Cation-induced superoxide generation in tobacco cell suspension culture is dependent on ion valence

There have been many reports suggesting the involvement of reactive oxygen species (ROS), including superoxide anion (O-2(-)), in salt stress. Herein, direct evidence that treatments of cell suspension culture of tobacco (Nicotiana tabacum L.; cell line, BY-2) with various salts of trivalent, divalent and monovalent metals stimulate the immediate production of O-2(-) is reported. Among the salts tested, LaCl3 and GdCl3 induced the greatest responses in O-2(-) production, whereas CaCl2 and MgCl2 showed only moderate effects; salts of monovalent metals such as KCl and NaCl induced much lower responses, indicating that there is a strong relationship between the valence of metals and the level of O-2(-) production. As the valence of the added metals increased from monovalent to divalent and trivalent, the concentrations required for maximal responses were lowered. Although O-2(-) production by NaCl and KCl required high concentrations associated with hyperosmolarity, the O-2(-) generation induced by NaCl and KCl was significantly greater than that induced simply by hyperosmolarity. Since an NADPH oxidase inhibitor, diphenyleneiodonium chloride, showed a strong inhibitory effect on the trivalent and divalent cation-induced generation of O-2(-), it is likely that cation treatments activate the O-2(-)-generating activity of NADPH oxidase.