Photosynthetic parameters as early indicators of ozone injury in apple leaves

Ozone may affect leaf photosynthesis even before visible symptoms become apparent. This study had the objective to test several parameters of chlorophyll fluorescence and leaf gas exchange for their usefulness as indicators of latent ozone injury in the field. Container-grown apple trees (Malus domestica Borkh. cv. Golden Delicious) were exposed to four different ozone levels in open-top chambers. Identical leaves were analyzed in fixed-time intervals for the characteristics of fast fluorescence induction kinetics in vivo. By using high-time resolution, characteristic parameters describing the early photochemical events could be calculated according to the JIP-test. Parameters responsive to the different ozone treatments showed clear dependence on the accumulated ozone dose. Ozone exposure immediately preceding the measurements was more important for the extent of the physiological effects than the total accumulated ozone dose. The most sensitive parameters were the turnover number N (indicating how many times Q_A has to be reduced for full reduction of all acceptors; positively correlated to ozone dose) and D₀, the density of reaction centres per leaf area (negatively correlated to ozone dose). Most parameters analyzed showed clearer responses to ozone on the adaxial than on the abaxial surface of the leaf. Changes in the parameter N were better correlated to ozone doses with low cut-offs (AOT00 and AOT20), whereas changes in D₀ and in the specific electron fluxes per reaction centre were mainly influenced by ozone doses with high cut-offs (AOT80 and AOT100). Leaf gas exchange analyses revealed a higher ozone sensitivity in carboxylation efficiency than in light utilization efficiency and in the rate of light-saturated net photosynthesis. All ozone-induced photosynthetic effects were observed in leaves showing no sign of visible leaf injury. This study identified fluorescence parameters that could be useful for rapid monitoring and early detection of latent leaf injury by ozone.     

The response of sensitive and tolerant clones of Populus tremuloides to dynamic ozone exposure under controlled environmental conditions

Both sensitive and tolerant clones of aspen ( Populus tremuloides ) were exposed to ozone using four different exposure regimes under controlled environmental conditions. Based on data on ambient ozone from 10 cities in the USA, three treatments of 4-wk exposure to the same SUM06 (an accumulation of hourly O₃ concentrations greater than 0.06 ml l⁻¹) were constructed. The regimes allowed us to investigate: (a) the importance of long (3 wk, treatment 1) versus short (1 wk, treatment 2) duration of regimes with high peaks; (b) the effect of treatments with variable peak occurrence (treatments 1 and 2) versus uniform peak occurrence (treatment 3) during the exposure period. Nonfumigated control plants were maintained at ozone concentrations <10 nl l⁻¹. Bifacial black necrosis, a typical symptom of ozone injury on aspen leaves, occurred on both clones after 2 wk exposure. Up to 60% of the leaves on the sensitive clone were injured, with an average of 6% of total leaf area injured. In the tolerant clone only 10% of the leaves were injured, with less than 1% of the total leaf area symptomatic. The severity of injury was consistently greatest in treatment 2, followed by treatments 1 and 3, respectively. The interval between peak exposures was less important than the occurrence of peaks versus a stable maximum concentration. Premature leaf abscission occurred in the sensitive clone. Measures of gas exchange demonstrated reduced photosynthesis under ozone fumigation, but exposure regime was not a significant factor. Concentrations of two antioxidants, ascorbic acid and glutathione, were almost always greater in the resistant than in the sensitive clone, but the differences were not statistically significant. The levels of these antioxidants in aspen leaves did not change with ozone fumigation or leaf age.     

