Journal Article

Mechanism of peroxidase actions for salicylic acid‐induced generation of active oxygen species and an increase in cytosolic calcium in tobacco cell suspension culture

Tomonori Kawano and Shoshi Muto

in Journal of Experimental Botany

Volume 51, issue 345, pages 685-693
Published in print April 2000 | ISSN: 0022-0957
Published online April 2000 | e-ISSN: 1460-2431 | DOI: http://dx.doi.org/10.1093/jexbot/51.345.685
Mechanism of peroxidase actions for salicylic acid‐induced generation of active oxygen species and an increase in cytosolic calcium in tobacco cell suspension culture

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Extracellularly secreted peroxidases in cell suspension culture of tobacco (Nicotiana tabacum L. cv. Bright Yellow‐2, cell line BY‐2) catalyse the salicylic acid (SA)‐dependent formation of active oxygen species (AOS) which, in turn, triggers an increase in cytosolic Ca2+ concentration. Addition of horseradish peroxidase (HRP) to tobacco cell suspension culture enhanced the SA‐induced increase in cytosolic Ca2+ concentration, suggesting that HRP enhanced the production of AOS. The mechanism of peroxidase‐catalysed generation of AOS in SA signalling was investigated with chemiluminescence sensitive to AOS and electron spin resonance (ESR) spectroscopy, using the cell suspension culture of tobacco, and HRP as a model system of peroxidase reaction. The results showed that SA induced the peroxidase inhibitor‐sensitive production of superoxide and H2O2 in tobacco suspension culture, but no production of hydroxy radicals was detected. Similar results were obtained using HRP. It was also observed that SA suppressed the H2O2‐dependent formation of hydroxy radicals in vitro. The results suggest that SA protect the cells from highly reactive hydroxy radicals, while producing the less reactive superoxide and H2O2 through peroxidase‐catalysed reaction, as the intermediate signals. The formation of superoxide was followed by that of H2O2, suggesting that superoxide was converted to H2O2. In addition, it was observed that superoxide dismutase‐insensitive ESR signal of monodehydroascorbate radical was induced by SA both in the tobacco suspension culture and HRP reaction mixture, suggesting that SA free radicals, highly reactive against ascorbate, were formed by peroxidase‐catalysed reactions. The formation of SA free radicals may lead to subsequent monovalent reduction of O2 to superoxide.

Keywords: Active oxygen species; calcium; Nicotiana tabacum; peroxidase; salicylic acid; signal transduction.; AOS, active oxygen species; [Ca2+]c, cytosolic free Ca2+ concentration; CL, chemiluminescence, CLA, Cripridina luciferin‐derived chemiluminescent agent; DMPO, 5,5′‐di‐methyl‐1‐pyrroline‐N‐oxide; DMTU, N,N′‐dimethylthiourea; ESR, electron spin resonance; GPX, guaiacol peroxidase; HO˙, hydroxyl radical; HRP, horseradish peroxidase; MDA, monodehydroascorbate; \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(O_{2}^{{\cdot}{-}}\) \end{document}, superoxide anion,; SA, salicylic acid; SA˙, SA free radical; SAR, systemic acquired resistance; SOD, superoxide dismutase.

Journal Article.  5764 words.  Illustrated.

Subjects: Plant Sciences and Forestry

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