Salicylic acid induces extracellular superoxide generation followed by an increase in cytosolic calcium ion in tobacco suspension culture: The earliest events in salicylic acid signal transduction

Addition of salicylic acid (SA) to tobacco (Nicotiana tabacum) suspension culture immediately induced a rapid and transient generation of superoxide anion (O₂^-), followed by a transient increase in cytosolic free calcium ion concentration ([Ca²⁺](c)). The level of SA-induced O₂^- was lowered by treatment with several scavengers of active oxygen species and a peroxidase inhibitor, but not with an NADPH oxidase inhibitor. The SA-induced [Ca²⁺](c) elevation was also lowered by inhibitors which effectively lowered the O₂^- level. Inhibition of [Ca²⁺](c) elevation by Ca²⁺ channel blockers and a Ca²⁺ chelator indicated that extracellular Ca²⁺ was responsible for the increased [Ca²⁺](c). Among the several SA analogs, only compounds that actively induced the O₂^- generation also elevated [Ca²⁺](c). In addition, the inhibitory effects of SA analogs on catalase activity correlated well with their effects on the O₂^- generation and the [Ca²⁺](c) elevation. SA-dependent O₂^- generation was shown to occur extracellularly, requiring both H₂O₂ and at least one proteinaceous factor excreted from the cells. This factor was determined to be a salicylhydroxamic acid-sensitive extracellular guaiacol-utilizing peroxidase.