetylphosphoramidothioate) (Whitmire Micro-Gen Research Laboratories, Inc., St. Louis, MO) on 31 May and 17 June 2002, and 6 June 2003; bifenthrin [(2-methyl-1,1-biphenyl-3-y1)-methyl-3-(2-chloro-3,3,3-trifluoro-1-propenyl)-2,2-dimethylcyclopropanecarboxylate] (Whitmire Micro-Gen Research Laboratories, Inc.) on 6 Aug. 2002, and 22 July and 6 Sept. 2003; and, imidacloprid (1-[(6-chloro-3-pyridinyl)methyl]-N-nitro-2-imidazolidinimine) (Bayer Corporation, Kansas City, MO) on 6 Sept. 2003. None of these insecticides has been reported to affect crop responses to elevated CO2 or O3.

Plants were exposed to mixtures of CO2 and O3 in cylindrical open-top chambers, 3 m in diameter by 2.4 m tall beginning on 30 May 2002 and 3 June 2003. Gas dispensing and monitoring were conducted as described for CO2 (Rogers et al., 1983) and O3 (Heagle et al., 1979). Supplementary O3 was generated by electrostatic discharge in dry O2 (model GTC-1A, Ozonia North America, Elmwood Park, NJ) and dispensed 12 h daily (0800–2000 h EST) in proportion to ambient O3 concentrations. Ozone concentration in the chambers was monitored at canopy height using UV photometric O3 analyzers (model 49, Thermo Environmental Instruments Co., Franklin, MA). The O3 analyzers were calibrated once every 2 wk (model 49 PS calibrator, Thermo Environmental Instruments Co.). Carbon dioxide was dispensed from a 12.7-Mg liquid receiver 24 h daily and was monitored at canopy height with infrared CO2 analyzers (model 6252, Li-Cor, Inc., Lincoln, NE). The CO2 monitors were calibrated once every 2 wk with CO2 standards. Dispensing of CO2 at night (1900–0700 h EST) was decreased by half to prevent concentrations from exceeding daytime target levels by more than 50%. Ground-level CO2 concentrations typically increase at night, and levels in the control treatment reached 500 µmol mol–1 at times.

The experiment consisted of all combinations of three CO2 treatments and three O3 treatments (Tables 1, 2). The CO2 treatments were ambient CO2, ambient plus 173 µmol CO2 mol–1, and ambient plus 355 µmol CO2 mol–1. The O3 treatments were charcoal-filtered (CF) air, NF air, and NF air plus 1.56 times ambient O3. Air filtration by activated charcoal lowered ambient O3 concentrations to levels considered nonphytotoxic for peanut (Heagle et al., 1983). An additional treatment of approximately 634 µmol CO2 mol–1 added to NF air was included to test the effects of a higher CO2 concentration. Plants were also grown in ambient air within chamber frames lacking plastic panels to assess chamber effects. All CO2 and O3 treatments were administered 7 d wk–1, and continued until 30 Sept. 2002 and 5 Oct. 2003, when plants were harvested.


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  		Table 1. Elevated CO2 and O3 treatments, treatment abbreviations, and number of replicate chambers per treatment in each year of the 2-yr experiment.

 

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  		Table 2. Average monthly and seasonal meteorological conditions, and CO2 and O3 treatment concentrations in the 2-yr experiment.{dagger}

 
Leaf and Plant Biomass Sampling
Once a month from late June through late September (21 June, 19 July, 26 August, and 19 September in 2002; 7 July, 28 July, 25 August, and 25 September in 2003), the third leaf down from the apex of a branch on four plants was obtained from each chamber. One plant in each quadrant of the chamber was sampled. Leaf petioles were removed and leaflet samples were pooled by chamber at each sampling date, frozen in liquid N2, and stored at –80°C for later analysis of leaf chemistry. Ten leaf disks (1.6-cm diameter) were also obtained from each sample of leaflets, freeze-dried, and weighed for determination of leaf mass per unit leaf area (LMPA). At harvest, two 2-m-long rows of plants measured from the center of each chamber were subdivided into 1-m segments. Twelve plants (three plants from one, randomly selected end of each 1-m-row segment) per chamber were sampled for biomass. Harvested plants were air-dried to a constant weight, and dry mass of leaves, stems, roots, pods, and culls was measured. Culls were defined as immature pods less than 1 cm in diameter and pods of any size exhibiting symptoms of disease.

