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Predominant triterpenes (free oleanolic acid, b-sitosterol and b-sitosterol-3-O-b-d-glucoside) in skin and cuticular wax of grape berries have been analysed by liquid chromatography-mass spectrometry (LC-APCI-MS). The validity of the developed method was established by determining linearity, recovery, precision, accuracy, limit of detection and quantification. Detection limits were in the range of 0.12-0.25 mg L )1 , and linearity values ranged up to 105 mg L )1 . The repeatability of the method was good. High variability was found among the measured grape varieties based on triterpene content, quantitites of oleanolic acid of cuticular wax ranged from 31.53 mg kg )1 to 162.01 mg kg )1 of the twelve analysed samples. The highest sitosterol content was measured in the sample 'Othello' (73.12 mg kg )1 ), while maximum sitosterol glucoside content was also found in 'Othello' variety (13.68 mg kg )1 ). The results showed that the study on triterpenes could be an informative tool to characterise grape varieties.

H(2)O(2) 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 H(2)O(2) formation. Cellular H(2)O(2) was measured by a modified titanium(IV) method, while that in the medium was detected with scopoletin fluorescence. Localization of H(2)O(2) production at the ultrastructural level was carried out by the CeCl(3) reaction. A fungal elicitor induced H(2)O(2) 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 H(2)O(2) concentration maxima over 48 h; however, their timing was slightly shifted. Fungal-elicitor, jasmonic acid, and salicylic acid treatments had different effects on the H(2)O(2) 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 H(2)O(2) 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 H(2)O(2) generation occurs locally.

Plant defense is based on a complex response triggered by unfavorable external impacts. The redox state of the cells and its temporal alteration, the oxidative burst, is an important regulatory element of this defense response. Data collected during the last years have caused us to change the previous, strongly simplified theory on signaling which had been based on a speculative, rather sequential mechanism. In the framework of signal transduction, H(2)O(2) signaling pathway(s) is/are only a special part of signal transduction but interacting with other pathways it/they influence the whole transducting system in several points. Our results show that in complexity and in basic regulatory mechanisms (transients, oscillation, tuning, signaling pattern) H(2)O(2) signaling is comparable with other pathways, of which we have more detailed cognition, and our present knowledge makes developing a new theory on this aspect necessary.

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