Oxidative Stress Response Mechanisms Sustain the Antibacterial and Antioxidant Activity of Quercus ilex.

The development of new natural antibiotics is considered as the heart of several investigations in the nutraceutical field. In this work, leaves of Quercus ilex L. treated by tropospheric ozone (O3) and nitrogen (N) deposition, exhibited a clear antimicrobial efficacy against five multi-drug resistant (MDR) bacterial strains (two gram-positive and three gram-negative). Under controlled conditions, it was studied how simulated N deposition influences the response to O3 and the antibacterial and antioxidant activity, and antioxidant performance. The extraction was performed by ultra-pure acetone using two different steps. A higher antioxidant activity was measured in the presence of interaction between O3 and N treatments on Quercus leaves. At the same time, all organic extracts tested have shown bacteriostatic activity against all the tested strains with a MIC comprised between 9 and 4 micrograms/mL, and a higher antioxidant efficacy shown by spectrophotometric assay. Stronger antimicrobial activity was found in the samples treated with O3, whereas N-treated plants exhibited an intermediate antibacterial performance. This performance is related to the stimulation of the non-enzymatic antioxidant system induced by the oxidative stress, which results in an increase in the production of antimicrobial bioactive compounds.

Germination, root elongation, and photosynthetic performance of plants exposed to sodium lauryl ether sulfate (SLES): an emerging contaminant.

The anionic surfactant SLES (sodium lauryl ether sulfate) is an emerging contaminant, being the main component of foaming agents that are increasingly used by the tunnel construction industry. To fill the gap of knowledge about the potential SLES toxicity on plants, acute and chronic effects were assessed under controlled conditions. The acute ecotoxicological test was performed on Lepidum sativum L. (cress) and Zea mays L. (maize). Germination of both species was not affected by SLES in soil, even at concentrations (1200 mg kg-1) more than twice higher than the maximum realistic values found in contaminated debris, thus confirming the low acute SLES toxicity on terrestrial plants. The root elongation of the more sensitive species (cress) was instead reduced at the highest SLES concentration. In the chronic phytotoxicity experiment, photosynthesis of maize was downregulated, and the photosynthetic performance (PITOT) significantly reduced already under realistic exposures (360 mg kg-1), owing to the SLES ability to interfere with water and/or nutrients uptake by roots. However, such reduction was transient, likely due to the rapid biodegradation of the surfactant by the soil microbial community. Indeed, SLES amount decreased in soil more than 90% of the initial concentration in only 11 days. A significant reduction of the maximum photosynthetic capacity (Pnmax) was still evident at the end of the experiment, suggesting the persistence of negative SLES effects on plant growth and productivity. Overall results, although confirming the low phytotoxicity and high biodegradability of SLES in natural soils, highlight the importance of considering both acute and nonlethal stress effects to evaluate the environmental compatibility of soil containing SLES residues.

Selection of tree species for forests under climate change: is PSI functioning a better predictor for net photosynthesis and growth than PSII?

A chlorophyll fluorescence (ChlF) assessment was carried out on oak seedlings (Quercus ilex L., Quercus pubescens Willd., Quercus frainetto Ten.) of Italian and Greek provenance, during the years 2017 and 2018, in a common garden in central Italy planted in 2017. This trial aimed to test the relative performances of the oak species in the perspective of assisted migration as part of the actions for the adaptation of forests to climate change. The assessment of the photosynthetic performance of the tree species included the analysis of the prompt chlorophyll fluorescence (PF) transient and the modulated reflection (MR) at 820 nm, leaf chlorophyll content, leaf gas exchange (net photosynthesis, stomatal conductance), plant growth (i.e., height) and mortality rate after 2 years from the beginning of the experiment. The assessment of the performance of the three oak species was carried out 'in vivo'. Plants were generated from seeds and exposed to several environmental factors, including changing seasonal temperature, water availability, and soil biological and physical functionality. The results of PF indicate a stable functionality of the photosynthetic system PSII (expressed as FV/FM) across species and provenances and a decline in photochemistry functionality at the I-P phase (ΔVIP) in Q. frainetto, thus indicating a decline of the content of PSI in this species. This result was confirmed by the findings of MR analysis, with the speed of reduction and subsequent oxidation of PSI (VRED and VOX) strongly correlated to the amplitude of ΔVIP. The photosynthetic rates (net photosynthesis, PN) and growth were correlated with the parameters associated with PSI content and function, rather than those related to PSII. The low performance of Q. frainetto in the common garden seems to be related to early foliar senescence with the depletion of nitrogen, due to suboptimal climatic and edaphic conditions. Chlorophyll fluorescence allowed discrimination of populations of oak species and individuation of the less (or/and best) suitable species for future forest ecology and management purposes.