Water Stress Reduces Ozone Injury via a Stomatal Mechanism

Various studies have shown that water-stressed plants are more tolerant of ozone exposures than are unstressed plants. Two probable explanations for this tolerance are (a) stomatal closure which reduces ozone uptake and (b) biochemical or anatomical changes within the leaves. Phaseolus vulgaris cv Pinto bean plants were established and transferred to membrane systems which controlled the osmotic potential around the roots at −35 or −80 kilopascals for 5 days prior to ozone treatment (0 or 1.0 microliters per liter for 2 hours). Both water-stressed and unstressed plants were sprayed with various concentrations of abscisic acid to close the stomata or with fusicoccin to induce stomata opening. The abaxial stomatal resistances of primary and trifoliate leaves were measured just prior to ozone exposure. Plant response to ozone was determined by stress ethylene production and chlorophyll loss. Both water stress and abscisic acid induced stomatal closure and reduced ozone injury. In water-stressed plants, fusicoccin induced stomatal opening and those plants were as sensitive to ozone as were the non-water-stressed plants. These data suggest that water stress protects plants from ozone injury mainly through its influence on stomatal aperture rather than through biochemical or anatomical changes.     

Depression of Photosynthesis, Growth, and Yield in Field-Grown Green Pepper (Capsicum annuum L.) Exposed to Acidic Fog and Ambient Ozone

The relationship among physiological, injury, growth, and yield responses was examined in field-grown green pepper (Capsicum annuum L. `California Wonder') subjected to two airborne environmental stresses. The primary objectives were to determine if the stresses could cause alterations in the plant responses, and to determine if any stress induced alterations in physiological or injury responses were correlated with effects on growth or yield. Responses were monitored in green pepper exposed to simulated acidic fog alone, or in combination with ambient concentrations of ozone in open-top field chambers. Both highly acidic fog and ambient ozone depressed green pepper growth and yield responses via the inhibition of photosynthesis. Applications of highly acidic fog (i.e. two exposures of pH 1.68 fog per week for 11 weeks) caused a significant depression of net photosynthesis, reduction in leaf buffering capacity, and an extensive amount of leaf injury. These alterations closely paralleled decreases in growth and yield on a percentage basis. In contrast, ambient ozone had similar impacts on net photosynthesis, growth and yield, but enhanced leaf buffering capacity, and caused no visible injury. The pollutant-specific differences in plant response are discussed with respect to whole-plant carbon metabolism and physiological compensation.     

Differential ozone sensitivity of rice cultivars as indicated by visible injury and grain yield

Surface ozone pollution may cause reductions in rice ( Oryza sativa L.) yield. Ozone sensitivity in rice cultivars is often evaluated based on visible leaf injury at an early growth stage. However, it is not clear whether reduction in grain yield is related to visible injury. Therefore, visible damage and grain yield reduction were examined in Japanese and Asian rice cultivars exposed to ozone. In experiment 1, 3-week-old rice seedlings were exposed to ozone (min.: 20 nl·l⁻¹, max.:120 nl·l⁻¹) for 12 h in open-top chambers (OTCs). Visible leaf injury was quantified according to a leaf bronzing score. In experiment 2, rice plants were exposed to ozone in OTCs throughout the cropping season until grain harvest. Daily mean ozone concentrations were maintained at 2, 23, 28, 42, and 57 nl·l⁻¹ with a regular diurnal pattern of exposure. After harvest, grain yield was determined. Based on visible injury to the uppermost fully expanded leaf, the indica cultivar 'Kasalath' was most tolerant, and the japonica cultivar 'Kirara 397' was most sensitive to ozone. However, grain yields for both 'Kasalath' and 'Kirara 397' were significantly decreased after ozone exposure. The indica cultivar 'Jothi' suffered severe injury after ozone exposure but had no reduction in grain yield. Therefore, ozone sensitivity of rice cultivars evaluated by visible injury did not coincide with that evaluated by the reductions in grain yield. These results suggest that mechanisms that induce acute leaf injury do not relate to chronic ozone toxicity that reduces yield.     

