Seasonal profiles of leaf ascorbic acid content and redox state in ozone-sensitive wildflowers

Cutleaf coneflower (Rudbeckia laciniata L.), crown-beard (Verbesina occidentalis Walt.), and tall milkweed (Asclepias exaltata L.) are wildflower species native to Great Smoky Mountains National Park (U.S.A.). Natural populations of each species were analyzed for leaf ascorbic acid (AA) and dehydroascorbic acid (DHA) to assess the role of ascorbate in protecting the plants from ozone stress. Tall milkweed contained greater quantities of AA (7-10 micromol g(-1) fresh weight) than crown-beard (2-4 micromol g(-1) fresh weight) or cutleaf coneflower (0.5-2 micromol g(-1) fresh weight). DHA was elevated in crown-beard and cutleaf coneflower relative to tall milkweed suggesting a diminished capacity for converting DHA into AA. Tall milkweed accumulated AA in the leaf apoplast (30-100 nmol g(-1) fresh weight) with individuals expressing ozone foliar injury symptoms late in the season having less apoplast AA. In contrast, AA was not present in the leaf apoplast of either crown-beard or cutleaf coneflower. Unidentified antioxidant compounds were present in the leaf apoplast of all three species. Overall, distinct differences in antioxidant metabolism were found in the wildflower species that corresponded with differences in ozone sensitivity.     

Ambient ozone effects on gas exchange and total non-structural carbohydrate levels in cutleaf coneflower (Rudbeckia laciniata L.) growing in Great Smoky Mountains National Park

Ozone-sensitive and -tolerant individuals of cutleaf coneflower (Rudbeckia laciniata L.) were compared for their gas exchange characteristics and total non-structural carbohydrates at Purchase Knob, a high elevation site in Great Smoky Mountains National Park, USA. Photosynthesis and stomatal conductance decreased with increased foliar stipple. Sensitive plants had lower photosynthetic rates for all leaves, except the very youngest and oldest when compared to tolerant plants. Stomatal conductance decreased with increasing leaf age, but no ozone-sensitivity differences were found. Lower leaves had less starch than upper ones, while leaves on sensitive plants had less than those on tolerant plants. These results show that ambient levels of ozone in Great Smoky Mountains National Park can adversely affect gas exchange, water use efficiency and leaf starch content in sensitive coneflower plants. Persistence of sensitive genotypes in the Park may be due to physiological recovery in low ozone years.     

Cutleaf coneflower (Rudbeckia laciniata L.) response to ozone and ethylenediurea (EDU)

Cutleaf coneflower (Rudbeckia laciniata L.) seedlings were placed into open-top chambers in May, 2004 and fumigated for 12 wks. Nine chambers were fumigated with either carbon-filtered air (CF), non-filtered air (NF) or twice-ambient (2×) ozone (O₃). Ethylenediurea (EDU) was applied as a foliar spray weekly at 0 (control), 200, 400 or 600 ppm. Foliar injury occurred at ambient (30%) and elevated O₃ (100%). Elevated O₃ resulted in significant decreases in biomass and nutritive quality. Ethylenediurea reduced percent of leaves injured, but decreased root and total biomass. Foliar concentrations of cell-wall constituents were not affected by EDU alone; however, EDU × O₃ interactions were observed for total cell-wall constituents and lignocellulose fraction. Our results demonstrated that O₃ altered the physiology and productivity of cutleaf coneflower, and although reducing visible injury EDU may be phytotoxic at higher concentrations.     

Interpreting spatial variation in ozone symptoms shown by cutleaf cone flower,Rudbeckia laciniata L

Visible injury caused by ozone is recorded every year in native plant species growing in Great Smoky Mountains National Park (USA). One of the most sensitive species, cutleaf coneflower (Rudbeckia laciniata L.), shows great variation in symptoms between and within populations but the causes of this variation and its ecological significance are currently unknown. This paper presents data relating to genetic variation, ozone concentrations, stomatal conductance and light (PAR) within populations. The data show that populations differ in genetic diversity, one consisting of only three genets while another was very diverse. In the former population, symptoms varied greatly within a single genet, pointing to a large micro-environmental influence. Measurements of ozone, stomatal conductance and PAR within plant canopies suggest that variation in symptom expression is unlikely to be due to differences in ozone flux and more likely to be due to variation in light. The variation in visible symptoms raises the question of what bioindicators actually indicate, and it suggests that symptoms should be interpreted with great caution until the underlying causes of that variation are fully understood.     

