Ozone Injury Research Articles

Foliar ozone injury and radial growth of ponderosa pine

Foliar injury symptoms and stem diameter growth were measured on well-watered and drought-stressed ponderosa pine (Pinusponderosa Dougl. ex Laws.) seedlings at the end of each of 3 years of exposure to three levels of ozone: charcoal-filtered air, nonfiltered air, and nonfiltered air plus 1.5 times ambient ozone. Ozone-injury indices were constructed by adding percent chlorotic mottle and percent necrosis for each needle age-class. Percent necrosis was weighted from 1 to 5 times to construct different indices. Seedlings grown in nonfiltered air plus 1.5 times ambient ozone developed severe foliar injury after 2 years of exposure and were the only seedlings with significant reductions in radial growth after three seasons of exposure to a mean seasonal ozone concentration of 88 ppb. Foliage that had developed >30% chlorotic mottle by September of the 2nd year had abscised by September of the following year. Reduction in radial growth was significantly correlated with amount of foliar injury in well-watered trees, and the best-fit regression equation occurred when percent necrosis was weighted by a factor of 4. Regressions between foliar-injury indices and radial growth in drought-stressed trees were not significant, nor were regressions between radial growth and foliar injury among well-watered trees with only 1 year of premature needle abscission. The low R2 (0.30) between foliar injury and radial growth in well-watered trees and the lack of a significant regression in drought-stressed trees suggest that detection of reductions in stem diameter growth of ponderosa pine in the field in response to ozone injury could be difficult, except for severely injured trees with fewer than 2 years of foliar retention.

Coordinated expression of a 45 kD protein and ozone toxicity in a human bronchial epithelial cell line.

The human bronchial epithelial cell line, BEAS-2B, which was immortalized by transformation with SV40 virus, when grown biphasically between 0.1 and 1.0 ppm of ozone and liquid medium showed increased release of Cr, decreased synthesis of various macromolecules, and decreased cell viability. Cell injury was a function of the concentration of ozone to which the cells were exposed. Furthermore, in proportion to the extent of cell injury, ozone exposure also induced and/or enhanced synthesis of a 45 kD protein but not any of the well-characterized heat shock proteins, e.g., HSP 70. Actinomycin D prevented enhanced synthesis of the 45 kD protein in cells exposed to ozone, suggesting transcriptional regulation of expression of the 45 kD protein. Enhanced synthesis of the 45 kD protein was not observed in cells treated with heat, cigarette smoke condensate, hydrogen peroxide, or bleomycin. High concentrations of glutathione added to the culture medium reduced ozone toxicity and ozone-enhanced synthesis of the 45 kD protein. These results suggest that ozone injury and enhanced expression of a gene encoding a 45 kD protein of as yet unknown function are coordinated in the SV40-immortalized bronchial epithelial cells.

Effects of air pollutant emissions from a rural motorway on Petunia and Trifolium

Potted plants of Petunia hybrida (indicator plant for ethene), Trifolium subterraneum (indicator plant for ozone), and Trifolium pratense were grown downwind from a motorway with approximately 30 000 vehicles/day. Petunia plants were grown with two different levels of nutrient supply. Pots with Petunia plants were placed at 10, 20, 40, 80 and 120 m distances from the motorway. The flowers of petunia were significantly smaller close to the motorway. The percentage of ripened fruits was higher close to the motorway, indicating that flowering proceeded faster close to the motorway. The number of aborted flower buds was also greater close to the motorway. Dry weight and nitrogen content did not vary systematically with the distance from the motorway, but were significantly higher in the plants treated with higher nutrient supply. Clover plants were placed at 10, 20, 40, 80 and 200 m distances from the motorway. Towards the end of the experiment, after ozone episodes, visible injury was recorded. This kind of injury was significantly less close to the motorway compared to the 200 m distance. This effect is explained by the fast reaction between nitrogen monoxide and ozone, causing a local depression in the ozone concentration in the vicinity of the motorway. The extent of ozone injury was less in Trifolium pratense compared to T. subterraneum. A type of visible leaf injury, clearly distinguishable from the ozone injury, was observed in Trifolium subterraneum. This second kind of visible injury declined strongly with increasing distance from the motorway, and was almost absent at 200 m distance.