Visible injury (percentage of chlorosis and necrosis) on the upper 13 leaves on the main stem of two plants per chamber was evaluated on 5 Aug. 2002 and 8 Aug. 2003. Visible injury estimates among leaves were averaged to yield a per chamber value.

Gas-Exchange Measurements
Net photosynthesis was measured at growth CO2 and O3 conditions in the chambers using a portable photosynthesis system (Model 6200, Li-Cor Inc.) and a 1-L cuvette. Measurements were made on the second leaf down from the apex of a branch on three plants per chamber between 1000 and 1300 h when ambient photosynthetic photon flux density (PPFD) > 1000 µmol m–2 s–1. Net photosynthesis was measured in all the treatments except the +173 µmol CO2 mol–1 and ambient air treatments due to time limitations for making the measurements. Average PPFD, relative humidity, and leaf temperature was 1746 µmol m–2 s–1, 46%, and 36°C, respectively, during the measurements.

In addition, midday leaf conductance was measured with a steady-state porometer (Model 1600M, Li-Cor, Inc.) on the abaxial and adaxial surfaces of a leaflet on the third leaf down from the apex of a branch between 1100 and 1300 h when ambient PPFD > 1000 µmol m–2 s–1. One leaf on each of four plants was measured in each of two replicate chambers per treatment. Average PPFD, relative humidity, and leaf temperature was 1387 µmol m–2 s–1, 45%, and 32°C, respectively, during the measurements. Leaf conductance measurements were corrected for the standard boundary layer conductance imposed by the instrument (2.7 mol m–2 s–1, Li-Cor 1600M Instruction Manual, Revision 6, 1989), and reported as stomatal conductance (gs).

Gas-exchange measurements were made during each week from July through September in 2002 and 2003 when weather conditions permitted (A and gs were measured on 34 and 42 occasions, respectively). Measurements were averaged on a weekly basis for subsequent data analysis.

Leaf Chemistry Assays
Five leaf disks (0.85 cm diameter) were obtained for determination of chlorophyll concentration. Leaf disks were extracted with 3 mL of 95% ethanol (2x) overnight at 4°C, and chlorophyll concentration was determined spectrophotometrically by the following equation:

Formula
(Lichtenthaler and Wellburn, 1983). The chlorophyll concentration, expressed as micrograms of chlorophyll per square centimeter leaf area, was then obtained.

For assays of nonstructural carbohydrates and total soluble phenolics, freeze-dried leaf tissue samples were ground to pass a 0.5-mm mesh screen. Starch and soluble sugars were determined enzymatically by the UV absorbance method (R-Biopharm, Inc., Marshall, MI). To solubilize starch, tissue samples (25 mg) were each mixed with 2.4 mL of dimethylsulfoxide and 600 µL of 8 M HCl in sealed polypropylene tubes for 1 h at 60°C. Samples were then neutralized with 600 µL of 8 M NaOH and diluted to 15 mL with 112 mM citrate buffer (pH 4). Solutions were filtered, and 50-µL aliquots were assayed according to kit instructions. Starch was hydrolyzed to D-glucose by amyloglucosidase. D-glucose was determined indirectly by first forming D-glucose-6-phosphate with ATP and hexokinase followed by the formation of D-gluconate-6-phosphate by glucose-6-phosphate dehydrogenase and NADP+. The amount of NADPH formed in the second reaction is stoichiometric to the amount of D-glucose formed by the hydrolysis of starch. The concentration of NADPH was determined by measuring the absorbance of the reaction solution at 340 nm. Results were expressed as D-glucose equivalents.

To determine total soluble phenolic concentrations, tissue samples (25 mg) were extracted with 1 mL of freshly prepared 250 mM sodium citrate containing 2% (w/v) sodium bisulfite (pH 7) (3x) for 5 min at room temperature with periodic mixing, centrifuged (16000 x g), and the supernatants pooled by sample (Blum, 1997). A 50-µL aliquot of each sample was mixed with 475 µL of 0.25 M Folin-Ciocalteu reagent (Sigma-Aldrich Chemical Co., St. Louis, MO) and 475 µL of 1 M Na2CO3, incubated at room temperature for 1 h, and absorbance of the solutions was measured at 724 nm. Results were expressed as 4-coumaric acid equivalents using a standard curve.