Exposure to Residential Greenness as a Predictor of Cause-Specific Mortality and Stroke Incidence in the Rome Longitudinal Study.

BACKGROUND: Living in areas with higher levels of surrounding greenness and access to urban green areas have been associated with beneficial health outcomes. Some studies suggested a beneficial influence on mortality, but the evidence is still controversial. OBJECTIVES: We used longitudinal data from a large cohort to estimate associations of two measures of residential greenness exposure with cause-specific mortality and stroke incidence. METHODS: We studied a population-based cohort of 1,263,721 residents in Rome aged [Formula: see text], followed from 2001 to 2013. As greenness exposure, we utilized the leaf area index (LAI), which expresses the tree canopy as the leaf area per unit ground surface area, and the normalized difference vegetation index (NDVI) within 300- and [Formula: see text] buffers around home addresses. We estimated the association between the two measures of residential greenness and the outcomes using Cox models, after controlling for relevant individual covariates and contextual characteristics, and explored potential mediation by air pollution [fine particulate matter with aerodynamic diameter [Formula: see text] [Formula: see text] and [Formula: see text]] and road traffic noise. RESULTS: We observed 198,704 deaths from nonaccidental causes, 81,269 from cardiovascular diseases [CVDs; 29,654 from ischemic heart disease (IHD)], 18,090 from cerebrovascular diseases, and 29,033 incident cases of stroke. Residential greenness, expressed as interquartile range (IQR) increase in LAI within [Formula: see text], was inversely associated with stroke incidence {hazard ratio (HR) 0.977 [95% confidence interval (CI): 0.961, 0.994]} and mortality for nonaccidental [HR 0.988 (95% CI: 0.981, 0.994)], cardiovascular [HR 0.984 (95% CI: 0.974, 0.994)] and cerebrovascular diseases [HR 0.964 (95% CI: 0.943, 0.985)]. Similar results were obtained using NDVI with 300- or [Formula: see text] buffers. CONCLUSIONS: Living in greener areas was associated with better health outcomes in our study, which could be partly due to reduced exposure to environmental hazards. Further research is required to understand the underlying mechanisms. https://doi.org/10.1289/EHP2854.

Impacts of air pollution on human and ecosystem health, and implications for the National Emission Ceilings Directive: Insights from Italy.

Across the 28 EU member states there were nearly half a million premature deaths in 2015 as a result of exposure to PM2.5, O3 and NO2. To set the target for air quality levels and avoid negative impacts for human and ecosystems health, the National Emission Ceilings Directive (NECD, 2016/2284/EU) sets objectives for emission reduction for SO2, NOx, NMVOCs, NH3 and PM2.5 for each Member State as percentages of reduction to be reached in 2020 and 2030 compared to the emission levels into 2005. One of the innovations of NECD is Article 9, that mentions the issue of "monitoring air pollution impacts" on ecosystems. We provide a clear picture of what is available in term of monitoring network for air pollution impacts on Italian ecosystems, summarizing what has been done to control air pollution and its effects on different ecosystems in Italy. We provide an overview of the impacts of air pollution on health of the Italian population and evaluate opportunities and implementation of Article 9 in the Italian context, as a case study beneficial for all Member States. The results showed that SO42- deposition strongly decreased in all monitoring sites in Italy over the period 1999-2017, while NO3- and NH4+ decreased more slightly. As a consequence, most of the acid-sensitive sites which underwent acidification in the 1980s partially recovered. The O3 concentration at forest sites showed a decreasing trend. Consequently, AOT40 (the metric identified to protect vegetation from ozone pollution) showed a decrease, even if values were still above the limit for forest protection (5000 ppb h-1), while PODy (flux-based metric under discussion as new European legislative standard for forest protection) showed an increase. National scale studies pointed out that PM10 and NO2 induced about 58,000 premature deaths (year 2005), due to cardiovascular and respiratory diseases. The network identified for Italy contains a good number of monitoring sites (6 for terrestrial ecosystem monitoring, 4 for water bodies monitoring and 11 for ozone impact monitoring) distributed over the territory and will produce a high number of monitored parameters for the implementation of the NECD.

