Localized ozone fumigation system for studying ozone effects on photosynthesis, respiration, electron transport rate and isoprene emission in field-grown Mediterranean oak species

We used a localized ozone (O-3) fumigation (LOF) system to study acute and short-term O-3 effects on physiological leaf traits. The LOF system enabled investigation of primary and secondary metabolic responses of similarly and differently aged leaves on the same plant to three different O-3 concentrations ([O-3]), unconfounded by other influences on O-3 sensitivity, such as genetic, meteorological and soil factors. To simulate the diurnal cycle of O-3 formation, current-year and 1-year-old Quercus ilex (L.) and Quercus pubescens (L.) leaves were fumigated with O-3 at different positions (and hence, different leaf ages) on the same branch over three consecutive days. The LOF system supplied a high [O-3] (300 +/- 50 ppb) on leaves appressed to the vents, and an intermediate, super-ambient [O-3] (varying between 120 and 280 ppb) on leaves less than 30 cm from the vent. Leaves more than 60 cm from the O-3 vent were exposed to an [O-3] comparable with the ambient concentration, with a 100 ppb peak during the hottest hours of the day. Only leaves exposed to the high [O-3] were affected by the 3-day treatment, confirming that Mediterranean oak are tolerant to ambient and super-ambient [O-3], but may be damaged by acute exposure to high [O-3]. Stomatal and mesophyll conductance and photosynthesis were all reduced immediately after fumigation with high [O-3], but recovered to control values within 72 h. Both the intercellular and chloroplast CO concentrations ([CO2]) remained constant throughout the experiment. Thus, although treatment with a high [O-3] may have induced stomatal closure and consequent down-regulation of photosynthesis, we found no evidence that photosynthesis was limited by low [CO2] at the site of fixation. One-year-old leaves of Q. ilex were much less sensitive to O-3 than current-year leaves, suggesting that the low stomatal conductance observed in aging leaves limited O-3 uptake. No similar effect of leaf age was found in Q. pubescens. Dark respiration decreased during the treatment period, but a similar decrease was observed in leaves exposed venting them from acting as strong sinks that recycle respiratory CO2 in the leaves. There was no evidence of photochemical damage in Q. ilex leaves, whereas Q. pubescens leaves exposed to a high [O-3] showed limited photochemical damage, but recovered rapidly. Biochemical markers were affected by the high [O-3], indicating accumulation of reactive oxygen species (ROS) and increased denaturation of lipid membranes, followed by activation of isoprene biosynthesis in Q. pubescens leaves. We speculate that the high isoprene emissions helped quench ROS and normalize membrane stability in leaves recovering from O-3 stress.