Eva J. Pell 211 Buckhout Laboratory, University Park, PA 16802 USA/Tel. 814–865–0323; fax: 814 863–7217; e-mail: firstname.lastname@example.org
Ozone (O3)-induced accelerated senescence of leaves was measured in four tree species: black cherry (Prunus serotina), hybrid poplar (Populus maximowizii x trichocarpa, clone 245), northern red oak (Quercus rubra) and sugar maple (Acer saccharum). Seedlings or ramets of the four species were subjected to chronic O3 exposures and designated leaves harvested periodically from emergence to senescence. Gas exchange was analysed, and concentrations of total soluble protein and ribulose-1,5-bisphosphate carboxylase/oxygenase were measured as indicators of leaf senescence. Total antioxidant potential and ascorbate peroxidase and glutathione reductase activities also were determined. Black cherry and hybrid poplar exhibited O3-induced accelerated leaf senescence, whereas sugar maple and northern red oak did not. When the O3 effects were related to cumulative uptake of the gas, black cherry was the most sensitive of the four species. Although hybrid poplar exhibited similar symptoms of O3-induced accelerated senescence after the same exposure period as did black cherry, this species took up much greater quantities of O3 to achieve the same response. The O3-induced increase in glutathione reductase activity in hybrid poplar was consistent with the capacity of this species to take up high concentrations of the gas. Relative tolerance of northern red oak and sugar maple could be explained only in part by lower cumulative O3 uptake and lower rate of uptake. Sugar maple had the highest antioxidant potential of all four species, which may have contributed to O3 tolerance of this species. Ascorbate peroxidase activity, when expressed on a fresh weight basis, could not account for differential sensitivity among the four species.
Ozone (O3) induces a variety of responses in vegetation including foliar injury, reduction in plant growth and productivity, and acceleration of foliar senescence. There are numerous reports of differences in O3 responses among and within species. Visible injury has been the criteria used in many interspecies comparisons. Although foliage which develop O3-induced lesions may also exhibit accelerated senescence, the relative sensitivity of different genotypes as measured by one symptom type are not always predictors of other responses (Landry 1992; Woo 1996).
One of the challenges facing plant biologists is to determine an explanation for differences in species sensitivity to O3. Harkov & Brennan (1979) found a positive correlation between species susceptibility to O3-induced foliar injury and growth rate. Many investigators have attempted to explain differential responses of plants to O3 in relation to stomatal conductance. Reich (1987) proposed that variation in O3-induced effects on net photosynthesis and growth could be explained by total uptake of the gas. Alternative explanations for differential plant responses have related to relative ability of species to detoxify O3-generated reactive oxygen intermediates (Alscher, Donahue & Cramer 1997). Plants have an extensive array of defence systems including antioxidant metabolites, e.g. ascorbate, glutathione, polyamines, α-tocopherol, and carotenoids, and enzymes including superoxide dismutase, glutathione reductase, ascorbate peroxidase and catalase (Foyer, Descourviéres & Kunert 1994; Alscher et al. 1997). These systems are spatially distributed throughout the cell environment, and may be either constitutively present or induced. When free radicals are produced in a specific cellular location, many antioxidants are necessary to provide the net scavenging protection from oxidation. While the status of each antioxidant could be measured separately, there is no known way to integrate the scavenging potential across disparate antioxidants. Cao, Alessio & Cutler (1993) developed a simple procedure, the oxygen-radical absorbance capacity (ORAC) assay, designed to measure the antioxidizing potential of blood serum. We modified this assay for use with leaf extract to pose some general questions about the antioxidizing potential of tissue for plant species with differential sensitivity to O3.