Modeling ozone uptake by urban and peri-urban forest: a case study in the Metropolitan City of Rome.

Urban and peri-urban forests are green infrastructures (GI) that play a substantial role in delivering ecosystem services such as the amelioration of air quality by the removal of air pollutants, among which is ozone (O3), which is the most harmful pollutant in Mediterranean metropolitan areas. Models may provide a reliable estimate of gas exchanges between vegetation and atmosphere and are thus a powerful tool to quantify and compare O3 removal in different contexts. The present study modeled the O3 stomatal uptake at canopy level of an urban and a peri-urban forest in the Metropolitan City of Rome in two different years. Results show different rates of O3 fluxes between the two forests, due to different exposure to the pollutant, management practice effects on forest structure and functionality, and environmental conditions, namely, different stressors affecting the gas exchange rates of the two GIs. The periodic components of the time series calculated by means of the spectral analysis show that seasonal variation of modeled canopy transpiration is driven by precipitation in peri-urban forests, whereas in the urban forest seasonal variations are driven by vapor pressure deficit of ambient air. Moreover, in the urban forest high water availability during summer months, owing to irrigation practice, leads to an increase in O3 uptake, thus suggesting that irrigation may enhance air phytoremediation in urban areas.

Functional indicators of response mechanisms to nitrogen deposition, ozone, and their interaction in two Mediterranean tree species.

The effects of nitrogen (N) deposition, tropospheric ozone (O3) and their interaction were investigated in two Mediterranean tree species, Fraxinus ornus L. (deciduous) and Quercus ilex L. (evergreen), having different leaf habits and resource use strategies. An experiment was conducted under controlled condition to analyse how nitrogen deposition affects the ecophysiological and biochemical traits, and to explore how the nitrogen-induced changes influence the response to O3. For both factors we selected realistic exposures (20 kg N ha-1 yr-1 and 80 ppb h for nitrogen and O3, respectively), in order to elucidate the mechanisms implemented by the plants. Nitrogen addition resulted in higher nitrogen concentration at the leaf level in F. ornus, whereas a slight increase was detected in Q. ilex. Nitrogen enhanced the maximum rate of assimilation and ribulose 1,5-bisphosphate regeneration in both species, whereas it influenced the light harvesting complex only in the deciduous F. ornus that was also affected by O3 (reduced assimilation rate and accelerated senescence-related processes). Conversely, Q. ilex developed an avoidance mechanism to cope with O3, confirming a substantial O3 tolerance of this species. Nitrogen seemed to ameliorate the harmful effects of O3 in F. ornus: the hypothesized mechanism of action involved the production of nitrogen oxide as the first antioxidant barrier, followed by enzymatic antioxidant response. In Q. ilex, the interaction was not detected on gas exchange and photosystem functionality; however, in this species, nitrogen might stimulate an alternative antioxidant response such as the emission of volatile organic compounds. Antioxidant enzyme activity was lower in plants treated with both O3 and nitrogen even though reactive oxygen species production did not differ between the treatments.

Phytoremediation applications in natural condition and in mesocosm: The uptake of cadmium by Lemna minuta Kunth, a non-native species in Italian watercourses.

Metal pollution in water and soil is an environmental and public health issue. Cadmium (Cd) is included in the list of priority hazardous substances in the European Water Framework Directive. Phytoremediation system is a cost-effective, plant-based approach that takes advantage of the ability of plants to concentrate elements and compounds from the environment and to metabolize various molecules in their tissues. We studied the presence and the importance of an invasive species, such as Lemna minuta, in the environment and the effects of Cd pollution on this species. Growth, removal, and tolerance were evaluated for different Cd concentrations and different times of plant exposure. Overall, the results show that L. minuta has a good capacity of growth, metal bioconcentration, and tolerance up to 3 days of exposure at 0.5 and 1.5 mg L-1 of Cd. In particular, L. minuta was able to accumulate Cd up to 3771 mg kg-1 on dry mass basis. We can conclude that L. minuta possesses a great capability of Cd absorption and accumulation, thus supporting a potential use of this species in designing a metal bioremediation system in phytoremediation field.

Particle deposition in a peri-urban Mediterranean forest.