Statistical Analysis
In each year of the experiment, the treatments consisted of all factorial combinations of three CO2 levels and three O3 levels along with an additional very high CO2–NF air treatment combination. The treatments were assigned to chambers in a completely randomized design. Chamber treatments were randomly reassigned in the second year of the experiment. There were three replicate chambers for each of the high and low CO2 x O3 combinations (n = 12) (Table 1). In addition, there were two replicate chambers for each of the +173 µmol CO2 mol–1 and NF air treatment combinations (n = 10) (Table 1). Two chambers were used for the very high CO2 (+634 µmol mol–1)–NF air treatment combination. Assay results from plant tissue samples obtained within a chamber were averaged for use as a chamber replicate value. Results from the 2-yr experiment were combined for the statistical analyses, and the effect of year was treated as a fixed variable. Data were checked for homogeneity of variance, and a ln transformation was applied to the gas-exchange data before analysis. Treatment effects and least-squared means for harvest biomass measurements in the 3 CO2 x 3 O3 x 2 yr factorial experiment were determined using analysis of variance techniques (SAS Proc Mixed, SAS System for Windows, Ver. 8.2; Littell et al., 1996). Treatment effects and least-squared means for periodically measured gas-exchange processes (A and gs), LMPA, and leaf chemical components were estimated using a repeated measures model in which chambers constituted the whole plots and sampling period was the repeated factor (SAS Proc Mixed; Littell et al., 1996). The model included interactions between the whole plot factors and the effect of sampling period. Effects of the very high CO2–NF air treatment as well as the ambient air treatment were evaluated in separate analysis of variance tests.


   RESULTS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Environmental Conditions
The 2002 field season was slightly hotter and drier from mid-May through September compared with the 2003 field season (Table 2). Mean daytime ambient CO2 concentration during the experiment was 375 µmol mol–1, and the elevated CO2 treatments concentrations averaged 548 (1.46 x ambient CO2) and 730 (1.95 x ambient CO2) µmol mol–1 (12 h average, 0800–2000 h EST) (Table 2). The additional high CO2 treatment concentration averaged 1009 µmol mol–1 (2.69 x ambient CO2). Average daytime ambient O3 concentration (AA) for the 2002 and 2003 growing seasons was 48 nmol mol–1 (12 h average), which was typical for the area. The average O3 concentration in the CF air, NF air, and added O3 (OZ) treatments was 22 (0.48 x ambient O3), 46 (0.96 x ambient O3), and 75 (1.56 x ambient O3) nmol mol–1 (12 h average) (Table 2).

Visible Injury
Both elevated CO2 and O3 increased visible foliar injury (Table 3), although symptoms differed among the treatments. Elevated CO2 caused chlorotic mottle and irregular patches of white necrotic areas while O3 produced diffuse chlorosis and brownish stipple. Visible injury was highest in the OZ-375 treatment compared with the control (CF-375). Elevated CO2 suppressed O3–induced visible injury, although symptoms of injury from both gases were apparent.


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  		Table 3. Foliar visible injury at midseason for peanut exposed to mixtures of CO2 and O3.{dagger}

 
Photosynthesis and Stomatal Conductance
Net photosynthesis in upper canopy leaves in the control treatment (CF-375) was fairly steady at 30 to 38 µmol CO2 m–2 s–1 from 8 through 19 wk after planting (WAP) in this indeterminate-growth plant (Fig. 1 ). Average A was 23% higher in the CF-730 treatment than in the control (Table 4, Fig. 1). In contrast, NF air (NF-375) and added O3 (OZ-375) suppressed A by 21 and 48%, respectively. The NF-375 effect on A was large in 2002, but marginal in 2003 (data not shown), leading to the significant year x O3 interaction reported in Table 4. Inhibitory effects of O3 on A were generally attenuated by elevated CO2, although the CO2 effect tended to decline toward the end of the growing season. Averaged over the season, A in the NF-730 and OZ-730 treatments was 19 and 10% higher, respectively, than in the control treatment. Average A was 30% higher in the NF-1009 treatment compared with the control, but the rate was not significantly different from the NF-730 treatment (P ≥ 0.05).