Response of subterranean clover (Trifolium subterraneum) to ozone in relation to plant age and light conditions during exposure

Potted subterranean clover ( Trifolium subterraneum ) plants of different ages were exposed to 70 nl l⁻¹ ozone for 6 h, either during the light or during the dark period in a laboratory-based climate chamber. There was limited visible leaf injury on plants which were 14–20 and 28–34 d old and no significant decrease in biomass after daytime ozone exposure. The oldest leaves of 22–26 d old plants exhibited severe visible injury, which was associated with a significant reduction in biomass in 24–26 d old plants. Thus, ozone-induced visible injury of different magnitude developed in all plants, but was associated with biomass reduction only during a limited period of the plant's life-span. Apart from modifying ozone uptake by plants, climatic conditions are important as growth modifiers. It is suggested that subterranean clover plants of defined developmental stages should be used in bioindication of ozone. Night ozone exposure injured significantly fewer leaves than day exposure. However, some leaves developed visible injury even after night ozone exposure. Night uptake of ozone may be of more importance in northern than in central and southern Europe, because summer nights are short and, for a certain period, never completely dark.     

EVIDENCE FOR THE REPAIR OF OZONE-INDUCED MEMBRANE INJURY

The increase in [¹⁴C]-2-deoxy-D-glucose uptake by leaf discs 24 hr after fumigation was used as a measure of ozone injury to pinto bean leaves (Phaseolus vulgaris). This method showed that the primary leaves were most affected by ozone when plants were 10 to 12 days old, which coincided with the time of maximum leaf necrosis. However, 8-day-old plants, which had no visible leaf injury, still showed a higher uptake rate than controls, indicating that injury occurred at the cellular level. In these younger plants, uptake was shown to return to normal over a 5 day period. Moreover, the rate of this “repair” was retarded by cold or continuous darkness, enhanced by continuous light, and very markedly increased by glucose applied to the leaves. The timing of the glucose application was not critical, nor did H₂O or mannitol have an effect. The results suggest that ozone injury at the cellular level can be repaired by energy-dependent processes so that necrosis of the leaf tissue does not occur. These experiments also show that conditions and treatments after ozone exposure can alter the degree of ozone injury.     

OZONE SENSITIVITY OF LEAVES: RELATIONSHIP TO LEAF WATER CONTENT,GAS TRANSFER RESISTANCE,AND ANATOMICAL CHARACTERISTICS

Relative water content, resistance to gas transfer, stomatal spacing, and other characteristics of primary bean leaves were studied in relation to ozone sensitivity and injury. Cells of primary bean leaves are maximally sensitive to ozone exposure 9–10 days after germination under our experimental conditions. The stage of maximum sensitivity was not correlated with changes in stomatal number or resistance on either adaxial or abaxial leaf surfaces. It was deduced that bean leaf sensitivity was a function of more internal circumstances, and gas exchange was never the limiting factor through the developmental period studied. Changes in resistance were not significantly altered by ozone levels that produced no visible injury. After exposure to high ozone doses, a decrease in adaxial resistance occurred apparently as a result of palisade and epidermal cell lysis. Normally most gas exchange occurs through the adaxial surface. A 10 % decrease in relative water content accompanying a 60-min ozone exposure of 0.55 ppm could not be explained physiologically on the basis of cell injury as no visible leaf injury occurred.     

Stable free radicals in ozone-damaged wheat leaves

Chlorophyll fluorescence measurements were performed on attached leaves of wheat plants (Triticum aestivum L. cv. Nandu) that were exposed to ambient air and to air supplemented with 80 and 120nmol mol^-1 ozone. Decreases in the “current photochemical capacity” were observed that were dependent on both the ozone concentration and duration of exposure. Electron paramagnetic resonance (EPR) spectra on freeze-dried samples from the same batches of plants showed the presence of an unidentified stable free radical, whose spectra had similarities to that of the ubisemiquinone radical. The intensity of this radical signal increased with the duration of ozone exposure in leaves that received an additional 120nmol mol^-1 ozone. In contrast, with exposure to air with 80nmol mol^-1 added ozone, there was little if any change in free radical signal intensity over the 4 week period of the experiment. The increase in intensity of the EPR signal occurred later than the chlorophyll fluorescence changes, which suggests that it is associated with permanent leaf damage.     