Urban and peri-urban forests provide a multitude of Ecosystem Services to the citizens. While the capacity of removing carbon dioxide and gaseous compounds from the atmosphere has been tested, their capacity to sequestrate particles (PM) has been poorly investigated. Mediterranean forest ecosystems are often located nearby or inside large urban areas. This is the case of the city of Rome, Italy, which hosts several urban parks and is surrounded by forested areas. In particular, the Presidential Estate of Castelporziano is a 6000 ha forested area located between the Tyrrhenian coast and the city (25 km downtown of Rome). Under the hypothesis that forests can ameliorate air quality thanks to particle deposition, we measured fluxes of PM1, 2.5 and 10 with fast optical sensors and eddy covariance technique. We found that PM1 is mainly deposited during the central hours of the day, while negligible fluxes were observed for PM 2.5 and 10. A Hybrid Single-Particle Lagrangian Integrated Trajectory model (HYSPLIT v4) simulated PM emission from traffic areas in the city of Rome and showed that a significant portion of PM is removed by vegetation in the days when the plume trajectory meets the urban forest.

Ecophysiological and phytochemical response to ozone of wine grape cultivars of Vitis vinifera L.

Vitis vinifera sensitivity to tropospheric ozone (O3) has been evidenced in several studies. In this work, physiological and metabolic effects of O3 on two wine cultivars of V. vinifera (i.e. Maturano and San Giuseppe) have been studied. Moreover, chlorogenic acid (CGA) production, in consideration of its importance in the biosynthetic pathway of polyphenols and as antioxidant, has been investigated. Maturano cultivar resulted more sensitive to O3, as evidenced by the gas exchange reduction at the early stage of treatment, and by the increase in Ci/Ca and the decoupling of net photosynthesis and the stomatal conductance at the end of the treatment. Unexpectedly, O3 did not activate stilbene production. Ozone induced an early CGA decrease, significantly more consistent in cv. Maturano, and an increase after 8 days, more consistent in cv. S. Giuseppe. These results suggest that CGA could be considered a biochemical marker of O3-induced stress in V. vinifera.

Effects of acute O3 stress on PSII and PSI photochemistry of sensitive and resistant snap bean genotypes (Phaseolus vulgaris L.), probed by prompt chlorophyll "a" fluorescence and 820 nm modulated reflectance.

The response of PSII and PSI photochemistry to acute ozone (O3) stress was tested in a "model plant system", namely the O3 sensitive (S156) and O3 resistant (R123) genotype pairs of Phaseolus vulgaris L., during a phenological phase of higher O3 sensitivity (pod formation). The modulation of the photosynthetic activity during O3 stress was analysed by measuring gas exchanges, Prompt Fluorescence (PF, JIP-test) and 820 nm Modulated Reflectance (MR), a novel techniques which specifically detects the changes in the redox state of P700 and plastocyanin. The results showed that, coherently with genotypic-specific O3 sensitivity, the response of the two snap bean genotypes differed for the intensity and time of onset of the considered physiological changes. In fact, despite leaf injury and gas exchanges reduction appeared concurrently in both genotypes, S156 showed a PSII down regulation already after the first day of fumigation (DOF), and an enhancement of Cyclic Electron Flow of PSI after the second DOF, whereas R123 showed only slight adjustments until the third DOF, when the activity of both photosystems was down-regulated. Despite these differences, it is possible to distinguish in both genotypes an early O3 response of the photochemical apparatus, involving PSII only, and a following response, in which PSI activity and content are also modulated. The measurement of the MR signal, performed simultaneously with the PF measurements and the JIP-test analysis, has allowed a better understanding of the role that PSI plays in the O3 stress response of the S156/R123 model plant system.

Comparison of drought stress response and gene expression between a GM maize variety and a near-isogenic non-GM variety.

Maize MON810, grown and commercialised worldwide, is the only cultivated GM event in the EU. Maize MON810, variety DKC6575, and the corresponding near-isogenic line Tietar were studied in different growth conditions, to compare their behaviour in response to drought. Main photosynthetic parameters were significantly affected by drought stress in both GM and non-GM varieties to a similar extent. Though DKC6575 (GM) had a greater sensitivity in the early phase of stress response as compared with Tietar (non-GM), after six days of stress they behaved similarly, and both varieties recovered from stress damage. Profiling gene expression in water deficit regimes and in a generalised drought stress condition showed an up-regulation of many stress-responsive genes, but a greater number of differentially expressed genes was observed in Tietar, with genes belonging to transcription factor families and genes encoding heat shock proteins, late embryogenesis abundant proteins and detoxification enzymes. Since induction of these genes have been indicated from the literature as typical of stress responses, their activation in Tietar rather than in DKC6575 may be reminiscent of a more efficient response to drought. DKC6575 was also analysed for the expression of the transgene CryIAb (encoding the delta-endotoxin insecticidal protein) in water deficit conditions. In all the experiments, the CryIAb transcript was not influenced by drought stress, but was expressed at a constant level. This suggests that though possessing a different pattern of sensitivity to stress, the GM variety maintains the same expression level for the transgene.

