Dynamics and localization of H2O2 production in elicited plant cells

H2O2 produced in plant cells plays a dual role. In addition to its antimicrobial effect, it also acts as a secondary messenger initiating and modulating responses of plants exposed to unfavorable external signals. A suspension culture of Rubia tinctorum cells challenged with elicitors was used as a model system to investigate H2O2 formation. Cellular H2O2 was measured by a modified titanium(IV) method, while that in the medium was detected with scopoletin fluorescence. Localization of H2O2 production at the ultrastructural level was carried out by the CeCl3 reaction. A fungal elicitor induced H2O2 production with transient maxima, the first of which appeared 4 min after treatment. Three subsequent maxima appeared in the cells up to 48 h after treatment. Exposure of cells to exogenous jasmonic acid and salicylic acid also changed the H2O2 concentration maxima over 48 h; however, their timing was slightly shifted. Fungal-elicitor, jasmonic acid, and salicylic acid treatments had different effects on the H2O2 concentration in the medium. Ultrastructural investigations revealed that electron-dense precipitates were present at the plasmalemma and in some nearby vesicular cytoplasmic structures 30 min after treatment. Later samples showed cytochemical-precipitate accumulation in the cell walls. These deposits appeared to be local and independent of the direction of the external signal. We could not detect the presence of H2O2 in peroxisomes, mitochondria, plastids, or the central vacuolar space. Electron energy loss spectroscopy investigations distinguished between the cerium-containing precipitates and other electrondense particles, thereby proving that H2O2 generation occurs locally.