Cell death behind invisible symptoms: early diagnosis of ozone injury

A simple histo-cytochemical method, combining Evans blue staining to assess cell death and in vivo 3,3′-diaminobenzidine uptake for H2O2 localisation, has been used to evaluate O3 damages in leaf tissues of three Phaseolus vulgaris L. cultivars (Cannellino, BLF, Saxa) with different sensitivity to the pollutant. Bean plants were exposed to a single pulse of O3 (150 ± 10 mm3 m−3 × 3 h) and leaves were examined at different time-span after fumigation. Cannellino proved to be the most sensitive, showing chlorotic spots 2 h after fumigation and chlorotic lesions 24 h later. In BLF, necrotic spots appeared 4 h after fumigation and reddish necrotic lesions (bronzing) developed in further 24 h. Saxa remained symptomless up to 10 d of observation, thus appearing tolerant. The early appearance of symptoms in Cannellino correlated with H2O2 accumulation in leaf tissues and consequent extensive cell death, involving both palisade and spongy mesophyll. H2O2 accumulation was observed also in BLF, though to a lesser extent and dead cells were rare at 2 h after fumigation. However, they increased in number 24 h later, forming small groups in the palisade mesophyll. These groups further enlarged in the next 24 h, again involving only palisade mesophyll. In Saxa leaves, H2O2 accumulation was found only in the epidermal cells, though the number of dead cells was very similar to BLF, at least up to 24 h after fumigation. However, in Saxa, dead cells have been always found singly scattered through the palisade mesophyll, or forming very small groups around substomatal cavity, thus remaining invisible at a macroscopic level