In situ histochemical monitoring of ozone- and TMV-induced reactive oxygen species in tobacco leaves

The pattern and time course of reactive oxygen species (ROS) production were monitored histochemically in vivo in tobacco (Nicotiana tabacum L.) cv. Bel W3 and cv. Bel B leaves, comparing the hypersensitive (HR)-like reaction induced by the abiotic stressor ozone and the response to tobacco mosaic virus (TMV). The applied assays (nitro blue tetrazolium, NBT; 2’,7’-dichlorofluorescin diacetate, DCFH-DA; 3,3’-diaminobenzidine, DAB) gave positive results, evidencing scattered ROS production in a spotted pattern. DCFH-DA and DAB predominantly evidenced localised, long-lasting hydrogen peroxide production in cells prone to die, producing very similar pictures in TMV infected leaves of both tobacco cultivars and in ozone-sensitive Bel W3. Nitro blue tetrazolium reaction pointed out that a transient oxidative burst involving superoxide anion is induced early by ozone also in the tolerant cv. Bel B, despite the low malondialdehyde level.