Figure 1
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  		Figure 1. Effects of CO2 and O3 on average net photosynthesis (A) of upper canopy leaves of peanut from 8 through 19 wk after planting in the 2-yr experiment (A, B). Seasonal average A is also shown (C). Treatments included charcoal-filtered air (CF)–ambient CO2 (CF-375) (control), CF air plus 355 µmol CO2 mol–1 (CF-730), nonfiltered air (NF)–ambient CO2 (NF-375), NF air plus 355 µmol CO2 mol–1 (NF-730), NF air plus 634 µmol CO2 mol–1 (NF-1009), 1.5 x ambient O3–ambient CO2 (OZ-375), and 1.5 x ambient O3 plus 355 µmol CO2 mol–1 (OZ-730). Values are means ± SE from two or three replicate chambers per treatment in each year of the experiment (see Table 1). Values above the bars in panel C indicate percentage of the control treatment. Significant treatment effects are indicated as P ≤ 0.05 (*), P ≤ 0.01 (**), and P ≤ 0.001 (***).

 

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  		Table 4. Probabilities of elevated CO2 and O3 treatment effects on net photosynthesis (A) and stomatal conductance (gs) in the 2-yr experiment. Net photosynthesis was measured from 8 through 19 wk after planting (WAP) in 2002 and 2003. Stomatal conductance was measured from 10 through 19 WAP in 2002 and 2003.

 
Increased CO2 concentration in the CF-548 and CF-730 treatments reduced average gs by 16 and 42%, respectively, compared with the control from 11 through 19 WAP (Table 4, Fig. 2 ). Average gs in the NF-375 treatment was not significantly different from the control. Likewise, gas treatment effects on gs in the NF-548 and NF-730 treatments were not significantly different from those in the respective CF air-elevated CO2 treatments (CF-548 and CF-730) (P ≥ 0.05). In the OZ-375 treatment, gs was suppressed by 18% relative to the control. The combined effect of elevated CO2 and O3 on gs in the OZ-548 and OZ-730 treatments was additive; gs was lower in these treatments than in the respective CF-548 and CF-730 treatments (P ≤ 0.05). In the OZ-548 and OZ-730 treatments, gs was reduced by 39 and 50%, respectively, compared with the control. Average gs in the NF-1009 treatment was 59% lower than the control.


Figure 2
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  		Figure 2. Effects of CO2 and O3 on average stomatal conductance (gs) of upper canopy leaves of peanut from 10 through 19 wk after planting in the 2-yr experiment (A, B). Seasonal average gs is also shown (C). Treatments were charcoal-filtered air (CF)–ambient CO2 (CF-375) (control), CF air plus 173 µmol CO2 mol–1 (CF-548), CF air plus 355 µmol CO2 mol–1 (CF-730), nonfiltered air (NF)–ambient CO2 (NF-375), NF air plus 173 µmol CO2 mol–1 (NF-548), NF air plus 355 µmol CO2 mol–1 (NF-730), NF air plus 634 µmol CO2 mol–1 (NF-1009), 1.5 x ambient O3–ambient CO2 (OZ-375), 1.5 x ambient O3 plus 173 µmol CO2 mol–1 (OZ-548), and 1.5 x ambient O3 plus 355 µmol CO2 mol–1 (OZ-730). Values are means ± SE from two or three replicate chambers per treatment in each year of the experiment (see Table 1). Values above the bars in panel C indicate percentage of the control treatment. Significant treatment effects are indicated as P ≤ 0.05 (*), P ≤ 0.01 (**), and P ≤ 0.001 (***).

 
Seasonal average water use efficiency (WUE) (A/transpiration) was approximately doubled in the twice-ambient CO2 treatments (Table 5). The higher CO2 level in the NF-1009 treatment resulted in the highest WUE value. In contrast, WUE was 18 and 40% lower in the NF-375 and OZ-375 treatments, respectively, compared with the control. Elevated CO2 fully compensated for O3 effects on WUE.


