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Recent studies showed, that ozone-induced gene expression occurs via at least two different signalling mechanisms that are ethylene-dependent (fi-1,3-glucanases) and ethylene-independent independent (stilbene synthase). To identify transacting factors involved in ozone-induced gene expression we analyzed a 150 bp PCR fragment of an ozone-responsive promoter segment of the grape vine resveratrol synthase gene (Vst1) in combination of a cDNA library prepared from ozone-treated tobacco plants, using the yeast one-hybrid screening system. Two cDNA clones that encode WRKY binding proteins were isolated by this screening technique. The open reading frame of NtWRKY10 and NtWRKYll showed an identity of 93.5 % and the deduced amino acid sequence an identity of 89.3 %. According to the WRKY domain classification in Arabidopsis, both proteins belong to subgroup II. Comparison with known tobacco WRKY proteins indicate that WRKY10 and WRKY11 belong to a new class of tobacco WRKY transcription factors. Electrophoretic mobility shift assays (EMSA) of yeast extracts, containing the WRKY fusion protein showed a weak binding to the radioaclively labelled 150 bp ozone-responsive Vst1 fragment. These results are consistent with an involvement of WRKY proteins in ozone-induced phytoalexin gene expression.
Selected cDNA clones of Arabidopsis thaliana, isolated previously by suppression subtractive hybridisation, were used to differentiate between abiotic stress factors. Changes in expression patterns of 79 genes were examined by array analysis in Arabidopsis thaliana after fumigation with ozone and after short- or long-term mercuric-ion exposure. Substantial changes in the abundance of 42 transcripts were recorded in response to the treatments, and 6 transcript clusters were observed. The abundance of 37 mRNAs was increased more then 1.5-fold, whereas that of 5 mRNAs was reduced. The abundances of 5, 6 and 9 mRNAs were specifically in creased by short-term mercury application, ozone fumigation, and long-term mercuric-ion exposure, respectively. The transcription of the other 5 transcripts was induced by both ozone and short-term mercuric-ion treatment. The abundance of 10 different mRNAs was in creased by the different mercuric-ion applications. Two transcripts were induced by ozone fumigation, as well as long-term, mercury treatment. Finally, 5 transcripts were repressed by ozone exposure, and 3 out of them by short-term mercuric-ion treatment. These results show that the array technique can be used to analyse the expression pattern in Arabidopsis thaliana under ozone and mercuric-ion stress. Searches against the Arabidopsis database furthermore provide a classification of most genes. In addition possible cis-acting regulatory elements were identified by an in silico approach using the MIPS Arabidopsis thaliana database.
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