Phenylethylamine-induced generation of reactive oxygen species and ascorbate free radicals in tobacco suspension culture: Mechanism for oxidative burst mediating Ca2+ influx

In the previous paper [Kawano et al, (2000a) Plant Cell Physiol. 41: 1251], we demonstrated that addition of phenylethylamine (PEA) and benzylamine ca n induce an immediate and transient burst of active oxygen species (AOS) in tobacco suspension culture. Detected AOS include H2O2, superoxide anion an d hydroxyl radicals. Use of several inhibitors suggested the presence of mo noamine oxidase-like H2O2-generating activity in the cellular soluble fract ion. It was also suggested that peroxidase(s) or copper amine oxidase(s) ar e involved in the extracellular superoxide production as a consequence of H 2O2 production. Since more than 85% of the PEA-dependent AOS generating act ivity was localized in the extracellular space (extracellular fluid + cell wall), extracellularly secreted enzymes, probably peroxidases, may largely contribute to the oxidative burst induced by PEA. The PEA-induced AOS gener ation was also observed in the horseradish peroxidase (HRP) reaction mixtur e, supporting the hypothesis that peroxidases catalyze the oxidation of PEA leading to AOS generation. In addition to AOS production, we observed that PEA induced an increase in monodehydroascorbate radicals (MDA) in the cell suspension culture and in HRP reaction mixture using electron spin resonan ce spectroscopy and the newly invented MDA reductase-coupled method, Here w e report that (MDA) production is an indicator of peroxidase-mediated gener ation of PEA radical species in tobacco suspension culture.