In this study we conducted an analysis of O3-induced accelerated foliar senescence in four hardwood species of importance in the North-east region of the USA, namely, black cherry (Prunus serotina), hybrid poplar (Populus maximowizii x trichocarpa, clone 245), northern red oak (Quercus rubra) and sugar maple (Acer saccharum). There is considerable literature describing the sensitivity of foliage of black cherry and hybrid poplar to O3 (Reich & Lassoie 1985; Neufeld et al. 1995; Pell et al. 1995; Fredericksen et al. 1995, 1996; Karnosky et al. 1996); similarly, the relative tolerance of leaves of northern red oak and sugar maple have been defined (Rebbeck & Loats 1997; Samuelson & Kelly 1997). Previous emphasis has been placed on measurements of growth and gas exchange parameters including net photosynthesis. However, quantification of accelerated foliar senescence is based on a complex of responses including loss of foliar protein, which provides some definition to the process of leaf senescence. The objectives of the study were to determine: (1) whether O3 induced accelerated foliar senescence in the four tree species studied, as defined by decline in net photosynthesis, quantity of ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco) and total soluble protein; (2) whether the magnitude of response could be related to total O3 uptake; and (3) whether antioxidant status in the foliage of each species might explain the magnitude of response.
Experimental sites and plant culture
Experiments were conducted at the Russell Larson Research Farm of the Pennsylvania State University (Pennsylvania Furnace, PA) during the field summers of 1993, 1994 and 1995, and in 1994 at the Tennessee Valley Authority field site in Norris, Tennessee. For experiments conducted in Pennsylvania cuttings of hybrid poplar clone 245, 1-year-old seedlings of black cherry, and 2-year old seedlings of northern red oak and sugar maple were cultured as described elsewhere (Pell, Sinn & Vinten Johansen 1995; Brendley & Pell 1998); plants received a nutrient supplement of 3·53 g L–1 Osmocote (N : P : K 14 : 14 : 14; Sierra Chemical Co., Milpitas, CA, USA) at planting. Side shoots were pruned as they emerged from all four species. Drip irrigation was used as described previously (Pell et al. 1993). Containers were positioned in double rings; thus the pruning practice and location of plants within the chamber minimized shading of older foliage. The cultural history of the oak seedlings used in the experiment conducted at the Norris, TN site have been described by Edwards et al. (1994). These seedlings were 3-year-old half-siblings grown from acorns.
All plants at the Pennsylvania site were grown in open-top chambers receiving charcoal-filtered air (Pell et al. 1993). For each species, three (1995) or four (1993 and 1994) replicate chambers received supplemental O3 from 1000 to 1800 h each day; companion plants did not receive this supplementation and served as the control treatment. Ozone exposures were conducted from 24 June to 22 September 1993, 24 June to 27 September 1994 and 13 June to 16 August 1995. When daily 10 h means for the exposure period were averaged for the experimental season, the O3 concentrations in the charcoal-filtered chambers averaged 0·04 μL L–1 in all three years whereas in the treatment chambers the concentrations averaged 0·08 μL L–1 in 1993 and 1994, and 0·07 μL L–1 in 1995.
The generation, delivery, and monitoring of O3 at the Norris, TN site were conducted from 26 April to 30 September 1994 as described by Edwards, Wullschleger & Kelly (1994; Wullschleger, Hanson & Edwards 1996). Groups of 30 seedlings were grown in each of six open-top chambers. Three replicate chambers received a daily 7 h exposure to charcoal-filtered, and twice-ambient O3, resulting in mean seasonal concentrations of 0·008–0·016 and 0·06–0·098 μL L–1 O3, respectively (Wullschleger et al. 1996).
In order to study senescence, we selected leaves which emerged early in the experiment and observed performance throughout development. At the Pennsylvania site, when black cherry seedlings and hybrid poplar ramets were approximately 18 cm in height, and when the second flush of sugar maple and northern red oak were initiated, a newly emergent leaf on each plant in every chamber was tagged for gas exchange and destructive harvest. Once every 1 to 2 weeks throughout the growing seasons, leaves were sampled from two plants per chamber. At the Norris, TN site, leaves of the second flush of northern red oak were destructively harvested following the same protocol as at the Pennsylvania site. Gas exchange analysis for this tissue was performed on fully expanded foliage.
