Ethylenediurea Research Articles

Effects of ozone (O3) and ethylenediurea (EDU) on the ecological stoichiometry of a willow grown in a free-air exposure system

Ground-level ozone (O3) concentrations have been elevating in the last century. While there has been a notable progress in understanding O3 effects on vegetation, O3 effects on ecological stoichiometry remain unclear, especially early in the oxidative stress. Ethyelenediurea (EDU) is a chemical compound widely applied in research projects as protectant of plants against O3 injury, however its mode of action remains unclear. To investigate O3 and EDU effects early in the stress, we sprayed willow (Salix sachalinensis) plants with 0, 200 or 400 mg EDU L−1, and exposed them to either low ambient O3 (AOZ) or elevated O3 (EOZ) levels during the daytime, for about one month, in a free air O3 controlled exposure (FACE); EDU treatment was repeated every nine days. We collected samples for analyses from basal, top, and shed leaves, before leaves develop visible O3 symptoms. We found that O3 altered the ecological stoichiometry, including impacts in nutrient resorption efficiency, early in the stress. The relation between P content and Fe content seemed to have a critical role in maintaining homeostasis in an effort to prevent O3-induced damage. Photosynthetic pigments and P content appeared to play an important role in EDU mode of action. This study provides novel insights on the stress biology which are of ecological and toxicological importance.

Ethylenediurea Induces Hormesis in Plants.

Tropospheric ozone levels are elevated throughout the northern hemisphere. The potential threat of ozone to vegetation urges for studying plant protection methods. The chemical ethylenediurea (EDU) is the most extensively utilized substance for protecting plants against ozone damage in research projects. This commentary provides collective evidence showing hormetic responses of plants to EDU and suggests that EDU may act as a conditioning agent against elevated ozone exposures. This article also suggests testing different substances in a hormetic framework for protecting plants against ozone damage. The concept of hormesis provides a significant perspective for reducing the economic cost for plant protection.

Physiological and Biochemical Response of Winter Wheat (Triticum Aestivum L.) to Ambient O3 and the Antiozonant Chemical Ethylenediurea (EDU) in Jeddah, Saudi Arabia

This study was to conducted to investigate the use of ethylenediurea (EDU) as a possible tool to evaluate O3 effects on wheat (Triticum aestivum L.) plants under field conditions in Jeddah. Wheat plants were expsoed to ambient O3 (AA) and the antiozonant chemical ethylenediurea (EDU) in closed fumigation chambers for the full growing season. Growth, yield and physiology were determined in response to O3 and/or EDU. EDU-treated plants had higher photosynthetic rates (24%) and stomatal conductance (25%), which were reflected in higher growth and yield in terms of number of grains. The present study revealed that EDU could be used as a promising tool to mitigate damaging effects of O3 on under field conditions. EDU protected wheat plants leading to increases in photosynthetic rates, growth and yield.

Impact of Ethylene diurea (EDU) on growth, yield and proteome of two winter wheat varieties under high ambient ozone phytotoxicity

The present study evaluated the impact of high ambient O3 on morphological, physiological and biochemical traits and leaf proteome in two high-yielding varieties of wheat using ethylene diurea (EDU) as foliar spray (200 and 300 ppm). Average ambient ozone concentration was 60 ppb which was more than sufficient to cause phytotoxic effects. EDU treatment resulted in less lipid peroxidation along with increased chlorophyll content, biomass and yield. EDU alleviated the negative effects of ozone by enhancing activities of antioxidants and antioxidative enzymes. Two dimensional electrophoresis (2DGE) analysis revealed massive changes in protein abundance in Kundan at vegetative stage (50% proteins were increased, 20% were decreased) and at flowering stage (25% increased, 18% decreased). In PBW 343 at both the developmental stages about 15% proteins were increased whereas 20% were decreased in abundance. Higher abundance of proteins related to carbon metabolism, defense and photorespiration conferred tolerance to EDU treated Kundan. In PBW343, EDU provided incomplete protection as evidenced by low abundance of many primary metabolism related proteins. Proteomic changes in response to EDU treatment in two varieties are discussed in relation to growth and yield.