Seasonal development of ozone-induced foliar injury on tall milkweed (Asclepias exaltata) in Great Smoky Mountains National Park

The goals of this study were to document the development of ozone-induced foliar injury, on a leaf-by-leaf basis, and to develop ozone exposure relationships for leaf cohorts and individual tall milkweeds (Asclepias exaltata L.) in Great Smoky Mountains National Park. Plants were classified as either ozone-sensitive or insensitive based on the amount of foliar injury. Sensitive plants developed injury earlier in the season and to a greater extent than insensitive plants. Older leaf cohorts were more likely to belong to high injury classes by the end of each of the two growing seasons. In addition, leaf loss was more likely for older cohorts (2000) and lower leaf positions (2001) than younger cohorts and upper leaves, respectively. Most leaves abscised without prior ozone-like stippling or chlorosis. Failure to take this into account can result in underestimation of the effects of ozone on these plants.     

Sub-canopy deposition of ozone in a stand of cutleaf coneflower

Although there has been a great deal of research on ozone, interest in exposure of native, herbaceous species is relatively recent and it is still not clear what role the pollutant has in their ecological fitness. The ozone exposure of a plant is usually expressed in terms of the concentration above the canopy or as a time-weighted index. However, to understand the physiological effects of ozone it is necessary to quantify the ozone flux to individual leaves as they develop, which requires knowing the deposition velocity and concentration of the pollutant as a function of height throughout the plant canopy. We used a high-order closure model of sub-canopy turbulence to estimate ozone profiles in stands of cutleaf coneflower (Rudbeckia laciniata L.) located in the Great Smoky Mountains National Park, USA. The model was run for periods coinciding with a short field study, during which we measured vertical concentration profiles of ozone along with measurements of atmospheric turbulence and other meteorological and plant variables. Predictions of ozone profiles by the model are compared with observations throughout the canopy.     

Temporal patterns of foliar ozone symptoms on tall milkweed (Asclepias exaltata L.) in Great Smoky Mountains National Park

Incidence and severity of ozone-induced foliar symptoms on tall milkweed (Asclepias exaltata L.) along selected trails in Great Smoky Mountains National Park (GRSM) were determined by two surveys/season conducted from 1992 through 1996. Overall incidence was 73%, and was 84%, 44%, 90%, 58%, and 82% for 1992-1996, respectively for the same clusters. Average incidence was 61% and 84% for the 1st and 2nd surveys, respectively. Seasonal comparisons showed two distinct injury groupings regarding incidence and severity of injury: 1992, 1994 and 1996 (high injury); 1993 and 1995 (low injury). No discernible patterns were observed between symptomatic and asymptomatic plants regarding height, herbivory or flowering. Regression analyses indicated no differentiation in foliar symptoms regarding topographic position, aspect, slope or elevation over the 5-year study period. Our findings indicate other micro-site or genetic factors may control ozone sensitivity of tall milkweed in GRSM.     

Concentrations of mercury in otters (Lutra lutra L.) in Scotland in relation to rainfall

The incidence and severity of visible foliar ozone injury on black cherry (Prunus serotina) seedlings and saplings and tall milkweed (Asclepias exaltata) plants in Great Smoky Mountains National Park (GRSM) were determined by surveys along selected trails conducted during late summer 1992. The incidence (% injured plants) of ozone injury on black cherry was 47% and the percent injured leaves/injured plant and average leaf area injured were 43 and 6%, respectively. Maximum severity (avg. leaf area of the most severely injured leaf) was 12%. Black cherry seedlings and saplings exhibiting ozone injury were taller than non-injured plants. When insect feeding was present, it occurred 96% of the time on plants with ozone injury. Significantly more injury (p=0.007) on black cherry (% injured leaves/injured black cherry) occurred in the NW section of GRSM compared with the other Park sections. Regression analyses showed no relationships in ozone injury with respect to aspect, slope or elevation. Tall milkweed was evaluated twice during August for ozone injury. The incidence (% injured plants) of ozone injury was 74 and 79% for the first and second survey, respectively. The percentage of injured leaves per plant from the first to second survey was 63 to 79%, respectively. Tall milkweeds showing ozone injury were taller than the non-injured plants. The percentage of insect-damaged plants was 50% among plants without ozone injury and 60% among ozone-injured plants. Non-injured tall milkweed had fewer flowers and/or pods than the injured plants. Mean leaf area injured increased over time, and mean maximum leaf area injured increased from 8 to 11% during the same period. Regression analyses showed no differences in ozone injury regarding aspect, slope or elevation. Our findings indicate that ozone injury is widespread throughout the Park on sensitive vegetation.     