In situ net photosynthesis and leaf conductance were measured at full sunlight by non-destructive gas exchange analysis with a Li-Cor 6200 closed-loop photosynthesis system (Li-Cor, Inc., Lincoln, NE) (Pell, Eckardt & Enyedi 1992; Hanson et al. 1994). Measurements were taken early in the day when conductance should have been at a maximum. After the gas exchange analysis, leaves were harvested in the field, frozen in liquid nitrogen and stored at –80 °C.
Cumulative O3 uptake was estimated for each sample for the duration of exposure to the pollutant. For the Pennsylvania site, daily stomatal conductance was estimated based on a linear interpolation of periodic stomatal conductance measurements of water vapour conductance. These values were converted to O3 conductance utilizing the molecular diffusivity of O3, and assuming leaf intracellular O3 concentrations close to zero (Laisk, Kull & Moldau 1989). The dose was calculated by multiplying the O3 concentration by the duration of treatment, for each day, from the measurements made in the open-top chambers every 30 min throughout the 8 h exposure period. The daily O3 uptake rate (mmol m–2 d–1) was calculated as the product of daily O3 conductance and daily O3 dose. Uptake rate was summed over time to give an estimate of the cumulative O3 uptake at the time of leaf harvest. At the Norris, TN site fewer stomatal conductance values were available. Analysis of stomatal conductance measurements from studies at this site in 1991–94 (Samuelson & Edwards 1993; Edwards et al. 1994; Hanson et al. 1994) revealed minimal variation, within or among seasons. Consequently, a water vapour conductance of 0·114 mol m–2 s–1, representing a multiple year average, was selected to estimate O3 uptake as described above. The daily O3 dose was determined using the monitoring data provided by Tennessee Valley Authority and described by Edwards et al. (1994).
Tissue extraction and analysis
Reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted, and solutions were treated with Chelex 100 (Biorad Corp., Hercules, CA) to remove potentially interfering metals. Total soluble protein (TSP) was extracted by grinding 0·2 g frozen tissue to a powder under liquid nitrogen in a mortar. The frozen powder was transferred to a glass vial and 10 volumes of cold extraction buffer were added. Extraction buffer (pH 7·8) for hybrid poplar and black cherry contained 90 mM potassium phosphate, 1 mM EDTA, 3% PVPP, 5 mM ascorbate, and 8% glycerol. For northern red oak and sugar maple extractions, the buffer was optimized by increasing the PVPP to 4% and including 1·5% PVP-40T. Tissue was homogenized on ice for 30 s with a Tissue-tearor (Biospec Products, Bartlesville, OK) and the homogenate subsequently centrifuged for 15 min at 15 850 g. Aliquots were immediately frozen in liquid nitrogen and stored at –80 °C until analysis. Total soluble protein was determined by the method of Bradford (1976), using BioRad protein dye concentrate and BSA as the standard. Rubisco quantity was determined as described by Eckardt & Pell (1994).
Enzyme assays were performed with a Beckman DU-64 spectrophotometer (Beckman Instruments, Inc., Fullerton, CA), using the Kinetics Soft-pac module to calculate rates of absorbance change over time. Enzyme activity measurements were run in triplicate, and TSP in duplicate.
Ascorbate peroxidase (APX) activity was measured by monitoring the consumption of ascorbate at 290 nm as described by Torsethaugen et al. (1997). The APX activity was measured during the first 60 s of the reaction. Final APX activity was calculated after correcting for non-enzymatic degradation of ascorbate (background) and ascorbate oxidase activity.
Glutathione reductase (GR) activity was measured using a method based on Smith, Vierheller & Thorne (1988). The reaction mixture contained 100 mM potassium phosphate, pH 7·8, 0·5 mM DTNB, 0·2 mM NADPH, 0·2 mM GSSG, and 100 μL crude plant extract in a final volume of 1000 μL. The change in absorbance of DTNB at 412 nm was monitored for 2 min. The reaction remained linear for more than 5 min. Each set of assays included measurements of a GR standard from wheat germ (Sigma Chemical Co.); periodic tests of a mix of all reagents except extract or extract plus all reagents except GSSG revealed no interfering ‘background’ reaction. On the basis of preliminary data we found that APX and GR activities were not affected by filtration of extracts with a Sephadex G-25 gel exclusion column (Pharmacia Biotech, Piscataway, NJ; PD-10).
