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 can induce an immediate and transient burst of active oxygen species (AOS) in tobacco suspension culture. Detected AOS include H₂O₂, superoxide anion and hydroxyl radicals. Use of several inhibitors suggested the presence of monoamine oxidase-like H₂O₂-generating activity in the cellular soluble fraction. It was also suggested that peroxidase(s) or copper amine oxidase(s) are involved in the extracellular superoxide production as a consequence of H₂O₂ production. Since more than 85% of the PEA-dependent AOS generating activity 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 generation was also observed in the horseradish peroxidase (HRP) reaction mixture, 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 resonance spectroscopy and the newly invented MDA reductase-coupled method. Here we report that MDA production is an indicator of peroxidase-mediated generation of PEA radical species in tobacco suspension culture.