Use of Ethylenediurea (EDU) in identifying indicator cultivars of Indian clover against ambient ozone

Three clover (Trifolium alexandrium L.) cultivars (Bundel, Wardan and JHB-146) were assessed for their responses to ambient ozone (O3) with respect to growth, physiological and biochemical parameters at two rural sites (R1 and R2) using ethylenediurea (EDU). EDU solution (300ppm) was applied as soil drench, 10 days after germination (DAG) at an interval of 10 days up to 80 DAG. The average O3 concentrations were 52.76 and 60.86 ppb at R1 and R2 sites, respectively during the experimental period. Ambient O3 induced visible symptoms in all the cultivars at both the sites, with more at R2 site having high ambient O3 levels. Visible injury was observed first in non-EDU treated plants of Wardan at R2 site. Wardan also showed maximum reduction in leaf injury under EDU treatment at both the sites with more at R2. Under EDU treatment, better adaptation to ambient O3 at initial age of observation and higher acquisition of resources at later ages of observation at both the sites led to better physiological and biochemical adaptations in Wardan. Bundel retained more biomass in shoot as is reflected with higher shoot/root ratio and thus focused more on repair and defense. Shoot/root ratio of JHB-146 did not respond to EDU treatment and thus showed insignificant variations except at initial age of observation at R1 site. This study clearly suggests that Wardan and Bundel are sensitive to ambient O3 and can be used as bioindicator species in areas having higher O3 levels using EDU as a research tool.

High doses of ethylenediurea (EDU) as soil drenches did not increase leaf N content or cause phytotoxicity in willow grown in fertile soil

Ground-level ozone (O3) levels are nowadays elevated in wide regions of the Earth, causing significant effects on plants that finally lead to suppressed productivity and yield losses. Ethylenediurea (EDU) is a chemical compound which is widely used in research projects as phytoprotectant against O3 injury. The EDU mode of action remains still unclear, while there are indications that EDU may contribute to plants with nitrogen (N) when the soil is poor in N and the plants have relatively small leaf area. To reveal whether the N content of EDU acts as a fertilizer to plants when the soil is not poor in N and the plants have relatively large total plant leaf area, willow plants (Salix sachalinensis Fr. Schm) were exposed to low ambient O3 levels and treated ten times (9-day interval) with 200mL soil drench containing 0, 800 or 1600mg EDU L−1. Fertilizer was added to a nutrient-poor soil, and the plants had an average plant leaf area of 9.1m2 at the beginning of EDU treatments. Indications for EDU-induced hormesis in maximum electron transport rate (Jmax) and ratio of intercellular to ambient CO2 concentration (Ci:Ca) were observed at the end of the experiment. No other EDU-induced effects on leaf greenness and N content, maximum quantum yield of photosystem II (Fv/Fm), gas exchange, growth and matter production suggest that EDU did not act as N fertilizer and did not cause toxicity under these experimental conditions.

Effects of the Antiozonant Ethylenediurea (EDU) on Fraxinus ornus L.: The Role of Drought

Ethylenediurea (EDU) is a synthetic chemical known to protect plants from the phytotoxic effects of tropospheric ozone (O3). Although many studies have proposed the use of EDU for studying the O3 effects under field conditions, its mechanism of action is not fully understood, and it is unclear whether it exerts a specific antiozonant action, or if it may also interact with other oxidative stresses. The aim of this work was to evaluate the effect of EDU on forest species in a Mediterranean environment where, during summer, vegetation is exposed to multiple oxidative stresses, such as O3 and drought. The experiment was conducted on Fraxinus ornus L. (Manna ash) plants growing in six mesocosms, three maintained under full irrigation, while the other three were subjected to drought for 84 days. In each mesocosm, three plants were sprayed every 15 days with 450 ppm EDU. Gas exchange and chlorophyll “a” fluorescence measurements carried out through the experimental period highlighted that EDU did not affect stomatal conductance and had an ameliorative effect on the functionality of drought-stressed plants, thus suggesting that it may act as a generic antioxidant. The implications of these findings for the applicability of EDU in field studies are discussed.