The ORAC assay was adapted from the method of Cao et al. (1993). The assay measures the antioxidant potential of tissue samples. It is based on the capacity of free radicals generated by 2, 2’-azobis (d-amidinopropane) dihydrochloride (AAPH) to oxidize β-phycoerythrin (BPE), an algal pigment which fluoresces at 565 nm (excitation at 540 nm). In the presence of free radical scavengers, the free radical-induced loss of fluorescence of BPE is reduced. The assay was performed with a soluble leaf extract derived by the method described above, but using an argon purged 75 mM sodium phosphate buffer, pH 7·0; all extraction steps were performed under argon to further minimize oxidation. After centrifugation, the extracts were diluted to 0·2% concentration for immediate use in the ORAC assay. Leaf extract of all species did not absorb light at 540 or 565 nm; similarly there was no fluorescence when extracts were excited at 540 nm.
The sample reaction mixture contained 75 mM sodium phosphate, pH 7·0, 16·7 nM BPE (CyanoTech Corp., Hawaii, USA), 3 mM AAPH (Wako Chemicals USA, Richmond, VA), and 20 μL dilute plant extract in a final volume of 200 μL. Blanks were comprised of BPE and AAPH in the absence of extract, to measure free radical-induced decay in fluorescence of the pigment. Standards contained no plant extract, but instead had the water-soluble antioxidant Trolox (Boehringer Mannheim, Indianapolis, IN) added to final concentrations of 1–3 μM. Triplicates of each blank, sample and standard were loaded onto 96 well untreated Costar microtitre plates; fluorescence was measured with a PerSeptives Cytofluor II fluorescence plate reader (PE Biosystems, Perkin Elmer, Inc., Norwalk, CT). The assay was conducted at 37 °C and followed to completion. ORAC values were calculated using the area under the fluorescence decay curves as in Cao et al. (1993):
ORAC value = (Fluorescencesample– Fluorescenceblank) / (Fluorescence1 μMtrolox– Fluorescence blank)
One ORAC unit equals the amount of protection provided to BPE by the 1 μM Trolox standard.
The reliability of the method was verified by conducting recovery assays in which 2 μM Trolox were added to the plant extract; recovery of Trolox protection was always between 80 and 90%. In addition we determined that in the absence of AAPH, plant extract did not interfere with fluorescence of BPE.
Enzymatic activities were analyzed on specific activity, dry weight and fresh weight bases. The ORAC data were calculated on both dry and fresh weight bases.
Statistical analysis of net photosynthesis, TSP and Rubisco content, and of measures of antioxidants were conducted for each species using an analysis of variance (ANOVA); significance was accepted at the P≤ 0·05 level (SAS Institute Inc. 1985). All these analyses were conducted utilizing days of O3 exposure as the independent variable, thus reflecting the experimental design.
The ozone treatment elicited minimal foliar symptoms until the leaves began to mature. At that time signs of accelerated senescence, particularly chlorosis and foliar abscission, became apparent on O3-treated leaves of black cherry and hybrid poplar. The leaves of black cherry first began to abscise after approximately 25 d of O3 exposure; leaves of hybrid poplar exhibited a similar response to O3 after 45 d of exposure. No macroscopic evidence of foliar senescence was detected for northern red oak or sugar maple foliage.
Relationship between ozone dose and measures of leaf longevity
Ozone induced an accelerated decline in net photosynthesis, TSP and Rubisco concentrations in leaves of black cherry seedlings as the tissue matured (Table 1; Fig. 1). Hybrid poplar responded similarly (Table 1; Brendley & Pell 1998). Ozone had no effect on any of the parameters of leaf ageing measured for sugar maple or northern red oak (Table 1). We note that in 1994 there was a significant O3 effect on TSP in sugar maple. When the data were examined it was clear that the significance reflected a higher value for TSP in O3-treated foliage at the first sampling point. No other significant differences were detected for sugar maple at any other sampling point in any of the three seasons studied.
