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Published on April 30, 2004; 10.1104/pp.104.038968

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Received January 12, 2004
Returned for revision March 22, 2004
Accepted March 24, 2004

Analysis of Nitric Oxide Signaling Functions in Tobacco Cells Challenged by the Elicitor Cryptogein

Olivier Lamotte , Kevin Gould , David Lecourieux , Anabelle Sequeira-Legrand , Angela Lebrun-Garcia , Jörg Durner , Alain Pugin , and David Wendehenne *

Unité Mixte de Recherche 1088/Centre National de la Recherche Scientifique 5184/Université de Bourgogne, Plante-Microbe-Environnement, Institut National de la Recherche Agronomique, BP 86510, 21065 Dijon cedex, France (O.L., D.L., A.L.-G., A.P., D.W.); Plant Sciences Group, School of Biological Sciences, University of Auckland, Auckland, New Zealand (K.G.); Centre Commun de Cytométrie en Flux, Université de Bourgogne, Faculté de Médecine-IFR100, 21000 Dijon, France (A.S.-L.); Institute of Biochemical Plant Pathology, Gesellschaft für Strahlenforschung-National Research Centre for Environment and Health, 85764 Oberschleissheim, Germany (J.D.)

* Corresponding author; email: wendehen{at}dijon.inra.fr.

Nitric oxide (NO) has recently emerged as an important cellular mediator in plant defense responses. However, elucidation of the biochemical mechanisms by which NO participates in this signaling pathway is still in its infancy. We previously demonstrated that cryptogein, an elicitor of tobacco defense responses, triggers a NO burst within minutes in epidermal sections from tobacco leaves (Nicotiana tabacum cv Xanthi). Here, we investigate the signaling events that mediate NO production, and analyze NO signaling activities in the cryptogein transduction pathway. Using flow cytometry and spectrofluorometry, we observed that cryptogein-induced NO production in tobacco cell suspensions is sensitive to nitric oxide synthase inhibitors and may be catalyzed by variant P, a recently identified pathogen-inducible plant nitric oxide synthase. NO synthesis is tightly regulated by a signaling cascade involving Ca2+ influx and phosphorylation events. Using tobacco cells constitutively expressing the Ca2+ reporter apoaequorin in the cytosol, we have shown that NO participates in the cryptogein-mediated elevation of cytosolic free Ca2+ through the mobilization of Ca2+ from intracellular stores. The NO donor diethylamine NONOate promoted an increase in cytosolic free Ca2+ concentration, which was sensitive to intracellular Ca2+ channel inhibitors. Moreover, NO appears to be involved in the pathway(s) leading to the accumulation of transcripts encoding the heat shock protein TLHS-1, the ethylene-forming enzyme cEFE-26, and cell death. In contrast, NO does not act upstream of the elicitor-induced activation of mitogen-activated protein kinase, the opening of anion channels, nor expression of GST, LOX-1, PAL, and PR-3 genes. Collectively, our data indicate that NO is intimately involved in the signal transduction processes leading to cryptogein-induced defense responses.




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