Protection of ash (Fraxinus excelsior) trees from ozone injury by ethylenediurea (EDU): roles of biochemical changes and decreased stomatal conductance in enhancement of growth

Treatments with ethylenediurea (EDU) protect plants from ozone foliar injury, but the processes underlying this protection are poorly understood. Adult ash trees (Fraxinus excelsior), with or without foliar ozone symptoms in previous years, were treated with EDU at 450ppm by gravitational trunk infusion in May-September 2005 (32.5ppmh AOT40). At 30-day intervals, shoot growth, gas exchange, chlorophyll a fluorescence, and water potential were determined. In September, several biochemical parameters were measured. The protective influence of EDU was supported by enhancement in the number of leaflets. EDU did not contribute its nitrogen to leaf tissue as a fertiliser, as determined from lack of difference in foliar N between treatments. Both biochemical (increase in ascorbate-peroxidase and ascorbic acid, and decrease in apoplastic hydrogen peroxide) and biophysical (decrease in stomatal conductance) processes regulated EDU action. As total ascorbic acid increased only in the asymptomatic trees, its role in alleviating O(3) effects on leaf growth and visible injury is controversial.     

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.     

Effects of ethylenediurea (EDU) on ozone-induced acceleration of foliar senescence in potato (Solanum tuberosum L.)

Experiments were conducted to examine the effects of the anti-ozonant ethylenediurea (EDU) and chronic ozone (O3) exposure on leaf physiology and senescence in an O3-sensitive potato cultivar (Solanum tuberosum L. cv. Norland). A dose-response experiment showed that an EDU concentration of 15 mg l(-1) soil (given as a soil drench) provided complete protection from accelerated foliar senescence induced by exposure to 0.1 microl l(-1) O3 for 5 h day(-1) for 11 days. EDU doses of 45 and 75 mg active ingredient l(-1) soil also gave protection but were associated with symptoms of toxicity and delayed senescence. In further experiments, plants were given 0 or 15 mg EDU l(-1) soil and exposed to clean air or 0.1 microl l(-1) O3 for 5 h day(-1) for 14 days. Chronic O3 exposure in the absence of EDU resulted in accelerated foliar senescence, characterized by early declines in net photosynthesis and Rubisco quantity in O3-treated plants relative to controls. EDU in the presence of O3 gave complete protection against symptoms of accelerated senescence. Senescence was not delayed in plants that received EDU in the absence of O3, and no symptoms of EDU toxicity were evident. The results suggest that EDU-induced tolerance to O3 was not based on 'anti-senescent' properties of this anti-ozonant.     

Ozone sensitivity of Fagus sylvatica and Fraxinus excelsior young trees in relation to leaf structure and foliar ozone uptake

During the summer of 2001, 2-year-old Fraxinus excelsior and Fagus sylvatica plants were subjected to ozone-rich environmental conditions at the Regional Forest Nursery at Curno (Northern Italy). Atmospheric ozone concentrations and stomatal conductance were measured, in order to calculate the foliar fluxes by means of a one-dimensional model. The foliar structure of both species was examined (thickness of the lamina and of the individual tissues, leaf mass per area, leaf density) and chlorophyll a fluorescence was determined as a response parameter. Stomatal conductance was always greater in Fraxinus excelsior, as was ozone uptake, although the highest absorption peaks did not match the peaks of ozone concentration in the atmosphere. The foliar structure can help explain this phenomenon: Fraxinus excelsior has a thicker mesophyll than Fagus sylvatica (indicating a greater photosynthesis potential) and a reduced foliar density. This last parameter, related to the apoplastic fraction, suggests a greater ability to disseminate the gases within the leaf as well as a greater potential detoxifying capacity. As foliar symptoms spread, the parameters relating to chlorophyll a fluorescence also change. PI (Performance Index, Strasser, A., Srivastava, A., Tsimilli-Michael, M., 2000. The fluorescence transient as a tool to characterize and screen photosynthetic samples. In: Yunus, M., Pathre, U., Mohanty, P., (Eds.) Probing Photosynthesis: Mechanisms, Regulation and Adaptation. Taylor & Francis, London, UK, pp. 445-483.) has proved to be a more suitable index than Fv/Fm (Quantum Yield Efficiency) to record the onset of stress conditions.     

