Ozone (O3) is a photochemically formed reactive gas responsible for a decreasing carbon assimilation in plant ecosystems. Present in the atmosphere in trace concentrations (less than 100 ppbv), this molecule is capable of inhibiting carbon assimilation in agricultural and forest ecosystems. Ozone-risk assessments are typically based on manipulative experiments. Present regulations regarding critical ozone levels are mostly based on an estimated accumulated exposure over a given threshold concentration. There is however a scientific consensus over flux estimates being more accurate, because they include plant physiology analyses and different environmental parameters that control the uptake—that is, not just the exposure—of O3. While O3 is a lot more difficult to measure than other non-reactive greenhouse gases, UV-based and chemiluminescence sensors enable precise and fast measurements and are therefore highly desirable for eddy covariance studies. Using micrometeorological techniques in association with latent heat flux measurements in the field allows for the partition of ozone fluxes into the stomatal and non-stomatal sinks along the soil-plant continuum. Long-term eddy covariance measurements represent a key opportunity in estimating carbon assimilation at high-temporal resolutions, in an effort to study the effect of climate change on photosynthetic mechanisms. Our aim in this work is to describe potential of O3 flux measurement at the canopy level for ozone-risk assessment in established long-term monitoring networks.

To investigate the interactive effects of increasing [CO₂] and heat wave occurrence on isoprene (IE) and methanol (ME) emissions, Platanus orientalis was grown for one month in ambient (380 mmol mol 1) or elevated (800 mmol mol 1) [CO₂] and exposed to high temperature (HT) (38 C/4 h). In pre-existing leaves, IE emissions were always higher but ME emissions lower as compared to newly-emerged leaves. They were both stimulated by HT. Elevated [CO₂] significantly reduced IE in both leaf types, whereas it increased ME in newly-emerged leaves only. In newly-emerged leaves, elevated [CO₂] decreased photosynthesis and altered the chloroplast ultrastructure and membrane integrity. These harmful effects were amplified by HT. HT did not cause any unfavorable effects in pre-existing leaves, which were characterized by inherently higher IE rates. We conclude that: (1) these results further prove the isoprene's putative thermo-protective role of membranes; (2) HT may likely outweigh the inhibitory effects of elevated [CO₂] on IE in the future.

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. EDU was successful as a tool in determining ozone effects on vegetation in Italy, but while progress was made, its mode of action remains unresolved.

An account of histo-cytological and ultrastructural studies on ozone effect on crop and forest species in Italy is given, with emphasis on induced cell death and the underlying mechanisms. Cell death phenomena possibly due to ambient O3 were recorded in crop and forest species. In contrast, visible O3 effects on Mediterranean vegetation are often unclear. Microscopy is thus suggested as an effective tool to validate and evaluate O3 injury to Mediterranean vegetation. A DAB-Evans blue staining was proposed to validate O3 symptoms at the microscopic level and for a pre-visual diagnosis of O3 injury. The method has been positively tested in some of the most important crop species, such as wheat, tomato, bean and onion and, with some restriction, in forest species, and it also allows one to gain some very useful insights into the mechanisms at the base of O3 sensitivity or tolerance. Ozone-induced cell death is a frequent phenomenon in Mediterranean conditions, not only in the most sensitive crops but also in forest species.

A rapid, quantitative bioassay for detecting phytotoxic air pollutants has been developed. The technique uses wheat Triticum aestivum L. or tomato Lycopersicon esculentum L., seedlings growing on an agar medium in test-tubes. The seedlings are exposed to a pollutant in the test-tube and stress-ethylene induced by the pollutant is quantitatively measured by gas chromatography. Increases in ethylene production from seedlings exposed to a phytotoxic air pollutant as compared with controls not exposed to pollutants were related to the pollutant concentration.