Large variability in ambient ozone sensitivity across 19 ethylenediurea-treated Chinese cultivars of soybean is driven by total ascorbate

The sensitivity of Chinese soybean cultivars to ambient ozone (O3) in the field is unknown, although soybean is a major staple food in China. Using ethylenediurea (EDU) as an O3 protectant, we tested the gas exchange, pigments, antioxidants and biomass of 19 cultivars exposed to 28ppm·hr AOT40 (accumulated O3 over an hourly concentration threshold of 40ppb) over the growing season at a field site in China. By comparing the average biomass with and without EDU, we estimated the cultivar-specific sensitivity to O3 and ranked the cultivars from very tolerant (<10% change) to highly sensitive (>45% change), which helps in choosing the best-suited cultivars for local cultivation. Higher lipid peroxidation and activity of the ascorbate peroxidase enzyme were major responses to O3 damage, which eventually translated into lower biomass production. The constitutional level of total ascorbate in the leaves was the most important parameter explaining O3 sensitivity among these cultivars. Surprisingly, the role of stomatal conductance was insignificant. These results will guide future breeding efforts towards more O3-tolerant cultivars in China, while strategies for implementing control measures of regional O3 pollution are being implemented. Overall, these results suggest that present ambient O3 pollution is a serious concern for soybean in China, which highlights the urgent need for policy-making actions to protect this critical staple food.

Diagnosing ozone stress and differential tolerance in rice (Oryza sativa L.) with ethylenediurea (EDU)

Rising tropospheric ozone concentrations in Asia necessitate the breeding of adapted rice varieties to ensure food security. However, breeding requires field-based evaluation of ample plant material, which can be technically challenging or very costly when using ozone fumigation facilities. The chemical ethylenediurea (EDU) has been proposed for estimating the effects of ozone in large-scale field applications, but controlled experiments investigating constitutive effects on rice or its suitability to detect genotypic differences in ozone tolerance are missing. This study comprised a controlled open top chamber experiment with four treatments (i) control (average ozone concentration 16 ppb), (ii) control with EDU application, (iii) ozone stress (average 77 ppb for 7 h daily throughout the season), and (iv) ozone stress with EDU application. Three contrasting rice genotypes were tested, i.e. the tolerant line L81 and the sensitive Nipponbare and BR28. The ozone treatment had significant negative effects on plant growth (height and tillering), stomatal conductance, SPAD value, spectral reflectance indices such as the normalized difference vegetation index (NDVI), lipid peroxidation, as well as biomass and grain yields. These negative effects were more pronounced in the a priori sensitive varieties, especially the widely grown Bangladeshi variety BR28, which showed grain yield reductions by 37 percent. EDU application had almost no effects on plants in the absence of ozone, but partly mitigated ozone effects on foliar symptoms, lipid peroxidation, SPAD value, stomatal conductance, several spectral reflectance parameters, panicle number, grain yield, and spikelet sterility. EDU responses were more pronounced in sensitive genotypes than in the tolerant L81. In conclusion, EDU had no constitutive effects on rice and partly offset negative ozone effects, especially in sensitive varieties. It can thus be used to diagnose ozone damage in field grown rice and for distinguishing tolerant (less EDU-responsive) and sensitive (more EDU-responsive) genotypes.

Protecting the photosynthetic performance of snap bean under free air ozone exposure

Tropospheric ozone (O3) is a major air pollutant and causes serious injury to vegetation. To protect sensitive plants from O3 damage, several agrochemicals have been assessed, including cytokinin (e.g., kinetin, KIN) and ethylenediurea (EDU) with cytokinin-like activity. In higher plant, leaves are primarily injured by O3 and protective agrochemicals are often applied by leaf spraying. To our knowledge, the mitigating abilities of EDU and KIN have not been compared directly in a realistic setup. In the present research, impacts of elevated O3 (2× ambient O3, 24hr per day, for 8days) on an O3 sensitive line (S156) of snap bean (Phaseolus vulgaris), which is often used for biomonitoring O3 pollution, were studied in a free air controlled exposure system. The day before starting the O3 exposure, plants were sprayed with a solution of EDU (300ppm), KIN (1mmol/L) or distilled water, to compare their protective abilities. The results demonstrated that 2× ambient O3 inhibited net photosynthetic rate and stomatal conductance, increased the minimal fluorescence yield of the dark-adapted state, decreased the maximal quantum yield of PSII photochemistry, and led to visible injury. KIN and EDU alleviated the reduction of the photosynthetic performance, and visible injury under O3 fumigation. The plants sprayed with EDU showed greater ability to mitigate the O3 damage than those sprayed with KIN. Chlorophyll fluorescence imaging may have detected more precisely the differences in O3 response across the leaf than the conventional fluorometer.