Stable free radicals in ozone-damaged wheat leaves

Chlorophyll fluorescence measurements were performed on attached leaves of wheat plants (Triticum aestivum L. cv. Nandu) that were exposed to ambient air and to air supplemented with 80 and 120nmol mol^-1 ozone. Decreases in the “current photochemical capacity” were observed that were dependent on both the ozone concentration and duration of exposure. Electron paramagnetic resonance (EPR) spectra on freeze-dried samples from the same batches of plants showed the presence of an unidentified stable free radical, whose spectra had similarities to that of the ubisemiquinone radical. The intensity of this radical signal increased with the duration of ozone exposure in leaves that received an additional 120nmol mol^-1 ozone. In contrast, with exposure to air with 80nmol mol^-1 added ozone, there was little if any change in free radical signal intensity over the 4 week period of the experiment. The increase in intensity of the EPR signal occurred later than the chlorophyll fluorescence changes, which suggests that it is associated with permanent leaf damage.     

Minor effects of long-term ozone exposure on boreal peatland species Eriophorum vaginatum and Sphagnum papillosum

The effects of long-term ozone fumigation on two common peatland plant species, a sedge Eriophorum vaginatum L. and a moss Sphagnum papillosum Lindb., were studied applying peatland microcosms. The peat cores with intact vegetation were cored from an oligotrophic pine fen and partially embedded into the soil of an open-air experimental field for four growing seasons. The open-air ozone exposure field consists of eight circular plots of which four were fumigated with elevated ozone concentration (doubled ambient) and four were ambient controls. The results showed that E. vaginatum and S. papillosum can tolerate ozone better than expected. Elevated ozone concentration did not affect overall relative length growth of E. vaginatum or S. papillosum. The leaf cross-section area of E. vaginatum leaves was 8% bigger in the ozone treatment compared to that in the ambient control. Ultrastuctural variables did not show any significant treatment effect in E. vaginatum or in S. papillosum. Total chlorophyll (a + b) concentration tended to increase in early growing season under ozone exposure. During the first growing season, elevated ozone concentration decreased methanol-extractable, UV-absorbing compounds in E. vaginatum. The results suggest that E. vaginatum and S. papillosum are ozone tolerant plant species and are likely able to cope with expected increase in tropospheric ozone concentration.     

Stomatal Closure During Tobacco Leaf Desiccation as Affected by Ozone

The effect of acute ozone exposure on the stomatal conductance and leaf water content during rapid desiccation was examined in leaves of two tobacco cultivars, ozone sensitive cv. BelW3 and ozone tolerant cv. Samsun. The relative rate of stomatal closure was constant during leaf desiccation in cv. Samsun but decreased in cv. BelW3 in both ozonated and control plants. Ozone exposure increased the relative rate of stomatal closure and transpiration rate (measured on the following day) in cv. Samsun, but reduced the respective parameters in cv. BelW3. As a result, the plants of ozone-sensitive cultivar, treated with ozone, lost more water during desiccation than control plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ozone increases the permeability of isolated pea chloroplasts

The effect of short-term exposure of chloroplasts isolated from the leaves of Pisum sativum to high concentrations of ozone was examined. The inhibitory effect of O₃ on endogenous photophosphorylation was apparently related to an increased permeability of the chloroplast limiting membranes induced by ozone exposure. A 5 min treatment with 50 ppm O₃ reduced the reflection coefficient of meso-erythritol from 0.84 to 0.58 and that of glycerol from 0.26 to 0.03. Such decreases in reflection coefficients indicate that ozone caused a marked increase in the permeability of the limiting membranes of the chloroplasts, which may result from an oxidation of membrane lipids. The decrease in the reflection coefficient of meso-erythritol was proportional both to ozone concentration (up to 30 ppm for 5 min of bubbling) and to time (up to 5 min at 30 ppm). Extrapolating these results to lower concentrations and longer times, ozone injury should be possible for a 2 hr exposure of plants to 0.3 ppm ozone, as is indeed the case.     

