Abstract
The in vivo induction of H2O2 production was tested on tobacco cell suspension cultures (Nicotiana tabacum cv. Bright Yellow-2). 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-aminoanti-pyrine. 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 (E50=46 to 70 μM, Emax=101 to 128 μmol/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 (I50=1.26μM) and acridine orange and with membrane-permeative thiol reagents (N-ethyl maleimide, N-pyrene maleimide, iodoacetate); whereas the nonpermeative thiol reagentp-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.
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