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 (O-2(-)), followed by a transient increase in cytosolic free calcium ion concentration ([Ca2+](c)). The level of SA-induced O-2(-) 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 O-2(-) 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 O-2(-) generation also elevated [Ca2+](c). In addition, the inhibitory effects of SA analogs on catalase activity correlated well with their effects on the O-2(-) generation and the [Ca2+](c) elevation. SA-dependent O-2(-) 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.