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  		Table 5. Seasonal average photosynthetic water use efficiency (WUE) of upper canopy peanut leaves exposed to mixtures of CO2 and O3.{dagger}

 
Harvest Biomass
Main treatment effects of elevated CO2 and O3 were statistically significant for all harvest biomass components measured except cull dry mass (Table 6). Elevated concentrations of CO2 increased biomass while O3 suppressed it. Among the various biomass components, stem biomass on an absolute mass basis was most affected by the gas treatments.


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  		Table 6. Harvest biomass of peanut exposed to mixtures of CO2 and O3 in the 2-yr experiment.{dagger}

 
Total biomass of plants in the CF-548 and CF-730 treatments was 12 to 15% higher than in the control treatment (Table 6). Stem dry mass was increased about 24% in these two elevated CO2 treatments, while root dry mass was increased 33% in the CF-730 treatment. Leaf, pod, and cull biomass in the CF-548 and CF-730 treatments was not significantly different from the control.

Average dry mass of harvest components in the NF-375 and NF-548 treatments was not significantly different from the control treatment (Table 6). Elevated CO2 stimulated biomass production in the NF-730 and NF-1009 treatments, but the difference in biomass between the two treatments was not statistically significant (P ≥ 0.05). Component and total biomass were reduced 34 to 44% (excluding culls) in the OZ-375 treatment relative to the control. An increase in the CO2 concentration by 50% partially compensated for the added O3 effect on biomass (compare OZ-375 vs. OZ-548). However, the biomass stimulation attributable to a 50% increase in CO2 in CF air was diminished by added O3 (compare CF-548 vs. OZ-548). Twice-ambient concentrations of CO2 completely ameliorated the effects of added O3 on biomass components, and CO2 x O3 interactions were statistically significant except for roots and culls (Table 6). Year x CO2 interactions for leaf and stem biomass were caused by larger increases in biomass between the +173 and +355 treatments in 2002 than in 2003.

Elevated CO2 concentrations slightly increased partitioning of biomass to stems at the expense of pods (Table 7). In addition, there were small differences between the 2002 and 2003 experiments for pod biomass partitioning ratio (significant year x CO2 interaction). Main effects of the O3 treatments on biomass partitioning were not statistically significant. However, pod mass ratio was 10% higher in the OZ-375 treatment compared with the control (P ≤ 0.05).


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  		Table 7. Partitioning of harvest biomass among organs (organ mass/total mass) of peanut as influenced by CO2 and O3 in the 2-yr experiment.{dagger}

 
Leaf Mass Per Area and Leaf Chemistry
Seasonal average LMPA increased 15 to 20% in all the elevated CO2 treatments and decreased 11% in the OZ-375 treatment compared with the control (Table 8). However, LMPA declined in the second half of the field season in all treatments, and the decline at elevated CO2 was greater in the NF and OZ treatments than in the CF treatments (data not shown). The increase in LMPA with increasing CO2 levels was greater in the 2002 experiment than in 2003, especially between the +173 and +355 CO2 treatments (significant year x CO2 interaction). The NF-375 treatment had no statistically significant effect on LMPA. The CO2 x O3 interaction for LMPA was not statistically significant.


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  		Table 8. Seasonal average leaf mass per unit leaf area and leaf chemistry of peanut exposed to mixtures of CO2 and O3 in the 2-yr experiment.{dagger}

 
The most noteworthy treatment effects of elevated CO2 and O3 on leaf chemistry were on chlorophyll and starch concentrations (Table 8, Fig. 3 ). Both elevated CO2 and added O3 suppressed average seasonal leaf chlorophyll concentrations by 17 to 28%, although elevated CO2 reduced chlorophyll concentration during vegetative growth while added O3 lowered it during reproductive stages. Starch concentrations were higher at elevated CO2 and lower in the NF-375 and OZ-375 treatments compared with the control. Stimulatory effects of elevated CO2 on starch mass were diminished by added O3 and vice versa. Elevated CO2 increased soluble sugar concentrations while O3 treatment effects were not statistically significant. Gas treatment effects on N and total phenolics concentrations were relatively small, although some differences were statistically significant.


Figure 3
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  		Figure 3. Effects of CO2 and O3 on chlorophyll (A) and starch (B) concentrations in upper canopy leaves of peanut from 6 through 19 wk after planting in the 2-yr experiment. Treatments shown are charcoal-filtered air (CF)–ambient CO2 (CF-375) (control), CF air plus 355 µmol CO2 mol–1 (CF-730), 1.5 x ambient O3–ambient CO2 (OZ-375), and 1.5 x ambient O3 plus 355 µmol CO2 mol–1 (OZ-730). Values are means ± SE from two or three replicate chambers per treatment in each year of the experiment (see Table 1).

