Early events in Populus hybrid and Fagus sylvatica leaves exposed to ozone.

This paper aims to investigate early responses to ozone in leaves of Fagus sylvatica (beech) and Populus maximowiczii x Populus berolinensis (poplar). The experimental set-up consisted of 4 open air plots (OA), 4 charcoal filtered open-top chambers (CF) and 4 non filtered open-top chambers (NF). Qualitative and quantitative analyses were carried out on non-symptomatic (CF) and symptomatic (NF and OA) leaves of both species. Qualitative analyses were performed applying microscopical techniques: Evans blue staining for detection of cell viability; CeCl3 staining of transmission electron microscope (TEM) samples to detect the accumulation of H2O2; multispectral fluorescence microimaging and microspectrofluorometry to investigate the accumulation of fluorescent phenolic compounds in the walls of the damaged cells. Quantitative analyses consisted in the analysis of the chlorophyll a fluorescence transients (fast kinetics). The early responses to ozone were demonstrated by Evans blue and CeCl3 staining techniques that provided evidence of plant responses in both species one month before foliar symptoms became visible. The fluorescence transients analysis too, demonstrated the breakdown of the oxygen evolving system and the inactivation of the end receptors of electrons at a very early stage, both in poplar and in beech. The accumulation of phenolic compounds in the cell walls, on the other hand, was a species-specific response detected in poplar, but not in beech. Evans blue and CeCl3 staining, as well as the multispectral fluorescence microimaging and microspectrofluorometry, can be used to support the field diagnosis of ozone injury, whereas the fast kinetics of chlorophyll fluorescence provides evidence of early physiological responses.