Impact of high ozone on isoprene emission, photosynthesis and histologyof developing Populus alba leaves directly or indirectly exposed to the pollutant.

The direct and indirect impact of ozone on Populus alba was studied by exposing leaves enclosed in specially designed cuvettes for 30 days to high ozone (150 ppb, 11 h per day), while leaves developing above the cuvettes were exposed to ambient ozone. Gas exchanges and histo-anatomical parameters were measured to specifically understand whether ozone indirectly affects the anatomy and physiology of leaves. Three leaf classes were investigated: (1) those expanding above the cuvettes (A leaves); (2) those already developed inside the cuvettes (B leaves) and (3) those developing inside the cuvettes, since the beginning of the ozone treatment (C leaves). The anatomy and morphology of the first leaf developing outside the cuvette (A1) were strongly affected by ozone, whereas photosynthesis was not perturbed. However, in leaves of ozone-treated plants developing after A1, a large reduction of starch accumulation was observed, which suggests a delayed biosynthesis, or a very rapid export of starch toward other sinks. Isoprene emission was higher and isoprene synthase messenger RNA was more expressed in ozone-treated A1 leaves than in control leaves with similar ontogeny. This indicates that isoprene synthesis is stimulated by ozone, and reveals that isoprene emission is controlled at a transcriptional level. Leaves already developed inside the cuvette (B leaves) rapidly sensed ozone stress, which inhibited photosynthesis, stomatal conductance and isoprene emission. The observation that new leaves were developing inside the cuvettes during the treatment (C leaves) suggests that resistance to ozone may be acquired by plants. Leaves C showed a more packed and thinner mesophyll than controls of similar development, which may help reduce ozone penetration inside cells. They also showed a lower photosynthesis in comparison to controls and to other leaf classes, probably because of ribulose 1,5-bisphosphate carboxylase/oxygenase activity limitation, as inferred from photosynthesis response to intercellular CO2. However, isoprene emission was slightly stimulated also in C leaves, confirming that a large fraction of carbon is invested into isoprene formation under ozone stress.