Ozone Injury to Alveolar Epithelium in Vitro Does Not Reflect Loss of Antioxidant Defenses

The objective of this study was to characterize an in vitro model of oxidant gas toxicity, using primary cultures of alveolar type II cells maintained in serum-free medium, by evaluating (1) epithelial barrier function, (2) the stability of cellular antioxidant defenses, and (3) the response of alveolar epithelial barrier properties to ozone exposure. Antioxidant enzyme activities and glutathione levels were measured in rat type II cells that were freshly isolated, cultured for 1 day in serum-supplemented medium, and subsequently grown in serum-free nutrient medium. After measurement of peak bioelectric properties on Day 4 in primary culture, alveolar epithelial monolayers were exposed to ozone at various concentrations and lengths of exposure. Ozone-induced alterations in monolayer bioelectric properties and impairment of cellular organization were used to evaluate oxidant injury. The primary effect of ozone exposure was a dose-dependent increase in monolayer permeability, which resulted from damage to intercellular junctions and/or loss of epithelial integrity. Extensive and persistent permeability increases correlated with focal areas of epithelial degradation. The focal nature of ozone injury to alveolar epithelium in vitro suggests that individual cell susceptibility to oxidant stress may account for the overall decrement in barrier function. However, this sensitivity does not result from overall loss of antioxidant defenses associated with cell culture, as these monolayers (when cultured in serum-free medium) maintained their antioxidant enzyme activities and glutathione content at levels found in freshly isolated cells. We conclude that the sensitivity of these monolayers to ozone injury in vitro reflects a disproportionate degree of oxidant stress on cell membranes relative to intracellular antioxidant defenses, i.e., cellular susceptibility to oxidant injury may depend on the ratio of the surface area of the cell to its cytoplasmic volume.

Ozonation of DNA forms adducts: a 32P-DNA labeling and thin-layer chromatography technique to measure DNA environmental biomarkers.

Little direct documented evidence of ozone's genotoxicity exists. Deoxyribonucleic acid (DNA) adducts are produced by environmental toxic agents, including ozone. We have described a modified thin-layer chromatography (TLC) technique that can assess adduct formation as a biomarker of ozone injury. This requires 32P-labeling DNA, digestion of deoxynucleotides (dNMPs), and separation in two-dimensional PEI-cellulose TLC. We have applied this technique to control DNAs, to control DNA in solution exposed to acute ambient ozone, and to control DNA exposed to acute bubbled-through ozone (2 ppm for 24 h). We detected stable DNA adducts, including hydroxymethyluracil (HMU), thymine glycol (TG), 8-hydroxyguanine (8-OHG), and demonstrated, as yet, unidentified adducts that may serve as a "fingerprint" pattern of DNA adduction. This technique quantifies low-molecular-mass DNA adducts, both in vivo and in vitro, with potential applications to environmental toxicology.

Elevated Levels of Superoxide Dismutase Protect Transgenic Plants Against Ozone Damage

To evaluate the feasibility of using engineered antioxidant enzymes as an approach to improve the tolerance of plants to ambient stress, we have constructed transgenlc tobacco plants that overproduce superoxide dismutase (SOD), an enzyme which converts superoxide radicals into hydrogen peroxide and oxygen, and is believed to play a crucial role in antioxidant defense. We have targeted the MnSOD from Nicotiana plumbaginifolia either to the chloroplasts or to the mitochondria, and evaluated the ozone tolerance of transgenic and control plants. Enhanced SOD activity in the mitochondria had only a minor effect on ozone tolerance. However, overproduction of SOD in the chloroplasts resulted in a 3–4 fold reduction of visible ozone injury.

Studies on the role of active oxygen in ozone injury to plant cells. I. Generation of active oxygen in rice protoplasts exposed to ozone

The generation of active oxygen in ozone-treated rice protoplasts was measured with a view to examining the initial phases of injury in plants exposed to ozone. The production of superoxide anions was determined in protoplast suspensions on the basis of the rate of formation of adrenochrome from epinephrine following treatment with air including 0 or 8.0 ppm ozone. Hydroxyl radicals were measured by determining the rate of bleaching of p-nitrosodimethylaniline. The rates of both formation of adrenochrome, and bleaching of p-nitrosodimethylaniline, were less in the ozone-treated protoplasts than in those treated with air alone. On the other hand, an increase in the production of malondialdehyde (MDA) and a decrease in sulfhydryl groups (SH) was more noticeable in the ozone-treated protoplasts, while the amount of protein and chlorophyll did not change. The change in the amount of MDA and SH groups is presumed to be due to the influence of direct reactions with the ozone molecule.