Inflammatory and Repair Pathways Induced in Human Bronchoalveolar Lavage Cells with Ozone Inhalation

Background

Inhalation of ambient levels of ozone causes airway inflammation and epithelial injury.

Methods

To examine the responses of airway cells to ozone-induced oxidative injury, 19 subjects (7 with asthma) were exposed to clean air (0ppb), medium (100ppb), and high (200ppb) ambient levels of ozone for 4h on three separate occasions in a climate-controlled chamber followed by bronchoscopy with bronchoalveolar lavage (BAL) 24h later. BAL cell mRNA expression was examined using Affymetrix GeneChip Microarray. The role of a differentially expressed gene (DEG) in epithelial injury was evaluated in an in vitro model of injury [16HBE14o- cell line scratch assay].

Results

Ozone exposure caused a dose-dependent up-regulation of several biologic pathways involved in inflammation and repair including chemokine and cytokine secretion, activity, and receptor binding; metalloproteinase and endopeptidase activity; adhesion, locomotion, and migration; and cell growth and tumorigenesis regulation. Asthmatic subjects had 1.7- to 3.8-fold higher expression of many DEGs suggestive of increased proinflammatory and matrix degradation and remodeling signals. The most highly up-regulated gene was osteopontin, the protein level of which in BAL fluid increased in a dose-dependent manner after ozone exposure. Asthmatic subjects had a disproportionate increase in non-polymerized osteopontin with increasing exposure to ozone. Treatment with polymeric, but not monomeric, osteopontin enhanced the migration of epithelial cells and wound closure in an α9β1 integrin-dependent manner.

Conclusions

Expression profiling of BAL cells after ozone exposure reveals potential regulatory genes and pathways activated by oxidative stress. One DEG, osteopontin, promotes epithelial wound healing in an in vitro model of injury.     

Use of Bel-W3 Tobacco as indicator plant for atmospheric ozone during different seasons in the coastal zone of Israel

Bel-W3 Tobacco, which is highly sensitive to ozone, was grown in two glass chambers and exposed to the ambient air at the periphery of Tel-Aviv, during winter, spring, summer and autumn 1978. During the exposure time, atmospheric ozone was continuously measured by a chemiluminescent monitor. Throughout the experiments, plants' height was measured and the number of leaves was determined three times weekly. The extent of injury to the tobacco plants was measured by the percentage of injured plants, the percentage of injured leaves and the percentage of leaves' area injured. Necrotic lesions, typical for ozone injury, appeared on the mature leaves of the exposed tobacco plants in three out of four exposures. Appearance of incipient injury differed among the experiments and depended not only on exposure duration and on ozone concentrations, but also on the exposure conditions (like light intensity, temperature and humidity), which considerably influenced the appearance of the injury. The percentage of injured leaves and the percentage of leaves' area injured, increased with the duration of exposure and with rising cumulative ozone concentrations.Presented at the Eighth International Congress of Biometeorology 9–14 September 1979, Shefayim, Israel.     

Assessment of Atmospheric ozone levels in Israel through foliar injury to Bel-W3 tobacco plants

Summary Nicotiana tabacum L. Bel-W3, which is highly sensitive to ozone, was grown in two glass chambers and exposed to the ambient air at the periphery of Tel-Aviv, during winter, spring, summer and autumn 1978. During the exposure time, atmospheric ozone was continuously measured by a chemiluminescent monitor. Throughout the experiments, plants' height was measured and the number of leaves was determined three times weekly. The extent of injury to the tobacco plants was measured by the percentage of injured plants, the percentage of injured leaves and the percentage of leaves' area injured. Necrotic lesions, typical for ozone injury, appeared on the mature leaves of the exposed tobacco plants in three out of four exposures. Appearance of incipient injury and the extent of injury differed among the experiments and depended not only on exposure duration and on ozone concentrations, but also on the exposure conditions. The percentage of injured leaves and the percentage of leaves' area injured, increased with the duration of exposure and with rising cumulative ozone concentrations.This work was partially supported by the Chief Scientist's Office—Israeli Ministry of Health—Contract No. 550     