Foliar injury, leaf gas exchange and biomass responses of black cherry (Prunus serotina Ehrh.) half-sibling families to ozone exposure

Open pollinated families of black cherry seedlings were studied to determine genotypic differences in foliar ozone injury and leaf gas exchange in 1994 and growth response following three growing seasons. An O(3)-sensitive half-sibling family (R-12) and an O(3)-tolerant half-sibling family (MO-7) planted in natural soil were studied along with generic nursery stock (NS) seedlings. Ozone exposure treatments were provided through open top chambers and consisted of 50, 75, and 97% of ambient ozone, and open plots from May 9 to August 26, 1994. Ambient ozone concentrations reached an hourly peak of 88 ppb with 7-hour averages ranging from 39 to 46 ppb. Seedlings in the 50 and 75% of ambient chambers were never exposed to greater than 80 ppb O(3). Visible foliar ozone injury (stipple) was significantly higher for R-12 seedlings than MO-7 seedlings and increased with increasing ozone exposures. For the chamber treatments averaged over all families, there was no significant difference in stomatal conductance and net photosynthetic rates, but there was a significant decrease in root biomass, and a significant decrease in root/shoot ratio between the 50 and 97% of ambient chambers. Stomatal conductance and net photosynthetic rates were significantly different between families with R-12 seedlings generally greater than MO-7 seedlings. The R-12 seedlings had a 7.5 mmol m(-2) increase in ozone uptake compared to MO-7, and at the same cumulative O(3) exposure R-12 exhibited 40.9% stippled leaf area, whereas MO-7 had 9.2% stippled leaf area. Significant differences were observed in stem volume growth and total final biomass between the open-top chambers and open plots. Although R-12 had the most severe foliar ozone injury, this family had significantly greater stem volume growth and total final biomass than MO-7 and NS seedlings. Root:shoot ratio was not significantly different between MO-7 and R-12 seedlings.     

Size-mediated foliar response to ozone in black cherry trees

Local ozone concentration and visible foliar injury were measured over the 1994 growing season on open-grown black cherry (Prunus serotina Ehrh.) trees of varying size (age) within forest stands and adjacent openings at a site in north-central Pennsylvania. Relationships were determined between visible ozone injury and ozone exposure, as well as calculated between injury and ozone uptake expressed as the product of stomatal conductance and ozone concentration. In addition, simultaneous measurements of visible symptoms and leaf gas exchange were also conducted to determine the correlation between visible and physiological injury and ozone exposure. By September, the amount of leaf area affected by visible foliar ozone injury was greatest for seedlings (46%), followed by canopy trees (20%) and saplings (15%). A large amount of variability in foliar ozone symptom expression was observed among trees within a size class. Sum40 and Sum60 (ozone concentration > 40 and > 60 nl liter(-1)) cumulative exposure statistics were the most meaningful indices for interpretation of foliar injury response. Seedlings were apparently more sensitive to ozone injury than larger trees because their higher rates of stomatal conductance resulted in higher rates of ozone uptake. Seedlings also had higher rates of early leaf abscission than larger trees with an average of nearly 30% of the leaves on a shoot abscised by 1 September compared to approximately 5% for larger trees. However, per unit ozone uptake into the leaf, larger trees exhibited larger amounts of foliar injury. The amount of visible foliar injury was negatively correlated (r(2) = 0.82) with net photosynthetic rates, but was not related to stomatal conductance. Net photosynthesis and stomatal conductance thus became uncoupled at high levels of visible foliar injury.     

Physiological and foliar symptom response in the crowns of Prunus serotina, Fraxinus americana and Acer rubrum canopy trees to ambient ozone under forest conditions