[Health risks associated with urban environmental pollution in Europe: results of the HEREPLUS multicentre study in Rome (Italy)]. / Rischi per la salute da inquinamento urbano nei sistemi urbani europei: approfondimento sui risultati dello studio multicentrico HEREPLUS nella città di Roma.

Urban air pollution is known to cause numerous health problems. The HEREPLUS project aims to evaluate the association between environmental air pollution and health and to provide guidelines for the management and organization of the urban environment. Risk maps were developed by georeferentiation, using hospitalization data and concentration levels of ozone and pollution matter. Data for four European cities involved in the project (Rome, Madrid, Dresden, and Athens) were analysed for the period 2003-2004. In this article, we report results for Rome only. Study findings demonstrated that the green area in a city reduces pollution in a city reduces pollution levels in the atmosphere, thus reducing health risks.

Photosynthetic performance and biochemical adjustments in two co-occurring Mediterranean evergreens, Quercus ilex and Arbutus unedo, differing in salt-exclusion ability.

The responses to mild root zone salinity stress were investigated in two co-occurring Mediterranean woody evergreens, Quercus ilex L. and Arbutus unedo L., which differ in morpho-anatomical traits and strategies to cope with water deficit. The aim was to explore their strategies to allocate potentially toxic ions at organism level, and the consequential physiological and biochemical adjustments. Water and ionic relations, gas exchange and PSII performance, the concentration of photosynthetic pigments, and the activity of antioxidant defences, were measured. Q. ilex displayed a greater capacity to exclude Na+ and Cl- from the leaf than A. unedo, in part as a consequence of greater reductions in transpiration rates. Salt-induced reductions in CO2 assimilation resulted in Q. ilex suffering from excess of light to a greater extent than A. unedo. Consistently, in Q. ilex effective mechanisms of nonphotochemical quenching, also sustained by the lutein epoxide-lutein cycle, operated in response to salinity stress. Q. ilex also displayed a superior capacity to detoxify reactive oxygen species (ROS) than A. unedo. Our data suggest that the ability to exclude salt from actively growing shoot organs depends on the metabolic cost of sustaining leaf construction, i.e. species-specific leaf life-span, and the relative strategies to cope with salt-induced water stress. We discuss how contrasting abilities to restrict the entry and transport of salt in sensitive organs relates with species-specific salt tolerance.

Urban ecosystem services: tree diversity and stability of tropospheric ozone removal.

Urban forests provide important ecosystem services, such as urban air quality improvement by removing pollutants. While robust evidence exists that plant physiology, abundance, and distribution within cities are basic parameters affecting the magnitude and efficiency of air pollution removal, little is known about effects of plant diversity on the stability of this ecosystem service. Here, by means of a spatial analysis integrating system dynamic modeling and geostatistics, we assessed the effects of tree diversity on the removal of tropospheric ozone (O3) in Rome, Italy, in two years (2003 and 2004) that were very different for climatic conditions and ozone levels. Different tree functional groups showed complementary uptake patterns, related to tree physiology and phenology, maintaining a stable community function across different climatic conditions. Our results, although depending on the city-specific conditions of the studied area, suggest a higher function stability at increasing diversity levels in urban ecosystems. In Rome, such ecosystem services, based on published unitary costs of externalities and of mortality associated with O3, can be prudently valued to roughly US$2 and $3 million/year, respectively.

Comparison of seasonal variations of ozone exposure and fluxes in a Mediterranean Holm oak forest between the exceptionally dry 2003 and the following year.