Elevated ozone modifies the feeding behaviour of the common leaf weevil on hybrid aspen through shifts in developmental, chemical, and structural properties of leaves

In this study, we tested the impact of moderately elevated ozone (O₃) – 1.5 × ambient, equivalent to predicted near-future ozone concentrations – on the feeding behaviour of the common leaf weevil Phyllobius pyri L. (Coleoptera: Curculionidae), on two hybrid aspen [ Populus tremula  × Populus tremuloides (Salicaceae)] clones (clones 55 and 110) differing in ozone sensitivity using the open-air ozone exposure site in Kuopio, Finland. Three host-selection tests (test between treatments, test between clones, and test between treatments* clones) with common leaf weevil females were carried out in the laboratory in the 2nd year of ozone exposure. The beetles were offered two (four for the tests between treatments and clones) freshly cut leaf discs from first flush leaves. After 24 h, the beetles were removed and the leaf disc area consumed was measured. In the field, the unfolding of the buds was followed and samples were taken for anatomical and chemical (salicylates, condensed tannins, nitrogen, and water content) leaf analyses. Phyllobius pyri significantly preferred leaves from clone 55 to those from clone 110 in the ambient air treatment, whereas this preference was less evident under elevated ozone. Leaves from ozone-exposed trees were significantly preferred to leaves grown in ambient air. Our results suggest that the preference of clone 55 and of ozone-exposed leaves can be explained by phenotypic properties of the plant and prevailing ozone concentration through shifts in leaf development process, phenolic composition, and leaf thickness.     

OZONE-INDUCED MEMBRANE PERMEABILITY CHANGES

The effect of 0.5 ppm ozone for 0.5-1 hr on plant cell membrane permeability was ascertained. Permeabilities to both water and solutes were estimated by measuring leaf disc weight changes and following tritiated water and ⁸⁶Rb fluxes. Measurements were made immediately after ozone exposure and 24 hr after exposure. The reflection coefficient, σ, an index of solute permeability, decreased in ozone-treated primary leaves of pinto bean (Phaseolus vulgaris). The latter indicates an increase in membrane solute permeability or internal solute leakage. Water and THO flux estimates both indicated a decrease in membrane permeability to water; both the hydraulic conductivity (L_p) and the water diffusional coefficient (L_D) apparently decreased, an anomaly which is discussed. These data indicate that ozone has a direct effect on membrane function by altering permeability characteristics. We assume from these data that cell membranes are primary target sites for ozone injury.     

Studies on cellular resilience and adaptation following acute and repetitive exposure to ozone in cultured human epithelial (HeLa) cells

Ozone is used to treat several medical conditions, while the underlying mechanisms of action are sometimes poorly understood. In the current study, we exposed cultured human epithelial (HeLa) cells acutely and repeatedly to ozone and investigated the effects thereof on cell viability. The involvement of anti-apoptotic pathways in observed adaptive responses to ozone were investigated by employing the Akt inhibitor (-)-deguelin. Cells were exposed to an ozone-saturated physiological solution using various dosing regimens, including acute exposure and various repetitive exposures. Cell viability was determined with Trypan Blue or MTT tests, or by a DNA-fragmentation (comet) assay. Acute ozone exposure compromised cell membrane integrity severely, while adaptation to reverse an initial reduction in mitochondrial activity was observed. Repetitive, short-duration exposures followed by a single long-duration exposure to ozone furnished a protective adaptation that was reversed by Akt inhibition. Extracellular and intracellular damage (and adaptation) occurs differentially. While acute ozone may decrease cell viability, multiple preexposures up-regulates cellular plasticity via induction of anti-apoptotic pathways in a treatment regimen-specific manner.