The crowns of five canopy dominant black cherry (Prunus serotina Ehrh.), five white ash (Fraxinus americana L.), and six red maple (Acer rubrum L.) trees on naturally differing environmental conditions were accessed with scaffold towers within a mixed hardwood forest stand in central Pennsylvania. Ambient ozone concentrations, meteorological parameters, leaf gas exchange and leaf water potential were measured at the sites during the growing seasons of 1998 and 1999. Visible ozone-induced foliar injury was assessed on leaves within the upper and lower crown branches of each tree. Ambient ozone exposures were sufficient to induce typical symptoms on cherry (0-5% total affected leaf area, LAA), whereas foliar injury was not observed on ash or maple. There was a positive correlation between increasing cumulative ozone uptake (U) and increasing percent of LAA for cherry grown under drier site conditions. The lower crown leaves of cherry showed more severe foliar injury than the upper crown leaves. No significant differences in predawn leaf water potential (psi(L)) were detected for all three species indicating no differing soil moisture conditions across the sites. Significant variation in stomatal conductance for water vapor (g(wv)) was found among species, soil moisture, time of day and sample date. When comparing cumulative ozone uptake and decreased photosynthetic activity (P(n)), red maple was the only species to show higher gas exchange under mesic vs. drier soil conditions (P < 0.05). The inconsistent differences in gas exchange response within the same crowns of ash and the uncoupling relationship between g(wv) and P(n) demonstrate the strong influence of heterogeneous environmental conditions within forest canopies.     

Use of ethylenediurea (EDU) to ameliorate ozone effects on purple coneflower (Echinacea purpurea)

Purple coneflower plants (Echinacea purpurea) were placed into open-top chambers (OTCs) for 6 and 12 weeks in 2003 and 2004, respectively, and exposed to charcoal-filtered air (CF) or twice-ambient (2x) ozone (O3) in 2003, and to CF, 2x or non-filtered (NF), ambient air in 2004. Plants were treated with ethylenediurea (EDU) weekly as a foliar spray. Foliar symptoms were observed in >95% of the plants in 2x-treated OTCs in both years. Above-ground biomass was not affected by 2x treatments in 2003, but root and total-plant biomass decreased in 2004. As a result of higher concentrations of select cell wall constituents (% ADF, NDF and lignin) nutritive quality was lower for plants exposed to 2x-O3 in 2003 and 2004 (26% and 17%, respectively). Significant EDU x O3 interactions for concentrations of cell wall constituents in 2003 indicated that EDU ameliorated O3 effects on nutritive quality. Interactions observed in 2004 were inconsistent.     

Stem injection of Populus nigra with EDU to study ozone effects under field conditions

EDU or ethylenediurea (N-[2-(2-oxo-1-imidazolidinyl)ethyl]-N'-phenylurea) has been used in experiments to assess ozone effects on vegetation under field conditions because it provides protection against oxidative damage. Tests have mainly been conducted on crop plants, but for woody species only few reports have provided evidence that it can be used in long-term experiments. In this study we tested the technique of stem injection of EDU to study the effects of ozone exposure on Populus nigra cv. Wolterson over one growing season. Cuttings of Populus nigra were grown in pots in the field and between mid-July and early September plants were repeatedly injected with EDU solution (5 mg/plant) or with water at 14-day intervals. Significant differences were found between EDU- and water-injected plants: water-treated plants had more foliar injury, more chlorotic leaves, and shedding of leaves started earlier, suggesting EDU was effective in preventing visible ozone injury and acceleration of senescence. Photosynthetic rates, measured for one leaf age, showed no differences but were mostly higher for the EDU-treated plants. At the end of the growing season diameter increment was 16% higher and there was a non-significant trend for above-ground biomass to be increased by 9% for the EDU-treated plants. This experiment has provided evidence that for this clone serious ozone damage occurs at relatively low concentrations and that EDU can provide protection against visible injury, as well as against longer term growth reductions.     

An assessment of the impact of ambient ozone on field-grown crops in New Jersey using the EDU method: part 1-white potato (Solanum tuberosum)

Six potato cultivars were grown to maturity in field plots in New Brunswick, New Jersey, according to standard commercial practices over a 5-year period. One-half of the plots were given a periodic soil drench of an antioxidant (EDU) which has the capacity to protect foliage against ozone toxicity. Based upon visible foliar injury and total tuber yield, the cultivars Norland and Norchip proved significantly more sensitive to ambient ozone pollution than Green Mountain, Irish Cobbler, Belrus or Superior. When foliar injury was less than 20%, no impact on tuber yield was detected. However, when 75% of the foliage exhibited O(3) toxicity symptoms, tuber yield was reduced 25% and 31%, respectively, in 'Norland' and 'Norchip'. A review of results from studies in the US and Canada utilizing different assessment methodologies provides evidence that ambient ozone causes significant tuber yield reduction in sensitive white potato genotypes when foliar injury exceeds 20 to 40%.     