Table 1. . Significance of analysis of variance for measures of accelerated senescence. Data for each species were analysed separately and significance is designated as *P≤ 0·05, **P≤ 0·01
All three indicators of leaf ageing, namely the decline in photosynthetic rate, quantity of Rubisco and TSP, were accelerated in black cherry and hybrid poplar in response to O3. We selected TSP to further characterize and contrast the response of the four species to O3 because this response was not directly linked to gas exchange. When the impact of O3 on loss in TSP was considered as a function of days of O3 exposure, hybrid poplar exhibited the greatest response followed closely by black cherry; however, northern red oak and sugar maple were unresponsive to the pollutant (Fig. 2).
Relationship between O3 uptake and measures of leaf longevity
It is well known that uptake of O3 is largely dependent on stomatal conductance. Using stomatal conductance data, the cumulative uptake of O3 was estimated for each species at the time of harvest in each experiment. The relative impact of O3 on TSP was plotted against the net uptake of O3 by foliage in O3-treated versus charcoal-filtered air (Fig. 3). From these graphs it is clear that much less O3 was necessary to induce a reduction in total soluble leaf protein in black cherry than any other species. While hybrid poplar foliage is very sensitive when O3 response is measured by duration of exposure (Fig. 2), it takes substantially more O3 to induce a response comparable with that of black cherry. From Fig. 2 it is clear that TSP declines in response to O3 throughout the experiments. However, in Fig. 3 there is no evidence that TSP declines as completely in hybrid poplar foliage as it does in black cherry. During ageing, the stomatal conductance declines more rapidly in O3-treated hybrid poplar foliage than in leaves grown in charcoal-filtered air (data not shown). There is some O3 in the charcoal-filtered air chambers; consequently, when leaf conductance in the O3-treated hybrid poplar foliage decreases sufficiently, the calculated net cumulative O3 uptake for treated tissue appears to cease or even decline. This represents an artifact of the calculation and in Fig. 3 we elected to omit points after which net cumulative O3 uptake no longer ‘increased’. This artefact did not occur with the other three species. Sugar maple and northern red oak did not exhibit significant O3-induced reduction in total soluble leaf protein (Table 1; Fig. 3). Despite the apparent insensitivity of these species, sugar maple and northern red oak did take up as much or more O3 than did black cherry foliage.
Measures of antioxidant capacity were determined for leaves as they developed, matured and senesced in the 1995 experiment. The ORAC assay was designed to measure the antioxidizing capacity of soluble leaf extract. When expressed on a fresh weight basis, extracts of sugar maple had higher mean ORAC values than the other three species studied (Fig. 4). There was no apparent effect of O3 on the ORAC measurement except in the case of black cherry where there were significant increases in response to the pollutant.
Specific activity of APX was initially relatively high in sugar maple and northern red oak, and very low in hybrid poplar (Fig. 5). Ozone induction of APX was significant for hybrid poplar (Table 2), probably because of the increase observed at the last sampling point (Fig. 5). On a fresh weight basis hybrid poplar still had lower activity of APX than did the other three species studied (Fig. 5). Black cherry foliage exhibited O3-induced increases in specific activity of APX but differences could not be detected when activity was expressed either on a dry or fresh weight basis (Table 2; Fig. 5). The GR activity was lowest in foliage of northern red oak (Fig. 6). There was some evidence of O3-induced increase in specific activity of GR in hybrid poplar foliage (Table 2; Fig. 6).
Table 2. . Significance of analysis of variance for analysis of oxygen-radical absorbance capacity (ORAC), ascorbate peroxidase (APX) and glutathione reductase (GR) activities in 1995. ORAC analyses were conducted on data calculated on a fresh weight basis. APX and GR analyses are presented for data on dry weight (DW), fresh weight (FW) and specific activity (Sp. act.). Data for each species were analysed separately and significance is designated as *P≤ 0·05, **P≤ 0·01
The data presented herein were derived from three experiments in Pennsylvania and one in Tennessee. There were small differences, year to year, in the length of exposure within and among species. The experiment in Tennessee was longer in duration than those conducted in Pennsylvania, and exposures were proportional to O3 concentrations in the air rather than constant square wave additions of the gas. Regardless of experimental year or geographic location, similar differential responses of the four species to O3 were detected. We only present data demonstrating O3-induced acceleration of loss of TSP with leaf age; similar effects were observed when net photosynthesis and Rubisco were quantified (unpublished results; Brendley & Pell 1998).