Effects of various ozone exposures on the susceptibility of bean leaves ( Phaseolus vulgaris L.) to Botrytis cinerea

The effects of various ozone exposures in predisposing bean leaves ( Phaseolus vulgaris L.) to Botrytis cinerea have been investigated under laboratory conditions. Seedlings of two bean cultivars were exposed to incremental ozone concentrations (120, 180 and 270 μg m −3 for 8-h day −1) for five days and primary leaves were subsequently inoculated with conidia suspended in water or in an inorganic phosphate solution (Pi), and with mycelium. Ozone injury increased with increasing ozone concentration and was much higher in the ozone-sensitive cultivar ‘Pros’ than in the ozone-insensitive ‘Groffy’. Ozone only increased the number of lesions on leaves of Pros after inoculation with either of the conidial suspensions. The Pi-stimulated infection in Groffy was reduced by the lower ozone concentrations. Ozone decreased lesion expansion after inoculation with mycelium. In a chronic fumigation experiment, plants of the two cultivars were exposed to 90 μg m −3 (7-h day −1) and the primary and the oldest tree trifoliate leaves were inoculated after five and seven weeks of exposure. Ozone enhanced the senescence-related injury only in Pros. The number of lesions was not influenced by ozone for either cultivar, conidial suspension or inoculation date. Lesion expansion after inoculation with mycelium was generally reduced in exposed plants. Thus, contrasting effects of ozone on the susceptibility of bean leaves to B. cinerea were observed depending on the cultivar, the conidial suspension, the disease parameter and the ozone exposure pattern. In extrapolating the laboratory results to the field, it is suggested that episodic and chronic exposures to ambient ozone are of minor importance in increasing the susceptibility of bean leaves to B. cinerea.

Studies on the role of active oxygen in ozone injury to plant cells. II. Effect of antioxidants on rice protoplasts exposed to ozone

The effects of antioxidants, ascorbic acid, hydroquinone and mannitol, on ATPase activity in cellular membranes and on the formation of malondialdehyde in rice protoplasts treated with air or ozone in vitro, were examined. All three substances restrained the increase in malondialdehyde (MDA) during the treatment with both air and ozone, and ascorbic acid and hydroquinone were more effective on the protoplasts treated with ozone than those treate With regard to mannitol, however, no difference was noticed in the decrease in MDA between protoplasts treated with ozone and those treated with air. Only ascorbic acid was effective in maintaining ATPase activity in cellular membranes. The addition of ascorbic acid during ozone treatment restrained the decrease the ATPase activity by half compared with treatment without ascorbic acid.

Variation in Antioxidant Enzyme Activities in Anatomic Subcompartments within Rat and Rhesus Monkey Lung

Antioxidant enzymes including catalase, superoxide dismutase, glutathione peroxidase, and glutathione S-transferases are thought to be the primary cellular defense against reactive oxygen species. Since pulmonary injury produced by oxidant air pollutants like ozone is highly focal, involving primarily the trachea and centriacinar areas of the lung, measurements of alterations in antioxidant enzyme activities in whole lung may substantially underestimate changes occurring in target areas of the respiratory tract. We have applied a technique for preparation of lung specimens from well-defined anatomic locations to determine whether the focal injury associated with ozone exposure is related to an uneven distribution of antioxidant enzyme activity in the respiratory tract. Our study compared enzyme activities in rat and monkey, species which differ considerably in sensitivity to ozone-induced injury (monkey > rat). The activities of glutathione S-transferase varied less than twofold between different airway subcompartments for both the rat and monkey. Pulmonary veins had approximately 50% of the activity of airways in both species. Glutathione peroxidase activity was slightly higher in proximal compared to distal airways of the rat but was evenly distributed at all airway levels in the monkey. In both species, activity in pulmonary veins was lower than that in airways. The activity of superoxide dismutase was similar in rat and monkey and marked differences were not observed in the various subcompartments studied. Similarly, catalase activity was relatively evenly distributed in rat airways but, in the monkey, the distal bronchiole and lobar bronchus had marginally higher activity than the trachea. We conclude that: (1) measurement of antioxidant enzyme activities in anatomic subcompartments within the lung is feasible using microdissected specimens, (2) antioxidant enzyme activity can vary in different subcompartments of the lung of the same species. (3) the pattern of variation in enzyme activity differs by the enzyme and by species, and (4) species and subcompartment differences in ozone injury are not due primarily to differences in the distribution of antioxidant enzyme activity.