 
Open-Top Chamber Effects
A comparison between plants grown in the NF-375 and AA treatments indicated that harvest biomass components, leaf soluble sugars, N, and total phenolics were not significantly different (Table 9). Visible foliar injury at midseason was lower in the AA treatment, as were seasonal average gs and chlorophyll concentration, compared with the NF-375 treatment. Leaf mass per area and starch concentrations were 8 and 33% higher, respectively, for plants grown in the AA treatment compared with the NF-375 treatment.


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  		Table 9. Open-top chamber effects on visible foliar injury at midseason, harvest biomass, stomatal conductance (gs), leaf mass per unit leaf area (LMPA), and leaf chemistry of peanut. Plants were exposed to nonfiltered air (NF-375) and ambient air (AA; chamber frames without side panels).{dagger}

 

   DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Amelioration of O3 damage at elevated CO2 has been found in a number of experiments with C3 crop plants (Booker and Fiscus, 2005; Booker et al., 2005; Craigon et al., 2002; Fiscus et al., 2002; Heagle et al., 1999, 2000; Olszyk et al., 2000; Plessl et al., 2005; Poorter and Pérez-Soba, 2001). Results of our study also showed that elevated CO2 generally counteracted the O3 stress we applied to NC-V11 peanut in terms of biomass production and yield (Table 6) (also see Burkey et al., 2007). This response was accompanied by significant increases in A and decreases in gs (Fig. 1, 2), suggesting likely causative relationships among treatment effects on gs, A, growth, and yield. However, elevated CO2 did not completely abate the detrimental effects of added O3. For example, A in the NF-730 and OZ-730 treatments declined in advance of that in the CF-730 treatment near the end of the experiment. This has been observed in previous CO2 x O3 experiments with radish (Raphanus sativus L.) and soybean (Barnes and Pfirrman, 1992; Mulchi et al., 1992; Reid and Fiscus, 1998). Also, biomass and yield in the OZ-548 treatment were about 20% lower than in the CF-548 and NF-548 treatments (Table 6) (also see Burkey et al., 2007). Thus, biomass production responses observed in this experiment indicated that CO2 concentrations expected by the year 2050 (approximately 550 µmol mol–1) (Prather et al., 2003) will not fully protect peanut against possibly higher tropospheric O3 levels. This scenario is particularly relevant for some major peanut-producing regions of the world, such as eastern China, central India, and central Africa (Rhoades and Nazarea, 2003), where ground-level O3 concentrations are predicted to increase dramatically in the next 50 yr (Dentener et al., 2005; Prather et al., 2003; Wang and Mauzerall, 2004). Similarly, modeled yield responses of an O3–sensitive wheat cultivar to various future scenarios of atmospheric CO2 and O3 concentrations indicated that potential yield increases due to elevated CO2 c oncentrations could be halved or more by increasing concentrations of tropospheric O3 (Hertstein et al., 1995). Therefore, evaluations of crop responses to elevated CO2 need to consider possible influences of ambient O3 in their assessments.

There were limitations in the stimulatory effect of elevated CO2 on peanut growth as well. The NF-1009 treatment indicated that average A and biomass components were not significantly different from the NF-730 treatment (Table 6). Seed yield was also not further increased in the NF-1009 treatment compared with the NF-730 treatment (Burkey et al., 2007). Growth constraints at high CO2 levels could be related to increased plant competition, limitations in water and mineral nutrient availability, and genetic potential of the plant. Stanciel et al. (2000) found that dry mass production of hydroponically grown ‘Georgia Red’ peanut declined at 1200 µmol CO2 mol–1 compared with 800 µmol CO2 mol–1. Our results indicate that there is a maximum potential for biomass and yield stimulation by elevated CO2 in NC-VII peanut.

