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Journal of Experimental Botany, Vol. 54, No. 381, pp. 291-301, January 2, 2003
© 2003 Oxford University Press

Early steps in the oxidative burst induced by cadmium in cultured tobacco cells (BY-2 line)

Received 17 April 2002; Accepted 12 September 2002

Enrique Olmos, Juan R. Martínez-Solano, Abel Piqueras and Eladio Hellín1

Departamento de Nutrición y Fisiología Vegetal, CEBAS-CSIC, PO Box 4195, 30080 Murcia, Spain

1 To whom correspondence should be addressed. Fax: +34 968 396213. E-mail: ehellin{at}natura.cebas.csic.es
Abbreviations: APX, ascorbate peroxidase; DAB, 3–3' diamino benzidine; DMSO, dimethylsulphoxide; DPI, diphenyleneiodonium; FDA, fluorescein acetate; GSH, reduced glutathione; GSSG, oxidized glutathione; POX, peroxidase; ROS, reactive oxygen species; SOD, superoxide dismutase.

The rapid generation of H2O2 by Cd2+-treated plant cells was investigated in cultured tobacco (Nicotiana tabacum L.) BY-2 cells. The starting point for the generation of H2O2 has been located at the cell plasma membrane using cytochemical methods. Treatment of the cells with diphenyleneiodonium (DPI) and imidazol, both inhibitors of the neutrophil NADPH oxidase, prevented the generation of H2O2 induced by Cd2+. These data suggest the involvement of an NADPH oxidase-like enzyme leading to H2O2 production through O2.– dismutation by superoxide dismutase enzymes. To investigate the implication of Ca2+ channels in a Cd2+-induced oxidative burst, different inhibitors of Ca2+ channels were used. Only La3+ totally inhibited the generation of H2O2 induced by Cd2+. However, verapamil and nifedipine, inhibitors of Ca2+ channels, were not effective. Calmodulin or a Ca2+-dependent protein kinase is also implicated in the signal transduction sequence, based on the results obtained with two types of calmodulin antagonists, fluphenazine and N-(-6-amino-hexyl)-5-chloro-1-naphthalenesulphonamide (W-7) and staurosporine, an inhibitor of protein kinases. However, neomycin, an inhibitor of the phosphoinositide cycle, did not inhibit the generation of H2O2 induced by Cd2+, suggesting mainly an induction of the oxidative burst mediated by calmodulin and/or calmodulin-dependent proteins.

Key words: BY-2 cells, cadmium, NADPH oxidase, oxidative burst.


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