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

Abstract

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+]cIn 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.