Influence of ozone and ethylenediurea (EDU) on growth and yield of bean (Phaseolus vulgaris L.) in open-top field chambers

Greenhouse and ambient air experiments have shown ethylene diurea (EDU) to be a strong and specific protective suppressant of ozone injury in plants. To examine how EDU affects plant responses to various ozone (O(3)) levels under controlled field conditions, Phaseolus vulgaris L. cv. Lit was treated with 150 ppm EDU every 14 days and exposed in open-top chambers to charcoal-filtered air (CF), nonfiltered air (NF) or two cf treatments with ozone added. The ozone treatments were proportional additions of one (CF1) and two (CF2) times ambient ozone levels. The mean ozone concentrations in the CF, NF, CF1 and CF2 treatments were 0.98, 14.1, 14.98 and 31.56 nl litre(-1). A two-way split plot ANOVA revealed that shoot dry weight was significantly reduced by ozone. EDU treatment was highly significant for leaf dry weight, root dry weight and shoot dry weight, but not for pod dry weight; leading to a higher biomass of EDU-treated plants. Ozone/EDU interactions were significant for root weight only, indicating that EDU reduced growth suppression by ozone. These results show that EDU action on plant biomass could be interpreted as a delay in senescence since EDU-treated plants showed a significant decreased biomass loss even in the CF treatment.     

Protection of plants from ambient ozone by applications of ethylenediurea (EDU): A meta-analytic review

A meta-analysis was conducted to quantitatively assess the effects of ethylenediurea (EDU) on ozone (O₃) injury, growth, physiology and productivity of plants grown in ambient air conditions. Results indicated that EDU significantly reduced O₃-caused visible injury by 76%, and increased photosynthetic rate by 8%, above-ground biomass by 7% and crop yield by 15% in comparison with non-EDU treated plants, suggesting that ozone reduces growth and yield under current ambient conditions. EDU significantly ameliorated the biomass and yield of crops and grasses, but had no significant effect on tree growth with an exception of stem diameter. EDU applied as a soil drench at a concentration of 200-400 mg/L has the highest positive effect on crops grown in the field. Long-term research on full-grown tree species is needed. In conclusion, EDU is a powerful tool for assessing effects of ambient [O₃] on vegetation.     

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.     

Differential protection of ethylenediurea (EDU) against ambient ozone for five cultivars of tropical wheat

The antiozonant EDU (ethylenediurea) was used to assess the impact of ambient O₃ under field conditions on five cultivars of tropical wheat (Triticum aestivum L.). EDU solution (0 ppm and 400 ppm) was applied as soil drench (100 ml plant^?1) 10 days after germination (DAG) at an interval of 12 days. EDU-treated plants showed significant increments in stomatal conductance, photosynthetic rate, variable fluorescence, total chlorophyll, ascorbic acid, proline and protein contents and protective enzymes (POX, SOD and APX) activities in HUW468, HUW510 and HUW234 cultivars, while, a reverse trend was observed for lipid peroxidation. EDU application restored grain yield significantly by maintaining higher levels of antioxidants, metabolites and enzymes in cultivars HUW468 and HUW510. Sonalika and PBW343 showed least response of measured parameters under EDU treatment suggesting their greater resistance to O₃. EDU, thus proved its usefulness in screening suitable wheat cultivars for areas experiencing elevated concentrations of O₃.     

Protection of palak (Beta vulgaris L. var Allgreen) plants from ozone injury by ethylenediurea (EDU): Roles of biochemical and physiological variations in alleviating the adverse impacts

Ameliorative effects of ethylenediurea (N-[2-(2-oxo-1-imidazolinidyl) ethyl]-N′ phenylurea, abbreviated as EDU) against ozone stress were studied on selected growth, biochemical, physiological and yield characteristics of palak (Beta vulgaris L. var Allgreen) plants grown in field at a suburban site of Varanasi, India. Mean eight hourly ozone concentration varied from 52 to 73 ppb which was found to produce adverse impacts on plant functioning and growth characteristics. The palak plants were treated with 300 ppm EDU at 10 days after germination at 10 days interval up to the plant maturity. Lipid peroxidation in EDU treated plants declined significantly as compared to non-EDU treated ones. Significant increment in F_v/F_m ratio in EDU treated plants as compared to non-EDU treated ones was recorded. EDU treated plants showed significant increment in ascorbic acid contents and reduction in peroxidase activity as compared to non-EDU treated ones. As a result of the protection provided by EDU against ozone induced stress on biochemical and physiological characteristics of palak, the morphological parameters also responded positively. Significant increments were recorded in shoot length, number of leaves plant⁻¹, leaf area and root and shoot biomass of EDU treated plants as compared to non-EDU treated ones. Contents of Na, K, Ca, Mg and Fe were higher in EDU treated plants as compared to non-EDU treated ones. The present investigation proves the usefulness of EDU in partially ameliorating ozone injury in ambient conditions.     