Ozone uptake (flux) as it relates to ozone-induced foliar symptoms of Prunus serotina and Populus maximowiziixtrichocarpa

Field studies were conducted during 2003 and 2004 from early June to the end of August, at 20 sites of lower or higher elevation within north-central Pennsylvania, using seedlings of black cherry (Prunus serotina, Ehrh.) and ramets of hybrid poplar (Populus maximowiziixtrichocarpa). A linear model was developed to estimate the influence of local environmental conditions on stomatal conductance. The most significant factors explaining stomatal variance were tree species, air temperature, leaf vapor pressure deficit, elevation, and time of day. Overall, environmental factors explained less than 35% of the variation in stomatal conductance. Ozone did not affect gas exchange rates in either poplar or cherry. Ozone-induced foliar injury was positively correlated with cumulative ozone exposures, expressed as SUM40. Overall, the amount of foliar injury was better correlated to a flux-based approach rather than to an exposure-based approach. More severe foliar injuries were observed on plants growing at higher elevations.     

Ozone exposure thresholds and foliar injury on forest plants in Switzerland

Canton Ticino in southern Switzerland is exposed to some of the highest concentrations of tropospheric ozone in Europe. During recent field surveys in Canton Ticino, foliar symptoms identical to those caused by ozone have been documented on native tree and shrub species. In Europe, the critical ozone level for forest trees has been defined at an AOT40 of 10 ppm.h O3 (10 ppm.h accumulated exposure of ozone over a threshold of 40 ppb) during daylight hours over a six-month growing season. The objective of this study was to determine the amount of ambient ozone required to induce visible foliar symptoms on various forest plant species in southern Switzerland. Species were grown within eight open-top chambers and four open plots at the Vivaio Lattecaldo Cantonal Forest Nursery in Ticino, Switzerland. Species differed significantly in terms of the ppb.h exposures needed to cause visible symptoms. The most to least symptomatic species grown within open-plots in this study rank as Prunus serotina, Salix viminalis, Vibrnum lantana, Rhamnus cathartica, Betula pendula, Rumex obtusifolius, Sambucus racemosa, Morus nigra, Prunus avium, Fraxinus excelsior, Rhamnus frangula, Alnus viridis, Fagus sylvatica and Acer pseudoplatanus. Similar rankings were obtained in the non-filtered chamber plots. The ranking of species sensitivity closely follows AOT values for the occurrence of initial symptoms and symptom progression across the remainder of the exposure season. Species that first showed evidence of foliar injury also demonstrated the most sensitivity throughout the growing season, with symptoms rapidly advancing over ca. 25-30% of the total plant leaf surfaces by the end of the observation period. Conversely, those species that developed symptoms later in the season had far less total injury to plant foliage by the end of the observation period (1.5 to < 5% total leaf area injured). The current European ambient ozone standard may be insufficient to protect native plant species from visible foliar injury, and many more native species may be sensitive to ozone-induced foliar injury than are currently known.     

Physiological and foliar injury responses of Prunus serotina,Fraxinus americana,and Acer rubrum seedlings to varying soil moisture and ozone

Sixteen black cherry (Prunus serotina, Ehrh.), 10 white ash (Fraxinus americana, L.) and 10 red maple (Acer rubrum, L.) 1-year old seedlings were planted per plot in 1997 on a former nursery bed within 12 open-top chambers and six open plots. Seedlings were exposed to three different ozone scenarios (ambient air: 100% O3; non-filtered air: 98% ambient O3; charcoal-filtered air: 50% ambient O3) within each of two different water regimes (nine plots irrigated, nine plots non-irrigated) during three growing seasons.During the 1998 and 1999 growing season, leaf gas exchange, plant water relations, and foliar injury were measured. Climatic data,ambient- and chamber-ozone-concentrations were monitored. We found that seedlings grown under irrigated conditions had similar (in 1998) but significantly higher gas exchange rates (in 1999) than seedlings grown within non-irrigated plots among similar ozone exposures. Cherry and ash had similar ozone uptake but cherry developed more ozone-induced injury (< 34% affected leaf area, LAA) than ash (<5% LAA), while maple rarely showed foliar injury, indicating the species differed in ozone sensitivity. Significantly more severe injury on seedlings grown under irrigated conditions than seedlings grown under non-irrigated conditions demonstrated that soil moisture altered seedling responses to ambient ozone exposures.     