A shift in biomass allocation toward pods was observed in the OZ-375 treatment but not in the NF-375 and elevated CO2 treatments (Table 7). Similar changes in pod mass ratio have been found in previous studies with soybean and other crop species (Booker and Fiscus, 2005; Cooley and Manning, 1987; Miller et al., 1998). Evidently, pods become strong sinks for photosynthate in O3–treated plants. Nonetheless, the net effect of O3 on pod biomass is typically not positive because of the overriding suppression of total plant biomass by O3 (Miller et al., 1998).

Concentrations of total soluble phenolics were not significantly affected by the gas treatments in our study (Table 8). However, changes in LMPA with elevated CO2 and O3 can make assays of total phenolics difficult to interpret unless treatment effects on specific compounds and nonstructural carbohydrate concentrations are taken into account. By expressing total phenolic concentrations on a leaf area basis, we eliminated the influence of gas treatments effects based on leaf dry mass, but a more comprehensive analysis of phenolic compound biosynthesis will be required to determine whether there are specific CO2 and O3 effects in peanut. Ozone-induced increases in insoluble, polymeric phenolic–iron–protein complexes are more commonly observed in injured leaves and are thought to be responsible for the brown-colored lesions (stipple) in leaves of many plant species exposed to O3 (Booker and Miller, 1998; Howell and Kremer, 1973). Peanut leaves exhibited chlorosis and stipple in response to O3 in our study (Table 3), suggesting that loss of cellular integrity and polymerization of preexisting phenolics occurred in response to O3.

It was somewhat surprising that the inhibitory effects of NF air on A, along with the increase in visible foliar injury, were not translated into statistically significant decreases in biomass and yield (also see Burkey et al., 2007). However, inhibition of A and starch accumulation in upper canopy leaves did not become apparent in the NF-375 treatment until late in the growing season, which suggests that growth and reproductive processes might have been only mildly affected in this treatment until the later developmental stages when cumulative exposure effects were sufficient to impair photoassimilation.

Effects of the open-top field chambers on visible injury, biomass production, gs, LMPA, and leaf chemistry were compared in the AA and NF-375 treatments. Few statistically significant differences between the treatments were indicated (Table 9). Stomatal conductance was 12% lower in the AA treatment compared with the NF-375 treatment, while LMPA and starch concentration values were higher. In a previous study (Heagle et al., 1983), peanut biomass and yield were 8 to 25% higher in an AA treatment compared with the NF treatment, although a statistical analysis of the results was not reported. It is known that open-top chambers impose higher daytime air temperatures, lower PPFD, continuous air movement, and other changes in environmental factors that differ from ambient conditions (Heagle et al., 1979; Kimball et al., 1997). Therefore, it is not unexpected that plant growth per se in ambient air is often different from that in chambers. However, a number of comparisons between AA and NF treatments indicated that relative plant responses to O3 were not significantly influenced by the chamber environment (Heagle, 1989). We recognize that plant responses to elevated CO2 and O3 in our experiment have the potential to be confounded by the use of CF and NF air in the treatments. This protocol was adopted from the USDA National Crop Loss Assessment Network program (Heagle, 1989). The use of CF and NF air was deemed acceptable because levels of other air pollutants such as NO, NO2, and SO2 were below phytotoxic levels at our location. Ambient air is also entrained in all chambers during the course of the experiment. Thus, we have no reason to presume that the use of CF and NF air would lead to unrecognized interactions in our experiment.

In conclusion, results of our experiment indicated that A, starch biosynthesis, and biomass production in NC-V11 peanut was suppressed by ambient and higher levels of O3. Increasing concentrations of CO2 should ameliorate these responses, although its effectiveness will likely depend on concurrent O3 concentrations and other changes in environmental conditions.


   ACKNOWLEDGMENTS
 
We thank Jeff Barton, Mike Durham, Yhenneko Jallah, Barbara Jones, Erin Silva, and Renee Tucker for their assistance with this project. We gratefully acknowledge Robert Philbeck for construction and maintenance of dispensing and monitoring systems. Dr. Marcia Gumpertz, Department of Statistics, North Carolina State University, is thanked for her assistance with the statistical analysis. Dr. David Jordan, Department of Crop Science, North Carolina State University, provided advice on peanut cultivation and harvesting. Meteorological data were provided by the State Climate Office of North Carolina at North Carolina State University.


   NOTES
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 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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Received for publication August 21, 2006.


   REFERENCES
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 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 





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