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 (O3) 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 O3 concentrations (O3]), unconfounded by other influences on O3 sensitivity, such as genetic, meteorological and soil factors. To simulate the diurnal cycle of O3 formation, current-year and 1-year-old Quercus ilex (L.) and Quercus pubescens (L.) leaves were fumigated with O3 at different positions (and hence, different leaf ages) on the same branch over three consecutive days. The LOF system supplied a high O3] (300+/-50 ppb) on leaves appressed to the vents, and an intermediate, super-ambient O3] (varying between 120 and 280 ppb) on leaves less than 30 cm from the vent. Leaves more than 60 cm from the O3 vent were exposed to an O3] comparable with the ambient concentration, with a 100 ppb peak during the hottest hours of the day. Only leaves exposed to the high O3] were affected by the 3-day treatment, confirming that Mediterranean oak are tolerant to ambient and super-ambient O3], but may be damaged by acute exposure to high O3]. Stomatal and mesophyll conductance and photosynthesis were all reduced immediately after fumigation with high O3], but recovered to control values within 72 h. Both the intercellular and chloroplast CO2 concentrations (CO2]) remained constant throughout the experiment. Thus, although treatment with a high O3] 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 O3 than current-year leaves, suggesting that the low stomatal conductance observed in aging leaves limited O3 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 to low O3], and therefore may not be an effect of O3 treatment. Light respiration, on the other hand, was mostly constant in ozone-treated leaves and increased only in leaves in which photosynthesis was temporarily inhibited by high O3], preventing 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 O3] showed limited photochemical damage, but recovered rapidly. Biochemical markers were affected by the high O3], 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 O3 stress.