Journal Article

Cryptogein-Induced Initial Events in Tobacco BY-2 Cells: Pharmacological Characterization of Molecular Relationship among Cytosolic Ca<sup>2+</sup> Transients, Anion Efflux and Production of Reactive Oxygen Species

Yasuhiro Kadota, Tatsuaki Goh, Hajime Tomatsu, Ryosuke Tamauchi, Katsumi Higashi, Shoshi Muto and Kazuyuki Kuchitsu

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 45, issue 2, pages 160-170
Published in print February 2004 | ISSN: 0032-0781
Published online February 2004 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pch020
Cryptogein-Induced Initial Events in Tobacco BY-2 Cells: Pharmacological Characterization of Molecular Relationship among Cytosolic Ca2+ Transients, Anion Efflux and Production of Reactive Oxygen Species

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Ion fluxes and the production of reactive oxygen species (ROS) are early events that follow elicitor treatment or microbial infection. However, molecular mechanisms for these responses as well as their relationship have been controversial and still largely unknown. We here simultaneously monitored the temporal sequence of initial events at the plasma membrane in suspension-cultured tobacco cells (cell line BY-2) in response to a purified proteinaceous elicitor, cryptogein, which induced hypersensitive cell death. The elicitor induced transient rise in cytosolic Ca2+ concentration ([Ca2+]cyt) showing two distinct peaks, followed by biphasic (rapid/transient and slow/prolonged) Cl efflux and H+ influx. Pharmacological analyses suggested that the two phases of the [Ca2+]cyt response correspond to Ca2+ influx through the plasma membrane and an inositol 1,4,5-trisphophate-mediated release of Ca2+ from intracellular Ca2+ stores, respectively, and the [Ca2+]cyt transients and the Cl efflux were mutually dependent events regulated by protein phosphorylation. The elicitor also induced production of ROS including O2 and H2O2, which initiated after the [Ca2+]cyt rise and required Ca2+ influx, Cl efflux and protein phosphorylation. An inhibitor of NADPH oxidase, diphenylene iodonium, completely inhibited the elicitor-induced production of O2 and H2O2, but did not affect the [Ca2+]cyt transients. These results suggest that cryptogein-induced plasma membrane Ca2+ influx is independent of ROS, and NADPH oxidase dependent ROS production is regulated by these series of ion fluxes.

Keywords: Keywords: Calcium ion — Cryptogein — Elicitor — Ion flux — Reactive oxygen species — Suspension-cultured tobacco BY-2 cells.; Abbreviations: BAPTA, 1,2-bis-(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; [Ca2+]cyt, cytosolic free Ca2+ concentration; [Cl–], Cl– concentration; DIDS, 4,4′-diisothiocyanostilbene-2,2′-disulfonate; DPI, diphenylene iodonium; IP3, inositol 1,4,5-trisphosphate; MCLA, 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo[1,2-a]pyrazin-3-one; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide; NPPB, 5-nitro-2-(3-phenylpropylamino)benzoic acid; •O2–, superoxide anion; ROS, reactive oxygen species.

Journal Article.  6743 words.  Illustrated.

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

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