Perspectives for elucidating the ethylenediurea (EDU) mode of action for protection against O3 phytotoxicity

Ethylenediurea (EDU) has been widely studied for its effectiveness to protect plants against injuries caused by surface ozone (O3), however its mode of action remains unclear. So far, there is not a unified methodological approach and thus the methodology is quite arbitrary, thereby making it more difficult to generalize findings and understand the EDU mode of action. This review examines the question of whether potential N addition to plants by EDU is a fundamental underlying mechanism in protecting against O3 phytotoxicity. Yet, this review proposes an evidence-based hypothesis that EDU may protect plants against O3 deleterious effects upon generation of EDU-induced hormesis, i.e. by activating plant defense at low doses. This hypothesis challenges the future research directions. Revealing a hormesis-based EDU mode of action in protecting plants against O3 toxicity would have further implications to ecotoxicology and environmental safety. Furthermore, this review discusses the need for further studies on plant metabolism under EDU treatment through relevant experimental approach, and attempts to set the bases for approaching a unified methodology that will contribute in revealing the EDU mode of action. In this framework, focus is given to the main EDU application methods.

Ethylenediurea as a potential tool in evaluating ozone phytotoxicity: a review study on physiological, biochemical and morphological responses of plants.

Present-day climate change scenario has intensified the problem of continuously increasing ground-level ozone (O3), which is responsible for causing deleterious effects on growth and development of plants. Studies involving use of ethylenediurea (EDU), a chemical with antiozonant properties, have given some promising results in evaluating O3 injury in plants. The use of EDU is especially advantageous in developing countries which face a more severe problem of ground-level O3, and technical O3-induced yield loss assessment techniques like open-top chambers cannot be used. Recent studies have detected a hormetic response of EDU on plants; i.e. treatment with higher EDU concentrations may or may not show any adverse effect on plants depending upon the experimental conditions. Although the mode of action of EDU is still debated, it is confirmed that EDU remains confined in the apoplastic regions. Certain studies indicate that EDU significantly affects the electron transport chain and has positive impact on the antioxidant defence machinery of the plants. However, the mechanism of protecting the yield of plants without significantly affecting photosynthesis is still questionable. This review discusses in details the probable mode of action of EDU on the basis of available data along with the impact of EDU on physiological, biochemical, growth and yield response of plants under O3 stress. Data regarding the effect of EDU on plant 'omics' is highly insufficient and can form an important aspect of future EDU research.

Tropospheric ozone pollution in India: effects on crop yield and product quality.

Ozone (O3) in troposphere is the most critical secondary air pollutant, and being phytotoxic causes substantial losses to agricultural productivity. Its increasing concentration in India particularly in Indo-Gangetic plains is an issue of major concern as it is posing a threat to agriculture. In view of the issue of rising surface level of O3 in India, the aim of this compilation is to present the past and the prevailing concentrations of O3 and its important precursor (oxides of nitrogen) over the Indian region. The resulting magnitude of reductions in crop productivity as well as alteration in the quality of the product attributable to tropospheric O3 has also been taken up. Studies in relation to yield measurements have been conducted predominantly in open top chambers (OTCs) and also assessed by using antiozonant ethylene diurea (EDU). There is a substantial spatial difference in O3 distribution at different places displaying variable O3 concentrations due to seasonal and geographical variations. This review further recognizes the major information lacuna and also highlights future perspectives to get the grips with rising trend of ground level O3 pollution and also to formulate the policies to check the emissions of O3 precursors in India.

Impacts of ethylenediurea (EDU) soil drench and foliar spray in Salix sachalinensis protection against O3-induced injury

