The cantharidin-induced oxidative burst in tobacco BY-2 cell suspension cultures

The in vivo induction of H2O2 production was tested on tobacco cell suspension cultures (Nicotiana tabacum cv. Bright Yellow-a). The measurement of H2O2 was based on the oxidation of 3,5- dichloro-2-hydroxybenzensulfonic acid by endogenous peroxidases and spectrophotometric detection after reaction with 4-aminoantipyrine. The phosphatase inhibitor cantharidin induced a transient increase in H2O2 synthesis. The timing of the H2O2 production, the level of induction by cantharidin and the background H2O2 production were dependent on the tobacco cell concentration used. A concentration curve of cantharidin revealed saturating kinetics for the H2O2 detection (E-50 = 46 to 70 mu M, E-max = 101 to 128 mu mol/h . g fresh weight). An inhibitor study with the tobacco BY-2 cells showed high inhibitions of the H2O2 induction with the flavin analogues diphenylene iodonium (I-50 = 1.26 mu M) and acridine orange and with membrane-permeative thiol reagents (N-ethyl maleimide, N-pyrene maleimide, iodoacetate); whereas the nonpermeative thiol reagent p-chloromercuribenzoic acid was ineffective. Therefore, the induction of H2O2 production with phosphatase inhibitors (cantharidin) showed comparable properties to the elicitor-induced oxidative-burst response in other plant cells.