Ozone and energy fluxes have been measured using the eddy covariance technique, from June to December 2004 in Castelporziano near Rome (Italy), and compared to similar measurements made in the previous year. The studied ecosystem consisted in a typical Mediterranean Holm oak forest. Stomatal fluxes have been calculated using the resistance analogy and by inverting the Penmann-Monteith equation. Results showed that the average stomatal contribution accounts for 42.6% of the total fluxes. Non-stomatal deposition proved to be enhanced by increasing leaf wetness and air humidity during the autumnal months. From a comparison of the two years, it can be inferred that water supply is the most important limiting factor for ozone uptake and that prolonged droughts alter significantly the stomatal conductance, even 2 months after the soil water content is replenished. Ozone exposure, expressed as AOT40, behaves similarly to the cumulated stomatal flux in dry conditions whereas a different behaviour for the two indices appears in wet autumnal conditions. A difference also occurs between the two years.

Impact of ozone on monoterpene emissions and evidence for an isoprene-like antioxidant action of monoterpenes emitted by Quercus ilex leaves.

Quercus ilex (L.) leaves emit monoterpenes, particularly alpha-pinene, beta-pinene and sabinene. Apart from the monoterpene pools that are stored in specialized structures and have a clear defensive or attractive role, the function of monoterpenes in Q. ilex leaves is unknown. We tested whether monoterpenes have an antioxidant role, as has previously been found for isoprene in isoprene-emitting leaves. We exposed Q. ilex leaves to either mild and repeated ozone exposure (Experiment I) or to a single acute ozone exposure (Experiment II) at temperatures ranging between 20 and 32 degrees C. Both ozone treatments rapidly stimulated monoterpene synthesis, but had no effect on photosynthesis and caused no visible damage to leaves maintained at 25, 30 or 32 degrees C. Ozone inhibited both photosynthesis and monoterpene synthesis in leaves maintained at 20 degrees C. To characterize the relationship between monoterpenes and ozone-induced damage, we fed detached leaves fosmidomycin, a selective inhibitor of isoprene synthesis. Fosmidomycin caused rapid and complete inhibition of monoterpene emissions in leaves maintained at 30 degrees C, confirming that monoterpenes are synthesized by the same biochemical pathway as isoprene. However, over the experimental period, fosmidomycin did not affect concentrations of compounds that are formed from chloroplastic isoprenoids and that might have conferred antioxidant protection, either directly (carotenoids) or indirectly (chlorophylls, xanthophylls). In leaves whose monoterpene synthesis had been inhibited by fosmidomycin, ozone rapidly and significantly inhibited photosynthesis and increased the production of hydrogen peroxide and malonyldialdehyde. We conclude that monoterpenes produced by Q. ilex leaves share the same biosynthetic pathway and function as isoprene. Furthermore, all volatile isoprenoids may have similar antioxidant properties and may be stimulated by the same stress-inducing conditions.

Integrated ambient ozone evaluation by passive samplers and clover biomonitoring mini-stations.

An ozone monitoring network was set up using passive samplers and biological mini-stations of two clones of white clover (Trifolium repens L. cv. Regal), NC-R (O(3)-resistant) and NC-S (O(3)-sensitive). This paper reports on a pilot study performed in the period June-October 1999 in the Rome municipal area by using five biomonitoring mini-stations and ozone passive samplers with a new nitrite based design. This combined methodology can be used to obtain information on the biological implications of the injury due to tropospheric ozone. The two techniques can integrate data for the short-medium period and can be placed in different urban and rural sites, proving to be a very useful tool for ozone concentration mapping.

Physiological response of Pinus halepensis needles under ozone and water stress conditions.

The aim of this study was to evaluate how physiological processes of potted Pinus halepensis plants, grown under controlled conditions, were affected by ozone (O3) and/or water stress, integrating the gas exchange and biochemical data with fluorescence OJIP polyphasic transient data. Plants submitted to only water stress (T1) and with ozone (T3) showed a strong decrease in stomatal conductance and gas exchange, coinciding with a reduction of maximum yield of photochemistry (varphipo) and very negative values of leaf water potential. Simultaneously, a great increase of both PSII antenna size, indicated by absorption per reaction centre, and electron transport per reaction centre were found. The reduction of photosynthesis in the O3-treated plants (T2) by a slowing down of the Calvin cycle was supported by the increase of related fluorescence parameters such as relative variable fluorescence, heat de-excitation constant, energy de-excitation by spillover, and the decrease of varphipo. We suggest an antagonistic effect between the two stresses to explain the delayed ozone-induced decrease of stomatal conductance values for T3 with respect to T1 plants, by an alteration of the physiological mechanisms of stomatal opening, which involve the increase of intra-cellular free-calcium induced by ABA under co-occurring water shortage. We emphasise the importance of considering the intensity of the individual stress factor in studies concerning the interaction of stresses.