Histological effects of ozone on slash pine ( Pinus elliotti var. Densa)

Ozone is present in much of the United States and has been shown to reduce growth and development of some coniferous tree species. Slash pine ( Pinus elliotti) is a unique species which occurs naturally only in southern Florida and several of the Lower Keys. The purpose of this research was to determine if mesophyll cells of slash pine needles respond to ozone in a manner similar to that of other conifers, to determine exposure—response relationships of mesophyll cell injury and ozone exposure level, and if needles of native trees that exhibit chlorotic mottle have more mesophyll cell death than asymptomatic needles. Fumigations were performed at four ozone levels with 7-hr mean values of 0, 55, 67, and 71 ppb for 84 days. Results show that ozone injury symptoms occur to mesophyll cells of needles before visible needle injury (chlorotic mottle). The predominant injury to needles was cell necrosis. Ozone exposure—response relationships of exposure (ppm-hr) percentage of dead cells showed a highly statistically significant ( P < 0.0001) linear relationship of y = 3.56+0.58 ( x) with a correlation coefficient of 0.86. These results support the conclusion that mesophyll cells of needles of slash pine are very sensitive to ozone. Results presented also indicate that needles from native slash pine trees at Everglades National Park show chlorotic mottle, similar to that exhibited by many conifers, and that such visible injury was generally related to a higher percentage of cell necrosis compared with needles without symptoms.

A comparison of sensitivity to ozone in seedlings and trees of Quercus rubra L.

Assessments of ozone sensitivity in tree species have been based primarily on studies of Seedlings without any knowledge of the similarities between seedling and mature tree responses. To determine if seedlings and trees responded similarly to ozone, 2-yr-old seedlings and 30-yr-old trees of Quercus rubra L. were fumigated with subambient, ambient and twice-ambient ozone for one growing season (cumulative doses of 18, 45 and 87 ppm h-1 , respectively). Carboxylation efficiency, apparent quantum yield and light-saturated net photosynthesis of leaves from the lower canopy of the 30-yr-old trees were reduced by ambient and twice-ambient ozone treatment. However, physiological characteristics of leaves from the upper canopy of trees showed no signs of ozone injury. Canopy leaf dry weight of trees was reduced by the twice-ambient ozone treatment through reductions in leaf area rather than specific leaf weight, but bole diameter and branch growth of trees were unaffected by ozone treatment. In contrast, ozone did not influence leaf physiology of second flush leaves, stem diameter, and height and biomass growth of 2-yr-old seedlings. These results indicate that for deciduous hardwood species, studies of seedlings may underestimate the ozone sensitivity of larger and more physiologically mature trees. For large trees with developed canopies, leaf position within the canopy may influence the sensitivity of physiological processes to ozone.

Changes in susceptibility of bean leaves (Phaseolus vulgaris) to Sclerotinia sclerotiorum and Botrytis cinerea by pre-inoculative ozone exposures

The effects of ozone on the susceptibility of leaves ofPhaseolus vulgaris toSclerotinia sclerotiorum andBotrytis cinerea have been investigated. Seedlings of one ozone-sensitive (‘Pros’) and five relatively ozone-insensitive cultivars (‘Gamin’, ‘Precores’, ‘Groffy’, ‘Narda’, ‘Berna’) were exposed to different ozone concentrations (0, 120, 180 and 270 μg m−3) for 8 h. One day after the exposures, primary leaves were detached and immediately inoculated with spores of either pathogen suspended in water or in a 62.5 mM KH2PO4 (Pi) solution. Visible ozone injury differed between the cultivars and increased with increasing ozone concentration. On the leaves of non-exposed plants, spores of the pathogens suspended in water caused very few lesions, whereas fungal pathogenicity was stimulated by addition of Pi to the inoculum. Ozone-injured leaves of all cultivars exhibited lesions after inoculation of the leaves with the pathogens suspended in water, and the number of lesions was positively correlated with the level of ozone injury for either pathogen and cultivar. The increase in susceptibility of bean leaves in response to increasing ozone concentrations was greater forB. cinerea than forS. sclerotiorum when spores were suspended in water, but was similar when the spores were suspended in Pi.

Cotton Yield Losses and Ambient Ozone Concentrations in California's San Joaquin Valley

AbstractBased on controlled experiments and simulation modeling, ozone (O3) has been estimated to cause significant yield losses to cotton. The study reported here was conducted to verify losses for ‘Acala’ cotton (Gossypium hirsutum L. ‘SJ2’) along a gradient of ambient O3 concentrations across the San Joaquin Valley in California. Cotton was grown in nonfiltered (NF) and charcoal‐filtered (CF) open‐top chambers, and ambient air (AA) at four sites during the 1988 and 1989 summer growing seasons. Cotton yields (weights of mature bolls m−2) were reduced in NF compared with CF air in general proportion to O3 concentrations across all sites and years. Greatest cotton yield losses were as Shafter in the southern part of the San Joaquin Valley (20% in 1989), and lowest losses were at Five Points in the western part of the valley (none in 1989). Ozone injury symptoms on cotton were most noticeable in areas with greatest yield losses. Linear O3 exposure vs. predicted relative yield loss models using four common exposure indices were constructed for each site and year based on the NF, CF, and AA data. All models except the second highest daily maximum concentration (2ndHDM, the current ambient air quality standard for O3) predicted yield losses comparable to those predicted with previously published models for cotton in the San Joaquin Valley. Ozone exposure indices giving more weight to higher O3 concentrations or the 7‐h daytime mean better predicted cotton yield responses over the growing season than 2ndHDM.