Translocation and persistence of EDU (ethylenediurea) in plants: the relationship with its role in ozone damage

Bean plants (Phaseolus vulgaris L. cv Lit) were treated with N-[2-(2-oxo-1-imidazolinidyl)ethyl]-N'-phenylurea (EDU) (150 microg ml(-1)) in hydroponic conditions. The EDU concentration in different plant tissues was measured by HPLC. EDU accumulated in leaves and persisted for more than 10 days showing a slow degradation. Using five different EDU concentrations, a significant relationship between EDU concentration in nutrient solution, ozone tolerance and EDU concentration in leaves was shown. Leaves which contained more EDU were less sensitive to ozone damage. Investigations on protoplasts and cell cultures showed that EDU did not enter the cells. Possible implications of EDU accumulation in the leaf apoplast are discussed.     

Effects of ethylenediurea and ozone on the antioxidative systems in beans (Phaseolus vulgaris L.)

To study the biochemical mechanism of EDU protection against ozone injury, peroxidase, ascorbate-dependent peroxidase, and catalase activities, and the contents of ascorbic acid, dehydroascorbic acid, malondialdehyde and soluble protein were measured in Phaseolus vulgaris L. cv. Lit exposed to ozone and ethylenediurea (EDU) in open-top chambers. Plants not treated with EDU showed foliar bronzing due to ozone, while EDU-treated plants were not affected. EDU application modified the reaction of biochemical parameters to ozone. Soluble protein content was elevated by EDU. Peroxidase activity increased with ozone exposure in untreated plants only, while ascorbate-dependent peroxidase activity was lower in EDU treated plants. Catalase activity decreased in EDU-untreated plants. The ratio of ascorbic acid to dehydroascorbic acid was significantly increased in EDU treated plants. These results suggest that EDU might induce ascorbic acid synthesis and therefore provide the plant with a very potent antioxidant. Or the content of hydrogen peroxide was reduced due to other unknown processes and caused a delay in foliar senescence, regardless of whether these processes were ozone-induced or due to natural aging processes.     

Use of the antiozonant ethylenediurea (EDU) in Italy: verification of the effects of ambient ozone on crop plants and trees and investigation of EDU's mode of action

Twenty-four experiments where EDU was used to protect plants from ozone (O₃) in Italy are reviewed. Doses of 150 and 450 ppm EDU at 2-3 week intervals were successfully applied to alleviate O₃-caused visible injury and growth reductions in crop and forest species respectively. EDU was mainly applied as soil drench to crops and by stem injection or infusion into trees. Visible injury was delayed and reduced but not completely. In investigations on mode of action, EDU was quickly (<2 h) uptaken and translocated to the leaf apoplast where it persisted long (>8 days), as it cannot move via phloem. EDU did not enter cells, suggesting it does not directly affect cell metabolism. EDU delayed senescence, did not affect photosynthesis and foliar nitrogen content, and stimulated antioxidant responses to O₃ exposure. Preliminary results suggest developing an effective soil application method for forest trees is warranted.     

Assessing the impact of ambient ozone on growth and yield of a rice (Oryza sativa L.) and a wheat (Triticum aestivum L.) cultivar grown in the Yangtze Delta, China, using three rates of application of ethylenediurea (EDU)

Foliar applications of ethylenediurea (abbreviated as EDU) were made at 0, 150, 300 or 450 ppm to field-grown rice and wheat in the Yangtze Delta in China. Rice and wheat responded differently to ambient ozone and EDU applications. For wheat, some growth characteristics, such as yield, seed number per plant, seed set rate and harvest index, increased significantly at 300 ppm EDU treatment, while for rice no parameters measured were statistically different regarding EDU application. The reason may be that the wheat cultivar used may be more sensitive to ozone than the rice cultivar. EDU was effective in demonstrating ozone effects on the wheat cultivar, but not on the rice cultivar. Cultivar sensitivity might be an important consideration when assessing the effects of ambient ozone on plants.     