It is widely accepted that elevated levels of surface ozone (O3) negatively affect plants. Ethylenediurea (EDU) is a synthetic substance which effectively protects plants against O3-caused phytotoxicity. Among other questions, the one still open is: which EDU application method is more appropriate for treating fast-growing tree species. The main aims of this study were: (i) to test if chronic exposure of Salix sachalinensis plants to 200–400mgEDUL−1, the usually applied range for protection against O3 phytotoxicity, is beneficial to plants; (ii) to evaluate the effects of chronic exposure to elevated O3 on S. sachalinensis; (iii) to assess the efficacy of two methods (i.e. soil drench and foliar spray) of EDU application to plants; (iv) to investigate the appropriate concentration of EDU to protect against elevated O3-induced damage in S. sachalinensis; and (v) to compare the two methods of EDU application in terms of effectiveness and EDU consumption. Current-year cuttings grown in infertile soil free from organic matter were exposed either to low ambient O3 (AOZ, 10-h≈28.3nmolmol−1) or to elevated O3 (EOZ, 10-h≈65.8nmolmol−1) levels during daylight hours. Over the growing season, plants were treated every nine days with 200mL soil drench of 0, 200 or 400mgEDUL−1 or with foliar spray of 0, 200 or 400mgEDUL−1 (in two separate experiments). We found that EDU per se had no effects on plants exposed to AOZ. EOZ practically significantly injured S. sachalinensis plants, and the impact was indifferent between the experiments. EDU did not protect plants against EOZ impact when applied as soil drench but it did protect them when applied as 200–400mgL−1 foliar spray. We conclude that EDU may be more effective against O3 phytotoxicity to fast-growing species when applied as a spray than when applied as a drench.Keymessage: Soil-drenched EDU was ineffective in protecting willow plants against O3-induced injury, whereas foliar-sprayed EDU was effective even at the concentration of 200mgL−1.

Olive Oil for Dressing Plant Leaves so as to Avoid O3 Injury

The persistence of high ground-level ozone (O3) concentration in most regions of the northern hemisphere has severe implications to crop production, wild plant conservation, and forest sustainability. Therefore, methods for plant protection against O3 and O3 biomonitoring are of high relevance; however, there is not a method that can be applied in cultivations, which are intended for human consumption. After spraying bean (Phaseolus vulgaris L. cv. Pinto) seedlings with ethylene diurea, olive oil, or myclobutanil and exposing them to O3-enriched air (90 nmol mol−1) for a week (8 h day−1), we found that commercial olive oil can be effectively used as a protectant of plants against O3. This protection is attributed to avoidance of O3 uptake into the mesophyll, via decreased stomatal conductance. Olive oil can be applied even in organic cultivations, either for biomonitoring purposes or for short-term protection of plants during O3 episodes. Further studies are needed in order to investigate potential direct reaction of O3 with olive oil. Yet, attention should be paid when myclobutanil is applied to plants which are used for O3 biomonitoring purposes due to potential confounding effects by increasing O3-caused visible injury to plant leaves.

Moderate drought did not affect the effectiveness of ethylenediurea (EDU) in protecting Populus cathayana from ambient ozone

A field study was conducted to evaluate the effects of ambient ozone (O3) on an O3-sensitive poplar (Populus cathayana) by using ethylenediurea (EDU) as a chemical protectant under two soil water treatments (well-watered (WW) and moderate drought (MD, 50–60% of WW in volumetric soil water content). EDU was applied as foliar spray at 0, 300, 450, and 600ppm. Photosynthetic parameters, pigment contents, leaf nitrogen, antioxidant capacity, growth, and biomass were measured. The 8h (9:00–17:00) average ambient O3 concentration was 71.7ppb, and AOT40 was 29.2ppmh during the experimental period (9 June to 21 September), which was high enough to cause plant injury. MD had significantly negative effects on P. cathayana, as indicated by reduced photosynthesis, growth, and biomass, and higher MDA contents. On the other hand, EDU significantly increased photosynthesis rate, chlorophyll a fluorescence, Vcmax and Jmax, photosynthetic pigments, total antioxidant capacity, tree growth and biomass accumulation, and reduced lipid peroxidation, but there was no significant interaction between EDU and drought for most parameters, indicating that EDU can efficiently protect Populus cathayana against ambient O3 and the protection was not affected by soil water contents when soil water reached moderate drought level. Among all doses, EDU at 450ppm provided maximum protection. Comparison of EDU-treated and non-treated P. cathayana could be used as a biomarker system in risk assessment of the effects of ambient O3 on forest health.

