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Tropospheric ozone acts as a phytotoxin which produces an oxidative stress in plants. Two ways of defense are used, either by preventing ozone input through the regulation of stomatal conductance, or by detoxifying ozone and ROS in cells. It is known that stomatal movements are alt ered by ozone. We performed fumigation experiment on three euramerican poplar genotypes ( Po pulus deltoides x Populus Nigra : ‘Carpaccio’, ‘Cima’ and ‘Robusta’ ), cultivated in pots in phytotronic chambers submitted to 120 ppb ozone or filtered air. We explor ed the effects of ozone on stomatal responses to four environmental parameters (blue light, red light, CO 2 and VPD). We also find out using a porometer that the decrease of stomatal conductance due to ozone is earlier on the adaxial face than on abaxial fa ce. Finally, to better understand these impacts, we studied ultrastructure of guard cells by TEM, stomatal density and size of stomata by SEM, and we performed X - ray microanalysis of guard cells mineral content. These approaches are coupled with the study on microdissected stomata of expression of genes involved in regulation of stomatal movements

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Tropospheric ozone is a major air pollutant that acts as a phytotoxin. It enters the leaf through the stomata before being dissolved in the apoplast by generating reactive oxygen species (ROS) causing oxidative stress. Two defenses exist to restrict the effects of ozone: (i) the stomata which can limit ozone uptake by regulating stomatal conductance and (ii) the detoxification processes of ROS generated by ozone.We studied the effects of ozone (120 ppb) on these two mechanisms of defense in three euramerican poplar genotypes (Populus deltoides x Populus nigra) under controlled conditions in phytotronic chambers. A direct effect of ozone on photosynthesis and stomatal movements in response to changes in environmental factors (by slowing the stomatal opening and closure) has been highlighted. Models of stomatal conductance, on which indicators of critical level of ozone for vegetation are based, must take them into account. In addition, these studies have highlighted the role of constitutive concentrations of antioxidants in tolerance to ozone as well as the complexity of these detoxification mechanisms. The notion of effective ozone flux must consider these two aspects to better characterize the intra-and inter-specific differences in sensitivity to ozone, L'ozone troposphérique est un polluant atmosphérique majeur qui agit comme une phytotoxine. Il pénètre dans les feuilles par les stomates avant d'être dissout dans l'apoplaste en générant des radicaux libres oxygénés (ROS) provoquant ainsi un stress oxydatif. Deux barrières existent pour restreindre les effets de l'ozone : (i) les stomates qui peuvent limiter les flux entrants par contrôle de la conductance stomatique et (ii) le système de détoxication des ROS issus de la dégradation de l'ozone. Nous avons étudié les effets de l'ozone (120 ppb) sur ces deux moyens de défense chez trois génotypes de peuplier euraméricain (Populus deltoides x Populus nigra) placés en conditions contrôlées dans des chambres phytotroniques. Un effet direct de l'ozone sur la photosynthèse et sur les mouvements stomatiques en réponse à des variations de facteurs environnementaux (ralentissement des phénomènes d'ouverture et de fermeture) a été mis en évidence. Les modèles de calcul de la conductance stomatique, sur lesquels se basent les indicateurs de seuil de risque à l'ozone pour la végétation, doivent donc les prendre en compte. De plus, ces travaux ont mis en évidence le rôle prépondérant des concentrations constitutives en antioxidants dans la tolérance à l'ozone ainsi que la complexité de ces mécanismes de détoxication. La notion de flux effectif d'ozone doit prendre en considération ces deux aspects afin de caractériser au mieux les différences de sensibilité à l'ozone intra et inter spécifique

Ozone is a phytotoxic air pollutant causing oxidative stress. We studied the effect of ozone on carotenoids, chlorophylls and polyisoprenoid alcohols in three euramerican poplar clones (Populus deltoides x Populus nigra: 'Carpaccio', 'Cima' and 'Robusta'). Poplars originating from cuttings were grown for 6 weeks and exposed to ozone in fumigation chambers (120 ppb each day for 13h). Leaf samples were collected 2, 4, 11, 15 and 17 days after the start of ozone treatment. Chemical analyses were made by HPLC-APCI-MS. The clones clearly differed in growth rate and ozone responses. 'Carpaccio' had the highest above-ground biomass, and it was also the most ozone tolerant genotype. Greatest growth reductions by ozone were measured for 'Cima ', while 'Robusta' showed the greatest number of visible injuries. Ozone and control samples were clearly separate in PCA based on analysed chemical profiles, so that ozone treatment explained 50% of the variance. Ozone reduced the concentrations of chlorophylls a and b, β-carotenes, luteins, neoxanthin and some unidentified isoprenoid derivatives in all clones. The difference between the ozone and control treatments was the strongest on the last two sampling dates. The genotypes differed from each other, but genotype explained only 12% of the variation.

It is well admitted that to apprehend the critical level of ozone susceptible to damage vegetation, the effective ozone flux, corresponding to the non-detoxified ozone molecules, needs to be determined (Musselman et al., 2006; Wieser and Matyssek, 2007; Dizengremel et al.2008). This concept firstly integrates the stomatal conductance to appreciate the amount of ozone entering in the leaf. Additionally it needs to consider the capacity of the cell to neutralise ROS which could be estimated by the levels of antioxidant molecules but also by the activity of enzymes generating NADPH, useful to regenerate ascorbate and glutathione (Dizengremel et al., 2009). In a preliminary experiment, we have selected 3 Populus x euramericanagenotypes (Carpaccio, Cima and Robusta) for their distinct response to chronic ozone exposure (extent of necrosis and leaf loss). Subsequently several metabolites (ascorbate, glutathione, carotenoids, polyphenols) as well as the activity of NADPH delivering enzymes (malic enzyme, isocitrate dehydrogenase, glucose-6-P-dehydrogenase, glyceraldehyde-3-P-dehydrogenase) have been measured in poplar genotypes exposed to 120 ppb ozone for 21 days. The results which were expressed relative to the instantaneous ozone flux through stomata, underlined intraspecific variability of these poplar genotypes in their capacity to detoxify