Apoplastic ascorbate (ASCapo) is an important contributor to the detoxification of ozone (O3). The objective of the study is to explore whether ASCapo is stimulated by elevated O3 concentrations. The detoxification of O3 by ASCapo was quantified in tobacco (Nicotiana L), soybean (Glycine max (L.) Merr.) and poplar (Populus L), which were exposed to charcoal-filtered air (CF) and elevated O3 treatments (E-O3). ASCapo in the three species were significantly increased by E-O3 compared with the values in the filtered treatment. For all three species, E-O3 significantly increased the malondialdehyde (MDA) content and decreased light-saturated rate of photosynthesis (Asat), suggesting that high O3 has induced injury/damage to plants. E-O3 significantly increased redox state in the apoplast (redox stateapo) for all species, whereas no effect on the apoplastic dehydroascorbate (DHAapo) was observed. In leaf tissues, E-O3 significantly enhanced reduced-ascorbate (ASC) and total ascorbate (ASC+DHA) in soybean and poplar, but significantly reduced these in tobacco, indicating different antioxidative capacity to the high O3 levels among the three species. Total antioxidant capacity in the apoplast (TACapo) was significantly increased by E-O3 in tobacco and poplar, but leaf tissue TAC was significantly enhanced only in tobacco. Leaf tissue superoxide anion (O2•-) in poplar and hydrogen peroxide (H2O2) in tobacco and soybean were significantly increased by E-O3. The diurnal variation of ASCapo, with maximum values occurring in the late morning and lower values experienced in the afternoon, appeared to play an important role in the harmful effects of O3 on tobacco, soybean and poplar.

Plants can respond physiologically to damaging ultraviolet-B radiation by altering leaf chemistry, especially UV absorbing phenolic compounds. However, the effects on terpene emissions have received little attention. We conducted two field trials in plots with supplemented UV-B radiation and assessed the influence of feeding by pine weevils, Hylobius abietis L., on volatile emissions from 3-year old Norway spruce trees (Picea abies L. Karst.). We collected emissions from branch tips distal to the feeding weevils, and from whole branches including the damage sites. Weevil feeding clearly induced the emission of monoterpenes and sesquiterpenes, particularly linalool and (E)-β-farnesene, from branch tips, and the sums of monoterpenes and sesquiterpenes emitted by whole branches were substantially increased. We discovered little effect of UV-B radiation up to 30% above the ambient level on volatile emissions from branch tips distal to damage sites, but there was a possible effect on bark emissions from damage sites. Chronic exposure to enhanced UV-B radiation has little effect on volatile emissions of Norway spruce.

Stomatal ozone uptake, determined with the Jarvis' approach, was related to photosynthetic efficiency assessed by chlorophyll fluorescence and reflectance measurements in open-top chamber experiments on Phaseolus vulgaris. The effects of O₃ exposure were also evaluated in terms of visible and microscopical leaf injury and plant productivity. Results showed that microscopical leaf symptoms, assessed as cell death and H₂O₂ accumulation, preceded by 3-4 days the appearance of visible symptoms. An effective dose of ozone stomatal flux for visible leaf damages was found around 1.33 mmol O₃ m⁻². Significant linear dose-response relationships were obtained between accumulated fluxes and optical indices (PRI, NDI, ΔF/Fm'). The negative effects on photosynthesis reduced plant productivity, affecting the number of pods and seeds, but not seed weight. These results, besides contributing to the development of a flux-based ozone risk assessment for crops in Europe, highlight the potentiality of reflectance measurements for the early detection of ozone stress. Ozone stomatal fluxes affect leaf cell viability, photosynthetic performance, optical properties and crop yield of bean.

Nitrate and ammonium concentration in wet deposition detrimentally impacted a sensitive pollution indicator species irrespective of the nitrogen dose.

An Open-Top Chambers experiment on Fagus sylvatica and Quercus robur seedlings was conducted in order to compare the performance of an exposure-based (AOT40) and a flux-based approaches in predicting the appearance of ozone visible injuries on leaves. Three different ozone treatments (charcoal-filtered; non-filtered; and open plots) and two soil moisture treatments (watered and non-watered plots) were performed. A Jarvisian stomatal conductance model was drawn up and parameterised for both species and typical South Alpine environmental conditions, thus allowing the calculation of ozone stomatal fluxes for every treatment. A critical ozone flux level for the onset of leaf visible injury in beech was clearly identified between 32.6 and 33.6 mmol O3m-2. In contrast, it was not possible to identify an exposure critical level using the AOT40 index. Water stress delayed the onset of the leaf visible injuries, but the flux-based approach was able to take it into account accurately. Ozone fluxes are more accurate than AOT40 exposure index in predicting ozone visible foliar injury onset on beech seedlings in South Alpine environmental conditions.