Assessing the risk of foliar injury from ozone on vegetation in parks in the U.S. National Park Service's Vital Signs Network

The risk of ozone injury to plants was assessed in support of the National Park Service's Vital Signs Monitoring Network program. The assessment examined bioindicator species, evaluated levels of ozone exposure, and investigated soil moisture conditions during periods of exposure for a 5-year period in each park. The assessment assigned each park a risk rating of high, moderate, or low. For the 244 parks for which assessments were conducted, the risk of foliar injury was high in 65 parks, moderate in 46 parks, and low in 131 parks. Among the well-known parks with a high risk of ozone injury are Gettysburg, Valley Forge, Delaware Water Gap, Cape Cod, Fire Island, Antietam, Harpers Ferry, Manassas, Wolf Trap Farm Park, Mammoth Cave, Shiloh, Sleeping Bear Dunes, Great Smoky Mountains, Joshua Tree, Sequoia and Kings Canyon, and Yosemite.     

Seedling insensitivity to ozone for three conifer species native to Great Smoky Mountains National Park

Field symptoms typical of ozone injury have been observed on several conifer species in Great Smoky Mountains National Park, and tropospheric ozone levels in the Park can be high, suggesting that ozone may be causing growth impairment of these plants. The objective of this research was to test the ozone sensitivity of selected conifer species under controlled exposure conditions. Seedlings of three species of conifers, Table Mountain pine (Pinus pungens), Virginia pine (Pinus virginiana), and eastern hemlock (Tsuga canadensis), were exposed to various levels of ozone in open-top chambers for one to three seasons in Great Smoky Mountains National Park in Tennessee, USA. A combination of episodic profiles (1988) and modified ambient exposure regimes (1989-92) were used. Episodic profiles simulated an average 7-day period from a monitoring station in the Park. Treatments used in 1988 were: charcoal-filtered (CF), 1.0x ambient, 2.0x ambient, and ambient air-no chamber (AA). In 1989 a 1.5x ambient treatment was added, and in 1990, additional chambers were made available, allowing a 0.5x ambient treatment to be added. Height, diameter, and foliar injury were measured most years. Exposures were 3 years for Table Mountain pine (1988-90), 3 years for hemlock (1989-91), and 1 and 2 years for three different sets of Virginia pine (1990, 1990-91, and 1992). There were no significant (p<0.05) effects of ozone on any biomass fraction for any of the species, except for older needles in Table Mountain and Virginia pine, which decreased with ozone exposure. There were also no changes in biomass allocation patterns among species due to ozone exposure, except for Virginia pine in 1990, which showed an increase in the root:shoot ratio. There was foliar injury (chlorotic mottling) in the higher two treatments (1.0x and 2.0x for Table Mountain and 2.0x for Virginia pine), but high plant-to-plant variability obscured formal statistical significance in many cases. We conclude, at least for growth in the short-term, that seedlings of these three conifer species are insensitive to ambient and elevated levels of ozone, and that current levels of ozone in the Park are probably having minimal impacts on these particular species.     

Responses of hybrid poplar clones and red maple seedlings to ambient O(3) under differing light within a mixed hardwood forest

The responses of ramets of hybrid poplar (Populus spp.) (HP) clones NE388 and NE359, and seedlings of red maple (Acer rubrum, L.) to ambient ozone (O(3)) were studied during May-September of 2000 and 2001 under natural forest conditions and differing natural sunlight exposures (sun, partial shade and full shade). Ambient O(3) concentrations at the study site reached hourly peaks of 109 and 98 ppb in 2000 and 2001, respectively. Monthly 12-h average O(3) concentrations ranged from 32.3 to 52.9 ppb. Weekly 12-h average photosynthetically active radiation (PAR) within the sun, partial shade and full shade plots ranged from 200 to 750, 50 to 180, and 25 to 75 micromol m(-2) s(-1), respectively. Ambient O(3) exposure induced visible foliar symptoms on HP NE388 and NE359 in both growing seasons, with more severe injury observed on NE388 than on NE359. Slight foliar symptoms were observed on red maple seedlings during the 2001 growing season. Percentage of total leaf area affected (%LAA) was positively correlated with cumulative O(3) exposures. More severe foliar injury was observed on plants grown within the full shade and partial shade plots than those observed on plants grown within the sun plot. Lower light availability within the partial shade and full shade plots significantly decreased net photosynthetic rate (Pn) and stomatal conductance (g(wv)). The reductions in Pn were greater than reductions in g(wv), which resulted in greater O(3) uptake per unit Pn in plants grown within the partial shade and full shade plots. Greater O(3) uptake per unit Pn was consistently associated with more severe visible foliar injury in all species and/or clones regardless of differences in shade tolerance. These studies suggest that plant physiological responses to O(3) exposure are likely complicated due to multiple factors under natural forest conditions.