Effects of ozone on the foliar histology of the mastic plant (Pistacia lentiscus L.)

An open-top chamber study was conducted to investigate the tissue and cellular-level foliar effects of ozone (O3) on a Mediterranean evergreen species, the mastic plant (Pistacia lentiscus L.). Plants were exposed at three different O3 levels, and leaf samples were collected periodically from the beginning of the exposure. Although no visible foliar injury was evident, alterations of the plastids and vacuoles in the mesophyll were observed. Senescence processes were accelerated with an anomalous stacking of tannin vacuoles, and a reduction in the size and number of the chloroplasts. Overall, most of the modifications induced by O3 were consistent with previously reported observations on deciduous broadleaf species, with the exception of alterations in the cells covering the secretory channels, reported here as a new finding. Comments on the feasibility of using microscopy to validate O3 related field observations and subtle foliar injury are also given.     

Assessing the impact of ambient ozone on growth and productivity of two cultivars of wheat in India using three rates of application of ethylenediurea (EDU)

Three rates of ethylenediurea were used to assess the impact of ambient ozone on growth and productivity of wheat (Triticum aestivum L) cultivars "Malviya 533" (M 533) and "Malviya 234" (M 234) at a suburban site near Varanasi, India, beginning in December. Wheat plants were treated with EDU at 0, 150, 300 and 450 ppm as soil drenches at 10-day intervals. EDU treatment affected plant growth, with effects varying with cultivar, age, and EDU concentration. Seed yield was improved for M 533 at 150 ppm EDU, while yield improved for M 234 at 300 and 450 ppm EDU. M 533 appears to be more resistant to ozone than M 234. Overall results confirmed that EDU is very useful in assessing the effect of ambient ozone in India.     

An assessment of the impact of ambient ozone on field-grown crops in New Jersey using the EDU method: part 2-soybean (Glycine max (L.) Merr.)

The yields of eleven commercially grown soybean cultivars were compared in ethylenediurea (EDU)-treated and non-treated field plots in New Brunswick, New Jersey, over a 4 year period. No statistically significant difference between treatments was found for any cultivar; the inference being ambient ozone did not adversely affect soybean yield. Succeeding field experiments supported this interpretation of the data. 'Sanilac' white bean, a legume known to be more sensitive to O(3) than soybean, was found to produce a significantly greater yield in EDU-treated than non-treated plots, unlike a companion planting of 'Williams 82' soybean which did not exhibit the differential response. The results indicated that the specific EDU protocol used in the soybean experiments is capable of detecting an ozone effect in a legume. Moreover, in a concurrent greenhouse experiment the yield of EDU-treated Sanilac white bean was not significantly different from non-treated plants in the absence of ozone pollution. In a dose-response field experiment during a year of unusually high O(3) pollution, yield of 'Williams 82' increased slightly with each EDU increment up to 500 ppm and decreased at 1000 ppm. The difference between non-treated and EDU-treated plants, however, was not statistically significant. There was no evidence to suggest that the EDU concentration (500 ppm) used in previous soybean experiments reduced seed yield. Fortuitously, the tolerance of commercially-grown soybean to ambient ozone is at least partially conditioned by the practce of not irrigating the crop. The New Jersey results are in agreement with reports from Maryland, Georgia and Tennessee in which an adverse impact of ambient O(3) was not found in soybean, but contrary to a current predictive model.     

Alteration of physiological parameters in needles of cloned spruce trees (Picea abies (L.) Karst.) by ozone and acid mist

Three-year-old clonal spruce trees, kept in growth chambers, were treated with ozone and acid mist during a period of 14 months. One half of the trees were grown on an acidic sandy soil, the other half on a calcareous soil rich in carbonate. At the end of the fumigation period, carbohydrates (glucose, fructose, sucrose, raffinose, starch, glucose-1-phosphate and fructose-6-phosphate) and parameters of the energy status (ATP-, AdN-(ATP + ADP + AMP)- levels, ATP/ADP-ratios and adenylate-energy-charge-(AEC)-values) were determined in the current-year's needles. The results indicate that the metabolic status of a plant tissue is not only influenced by the nature of the air pollutants. Soil factors play an important role in metabolic changes within the plant and are thus of relevance in the manifestation of damage symptoms.     

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.