High doses of ethylene diurea (EDU) are not toxic to willow and act as nitrogen fertilizer

Ethylene diurea (EDU) is synthetic chemical which protects plants against damage caused by ground level O3 and is used experimentally as a biomonitoring tool at doses usually ranging from 200 to 400mgL−1 a.i. Although several studies have investigated the protective action of EDU, this mechanism remains unclear. Important uncertainties in EDU action are whether EDU acts as a source of nitrogen (N) to plants and whether high doses are phytotoxic. In order to answer these questions, we conducted an open-field experiment where potted willow (Salix sachalinensis Fr. Schm) plants were exposed to ambient O3 conditions and treated with 0, 800 or 1600mgL−1 EDU as a soil drench, every nine days, for about 2.5months. We examined approximately 50 response variables. Based on N content in different plant organs, we found that (a) all EDU was transferred to the leaves and (b) high doses of EDU increased the leaf N content. However, EDU did not affect the C content and distribution within the plant body. Still, even at the highest dose, EDU was not toxic to this fast-growing species (however such a high dose should not be applied in uncontrolled environments); and there was no EDU persistence in the soil, as indicated by soil N content. Notably, our soil was free from organic matter and N-poor.Key message: EDU per se does not cause toxicity to willow plants when applied as drench to a soil with no organic matter, rather, high EDU doses may act as nitrogen fertilizer in a nitrogen-poor soil.

Effects of different routes of application on ethylenediurea persistence in tobacco leaves

Ethylenediurea (EDU) is a common research tool for investigating ozone impacts on vegetation, although the role of different application routes (foliar spray vs soil drench) on EDU persistence in the leaves is unknown. We quantified EDU concentrations in leaves of the O3-sensitive Bel-W3 cultivar of tobacco treated with EDU as either foliar spray or soil drench. Foliar EDU concentrations were measured by Q-TOF LC/MS. When EDU was applied as foliar spray, 1 h was enough for reaching a measurable concentration within the leaf. EDU concentration increased over the 21-day period when the leaf was not washed after the application (treatment #1), while it decreased when the leaf was washed after the application (treatment #2). These results suggest that: a) dry deposition of EDU onto the leaf surface was gradually absorbed into the unwashed leaf, although the mechanisms of such uptake were unclear; b) concentration of EDU was decreased quickly (−35%) during the first 24 h from application and more slowly during the following three days (−20%) in the washed leaves. Degradation did not involve enzymatic reactions and was not affected by the presence of ROS. When EDU was applied as soil drench, foliar concentrations increased over time, likely due to adsorption onto soil organic matter and gradual re-solubilization by irrigation water. An analysis of EDU concentration in protoplast and intercellular washing fluid showed that EDU did not enter the cells, but was retained in the apoplast only. Possible implications of EDU in the apoplast and recommendations for EDU application are discussed.

The first toxicological study of the antiozonant and research tool ethylene diurea (EDU) using a Lemna minor L. bioassay: Hints to its mode of action

The antiozonant and research tool ethylene diurea (EDU) is widely studied as a phytoprotectant against the widespread pollutant ground-surface ozone. Although it has been extensively used, its potential toxicity in the absence of ozone is unknown and its mode of action is unclear. The purpose of this research was to toxicologically assess EDU and to further investigate its mode of action using Lemna minor L. as a model organism. Application of EDU concentrations greater than 593 mg L−1 (practically 600 mg L−1) resulted in adverse inhibition of colony growth. As no-observed-toxic-effects concentration (NOEL) we recommend a concentration of 296 mg L−1 (practically 300 mg L−1). A hormetic response was detected, i.e. stimulatory effects of low EDU concentrations, which may indicate overcompensation in response to disruption in homeostasis. Growth inhibition and suppressed biomass were associated with impacted chlorophyll a fluorescence (ΦPSII, qP and ETR). Furthermore, EDU increased mesophyll thickness, as indicated by frond succulence index. Applications of concentrations ≥593 mg L−1 to uncontrolled environments should be avoided due to potential toxicity to sensitive organisms and the environment.

Effects of long-term ambient ozone exposure on biomass and wood traits in poplar treated with ethylenediurea (EDU)

This is the longest continuous experiment where ethylenediurea (EDU) was used to protect plants from ozone (O3). Effects of long-term ambient O3 exposure (23 ppm h AOT40) on biomass of an O3 sensitive poplar clone (Oxford) were examined after six years from in-ground planting. Trees were irrigated with either water or 450 ppm EDU. Above (−51%) and below-ground biomass (−47%) was reduced by O3 although the effect was significant only for stem and coarse roots. Ambient O3 decreased diameter of the lower stem, and increased moisture content along the stem of not-protected plants (+16%). No other change in the physical wood structure was observed. A comparison with a previous assessment in the same experiment suggested that O3 effects on biomass partitioning to above-ground organs depend on the tree ontogenetic stage. The root/shoot ratios did not change, suggesting that previous short-term observations of reduced allocation to tree roots may be overestimated.