Interpretation of differential sensitivity of species is contingent upon the independent variable against which the responses are measured. If exposure dose is the determinant of sensitivity as measured by percentage drop in TSP, black cherry and hybrid poplar are more sensitive than sugar maple and northern red oak; hybrid poplar appears to be somewhat more sensitive than black cherry (Fig. 2). Using exposure dose as a measure of sensitivity provides useful information when identifying effects that will be observed in the environment. However, at a mechanistic level it may be more instructive to consider O3 uptake as the independent variable.
Differential species uptake of O3 has been well documented (Reich 1987). Reich (1987) proposed that uptake was an important factor determining the ozone responses based upon an analysis of hardwoods, pines and crop plants. Hanson et al. (1994) compared the effects of O3 on maximum photosynthesis in foliage of mature trees and seedlings of northern red oak; they concluded that internal uptake could not fully account for differences in the responses between these two growth forms. The results of our analyses suggest that differential species responses cannot be fully explained by cumulative uptake of O3. We note that the uptake values we present are an estimate and probably represent the upper limit. Stomatal conductance was only measured once each day, probably at the time when uptake was greatest. We did not account for leaf and/or canopy boundary layer resistance. However, dimensional analyses based upon Gates (1980) were used to estimate the leaf boundary layer resistance for the species studied and little variation among species could be detected.
When O3 uptake is the independent variable against which loss in TSP, a measure of leaf senescence, is considered, black cherry exhibited the most dramatic response to the gas (Fig. 3). Sugar maple and northern red oak took up as much or more O3 than did black cherry while appearing tolerant to O3-induced acceleration of leaf senescence. Interpretation of the relationship between total uptake of O3 and accelerated leaf senescence in hybrid poplar is less clear. A conservative interpretation is that leaves of hybrid poplar do take up more gas than the other species studied and this explains the greater sensitivity of this species to O3. However, northern red oak and sugar maple leaves took up as much gas as some of the hybrid poplar leaves and yet showed no biochemical, physiological, or visual signs of accelerated foliar senescence. When the rate of O3 uptake was calculated for these species, it was clear that hybrid poplar leaves took up O3 at a much higher rate than the other three species, the greatest uptake occurring early in development (Fig. 7). Thus, since northern red oak and sugar maple took up O3 at a slower rate they may have had a better chance of detoxifying the gas. We note that clone 245 of hybrid poplar is a rapid-growth genotype (Pell unpublished results) and there is significant diversity in available hybrid poplar germplasm (Hinckley et al. 1989). It is possible that other clones of hybrid poplar might perform differently.
The more surprising observation was the relatively low cumulative uptake of O3 in black cherry foliage that was needed to achieve a response to the pollutant comparable with that of hybrid poplar. The uptake rate of black cherry was much lower than that of hybrid poplar (Fig. 7). In 1993, which was one of the longest experiments conducted in Pennsylvania, sugar maple and black cherry were exposed to O3 for equivalent lengths of time. In that experiment the sugar maple leaves actually took up more O3 than did the leaves of black cherry. Uptake rates varied from year to year (data not shown) but black cherry consistently had somewhat greater rates than sugar maple or northern red oak early in the growing season, with rates declining more, later in the life of the leaf. However, the differences in uptake rates between black cherry, northern red oak and sugar maple were much less than between all three species and hybrid poplar.
