Journal Article

Phenylethylamine-Induced Generation of Reactive Oxygen Species and Ascorbate Free Radicals in Tobacco Suspension Culture: Mechanism for Oxidative Burst Mediating Ca<sup>2+</sup> Influx

Tomonori Kawano, Reinhard Pinontoan, Nobuyuki Uozumi, Yasujiro Morimitsu, Chikahiro Miyake, Kozi Asada and Shoshi Muto

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 41, issue 11, pages 1259-1266
Published in print November 2000 | ISSN: 0032-0781
Published online November 2000 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pcd053
Phenylethylamine-Induced Generation of Reactive Oxygen Species and Ascorbate Free Radicals in Tobacco Suspension Culture: Mechanism for Oxidative Burst Mediating Ca2+ Influx

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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.

Keywords: Key words: Active oxygen species — Monoamine oxidase — Monodehydroascorbate — Nicotiana tabacum — Peroxidase — Phenylethylamine.; Abbreviations: AMAs, aromatic monoamines and their derivatives; AOS, active oxygen species; AsA, ascorbic acid; [Ca2+]c, cytosolic free Ca2+ concentration; CL, chemiluminescence; CLA, 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one; Cu-AO, copper-amine oxidase; DPI, diphenyleneiodonium chloride; ESR, electron spin resonance; HRP, horseradish peroxidase; MAO, monoamine oxidase; MDA, monodehydroascorbate; MDAR, monodehydroascorbate reductase; O2.–, superoxide anion; PEA, phenylethylamine; rlu, relative luminescence units; SHAM, salicylhydroxamic acid; SOD, CuZn-superoxide dismutase.

Journal Article.  5285 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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