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 transientgeneration of superoxide anion (O^–₂), followed by a transientincrease in cytosolic free calcium ion concentration (Ca²⁺]_c).The level of SA-induced O^–₂ was lowered by treatment withseveral scavengers of active oxygen species and a peroxidaseinhibitor, but not with an NADPH oxidase inhibitor. The SA-inducedCa²⁺]_c elevation was also lowered by inhibitors which effectivelylowered the O^–₂ level. Inhibition of Ca²⁺]_c elevationby Ca²⁺ channel blockers and a Ca²⁺ chelator indicated thatextracellular Ca²⁺ was responsible for the increased Ca²⁺]_c.Among the several SA analogs, only compounds that actively inducedthe O^–₂ generation also elevated Ca²⁺]_cIn addition, theinhibitory effects of SA analogs on catalase activity correlatedwell with their effects on the O^–₂ generation and theCa²⁺]_c elevation. SA-dependent O^–₂ generation was shownto occur extracellularly, requiring both H₂O₂ and at least oneproteinaceous factor excreted from the cells. This factor wasdetermined to be a salicylhydroxamic acid-sensitive extracellularguaiacol-utilizing peroxidase. ⁴Present address: Isehara Research Laboratory, Kanto ChemicalCo., Inc., Suzukawa, Isehara, 259-1146 Japan.