One facet of O3 toxicity must be related to uptake of the gas. However, once the gas is taken up other factors come into play. The role of antioxidants as detoxifiers of O3 and/or its by-products has been explored by many investigators (Guri 1983; Guzy & Heath 1993; Bender et al. 1994; Alscher et al. 1997; reviewed by Tanaka et al. 1985; Peters, Castillo & Heath 1989; Sen Gupta, Alscher & McCune 1991; Kangasjärvi et al. 1994; Polle, Wieser & Havranek 1995; Ranieri et al. 1996; Rank 1997). In this study we examined the total antioxidant potential of leaves, using the ORAC assay which measures the ability of total leaf extracts to protect a model protein from oxidative damage. Sugar maple had the highest ORAC values (Fig. 4) which correlated with the greater resistance to O3-induced acceleration of foliar senescence. The ORAC results are reported based on extracts from fresh tissue. This is the most relevant way to report the data given the environment in which leaves receive oxidizing stress. However, we note that the percentage dry weight is much higher for maple and oak than for cherry and poplar; as a result if the ORAC results are calculated on a dry weight basis the ORAC values are no longer higher for maple (data not shown). This suggests that if the ORAC measurement is an indication of antioxidizing potential, it is highest in maple because of greater concentration rather than quantity of antioxidizing constituents. Total leaf activities of two enzymes with antioxidant functions, namely GR and APX, neither explained relative tolerance of sugar maple to O3 nor reflected a response to the gas.
Relative tolerance of northern red oak is not readily explained by the results of the antioxidant analyses. The ORAC values were similar for black cherry, northern red oak and hybrid poplar foliage when expressed on fresh weight (Fig. 4), and actually were lower in northern red oak when expressed on a dry weight basis (data not shown). While specific activity of APX was somewhat higher for northern red oak than the other three species, when enzyme activity was expressed on a fresh weight basis only hybrid poplar had lower activity (Fig. 5). Glutathione reductase activity in northern red oak was lowest when compared with the other three species regardless of the form in which data were expressed (Fig. 6).
The sensitivity of black cherry could be explained neither by the rate and cumulative uptake of O3, nor by lower antioxidant potential as determined by the indicators in this study. Ozone induction of higher ORAC values and increased specific activity of APX might be a reflection of an O3 response of this sensitive species to the air pollutant. Despite the relatively high specific activity of APX in black cherry foliage toward the end of the experiment, the activity on a fresh weight basis was low, reflecting the decline of the leaf. Since black cherry foliage exhibited adverse O3 responses, the changes in ORAC values and APX specific activity were clearly insufficient to provide adequate protection.
The responsiveness of hybrid poplar to O3 could be interpreted as a result of the large net uptake and rapid rate of uptake of the gas. An alternative perspective could lead to the question of how hybrid poplar takes up so much O3 before exhibiting responses. While the ORAC data do not explain the hybrid poplar response, the O3-induced increases in GR activity may provide a partial explanation for the relative tolerance observed in these plants (Figs 4 & 6). Previously, Aono et al. (1993) were unable to induce increased O3 tolerance in tobacco by overexpressing GR in the chloroplast. Since we measured total GR the O3-induced increase may in fact, have reflected elevated activity elsewhere in the cell.
Although the ORAC assay allowed us to look at net antioxidizing potential of foliage, the method is limited because it is an average across all locations within the cell and across all aqueous antioxidants. It is well known that there is a relationship between ascorbate in the apoplast and O3 responses (Polle et al. 1995; Ranieri et al. 1996). Smaller differences in antioxidants, either metabolites or enzymes, in key locations may be sufficiently diluted in a whole leaf assay as to go undetected. The procedure we used examined total soluble extract. It is also possible that the critical defence antioxidants are located in an insoluble form in membranes or the cell wall. What is clear from this research is that differential sensitivity among species to O3 cannot be explained solely on the basis of uptake of the gas.
Funding for this study was provided in part by the Pennsylvania Agricultural Experiment Station and the Environmental Resources Research of the Pennsylvania State University. Contribution no. 2060, Department of Plant Pathology. External funding was provided by Forest Service Agreement no. 32–651-A3, NSF Training Grant No. DBI-9413204, Tennessee Valley Authority cooperative agreement number TV 92719 V and USDI Cooperative Agreement 4000–3-2012.