LIPID PEROXIDATION AND PLANT TISSUE DISORDERS

Increased solute leakage resulting from ozone injury, senescence, wounding and virus or bacteria-induced hypersensitive reaction [HR] have been related to membrane lipid peroxidation [LPX]. The genesis of LPX in various situations may derive from several different processes: a) the direct addition of O2 to unsaturated fatty acids forming hydroperoxide in an enzymatic reaction catalyzed by lipoxygenase, an enzyme widely distributed in plant seed, root, and leaf tissue or b) peroxidation by active oxygen species [AO], e.g. O2 and H2O2, that are generated during normal cellular metabolic activities. Increased production of AO could occur as the result of a metabolic change or a reduction in the amount or activities of antioxidants, i.e. ascorbic acid, vitamin E, and glutathione, and the detoxifying enzymes catalase and superoxide dismutase. The HR against plant pathogenic bacteria and viruses will be used as case histories to discuss the genesis of lipid peroxidation.

Evaluation of Vegetation Near Coal-burning Power Plants in Southwestern Pennsylvania. II. Ozone Injury on Foliage of Hybrid Poplar

The objective of this study was to determine if the incidence or severity of foliar injury induced by regional, ambient ozone was influenced by local emissions from a complex of coal-burning power plants in southwestern Pennsylvania. Plantings of an ozonesensitive hybrid poplar clone {Populus maximowizii x trichocarpa, clone NE 388) were established in 1972 at various distances and directions from the power plants. Foliar injury caused by ambient ozone was evaluated annually from 1973 to 1990 in early to mid- August. Data are presented for the 12-year period, 1979 to 1990 inclusive, for which the most complete data sets were available. Injury from ambient ozone varied spatially and temporally, but with little relationship to power plant location. There was an apparent negative relationship between emission trends and ozone-induced symptoms, but only for one power plant. The correlation between annual mean levels of ozone-induced stipple and frequency of days (per year) with a 1-hr ozone maximum exceeding ...

Interactive Effects of Ozone and Powdery Mildew on Pea Seedlings

We assessed the effects of ozone and powdery mildew infection on plant growth, ozone injury, colonization of leaves by the fungus, and pisatin content of pea (Pisum sativum) seedlings. Seedlings of cultivars Alaska and Bounty grown in the greenhouse were moved to controlled-environment growth chambers and exposed to ozone concentrations of 0.00, 0.06, and 0.12 μl L -1 for 6 h/day (1 μl L -1 =1,960 μg m -3 at standard temperature and pressure). Fumigations were performed for 5 days before, after, or both before and after inoculation with conidia of Erysiphe polygoni f. sp. pisi, the causal agent of pea powdery mildew [...]

The False Positive in Bioindicators of Air Pollution

Yellow-poplar {Liriodendron tulipifera) seedlings were fumigated with a gradient of ozone to determine what effect ozone has on the species. The fumigated seedlings displayed physical symptoms of ozone injury but a large number of the individuals from the charcoal-filtered treatment showed physical symptoms of ozone when indeed no ozone was present. This is an indication that what appears to be ozone damage on yellow-poplar may not be caused by ozone.

Protective action of acetylcysteine in experimental ozone injuries on respiratory organs

Protective action of acetylcysteine in experimental ozone injuries on respiratory organs

Ozone effects on watermelon plants at the Ebro Delta (Spain): symptomatology

Symptoms thought to be of ozone (O 3) injury were first observed at the Migjorn farm on the Ebro Delta in 1976. Since then, O 3 symptoms have been observed every growing season, including 1990. In 1987 the first account of the suspected symptoms was published by Salleras. In order to confirm the cause of the symptoms and to determine the effects on crops, an experimental field with open-top chambers was established on the farm in 1988. There were three treatments: ambient air (AA), charcoal-filtered air (CF) and non-filtered plots (NF), with five replicates each. Visible injury was followed during the growing season and the symptoms were much less developed in the CF treatment than in AA or NF treatments. It was concluded from these observations, and data on ozone concentrations, that the Spanish Mediterranean coast has a significant air pollution problem.