Mercuric-ion-induced gene expression was studied in Arabidopsis thaliana Columbia wild type. Rosettes of plants grown for 21 d on agar medium supplemented with 20, 30 and 40 µ µ µ µ M HgCl 2 were pooled and used to isolate cDNAs of induced genes by suppression subtractive hybridization. Of the 576 clones isolated initially, 31 turned out to be mercury-induced by Northern hybridization. However, kinetic studies using cDNA arrays clearly showed that seven genes were exclusively mercuric-ion-induced, 14 were induced by mercury but also affected by a diurnal rhythm, and 10 clones were only modulated by the day-night cycle. The expression levels of the metal-induced genes increased from 1·5-fold to 10-fold. Functional classification resulted in genes encoding proteins for the photosynthetic apparatus and for the antioxidative system. In addition, unexpected genes, whose connection to mercury ion stress is not evident, were identified.

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Detection of foodborne pathogenic and spoilage bacteria by RNA-DNA hybridization is an alternative to traditional microbiological procedures. To achieve high sensitivity with RNA-DNA-based methods, efficient bacterial lysis and release of nucleic acids from bacteria are needed. Here we report the specific detection of the hygiene indicator microorganism Escherichia coli in meat by use of electrochemical biochips. We improved RNA isolation from bacteria in meat juice from pork and beef. Samples, either naturally or artificially contaminated by E. coli, were enriched by incubation in full or minimal medium. A combined treatment of the samples with lysozyme, proteinase K, and sonication resulted in efficient cell disruption and high total RNA yields. Together with optimization of enrichment time, this ensures high sensitivity of electrochemical measurements on biochips. A short enrichment period and the triple-lysis regimen in combination with electrochemical biochip measurement were tested with 25 meat samples. The lower limit of detection of the biochip was approximately 2,000 CFU of E. coli per ml. The entire analysis procedure (5 h of enrichment, triple lysis, and biochip detection) has a lower limit of detection of 1 CFU of E. coli per ml within a total time needed for analysis of 7 h.

Recent studies suggest that ethylene is involved in signalling ozone-induced gene expression. We show here that application of ozone increased glucuronidase (GUS) expression of chimeric reporter genes regulated by the promoters of the tobacco class I beta-1,3-glucanases (GLB and Gln2) and the grapevine resveratrol synthase (Vst1) genes in transgenic tobacco leaves. 5'-deletion analysis of the class I beta-1,3-glucanase promoter revealed that ozone-induced gene regulation is mainly mediated by the distal enhancer region containing the positively acting ethylene-responsive element (ERE). In addition, application of 1-methylcyclopropene (1-MCP), an inhibitor of ethylene action, blocked ozone-induced class I beta-1,3-glucanase promoter activity. Enhancer activity and ethylene-responsiveness depended on the integrity of the GCC boxes, cis-acting elements present in the ERE of the class I beta-1,3-glucanase and the basic-type pathogenesis-related PR-1 protein (PRB-1b) gene promoters. The minimal PRB-1b promoter containing only the ERE with intact GCC boxes, was sufficient to confer 10-fold ozone inducibility to a GUS-reporter gene, while a substitution mutation in the GCC box abolished ozone responsiveness. The ERE region of the class I beta-1,3-glucanase promoter containing two intact GCC boxes confered strong ozone inducibility to a minimal cauliflower mosaic virus (CaMV) 35S RNA promoter, whereas two single-base substitution in the GCC boxes resulted in a complete loss of ozone inducibility. Taken together, these datastrongly suggest that ethylene is signalling ozone-induced expression of class I beta-l,3-glucanase and PRB-1b genes. Promoter analysis of the stilbene synthase Vst1 gene unravelled different regions for ozone and ethylene-responsiveness. Application of 1-MCP blocked ethylene-induced Vst1 induction, but ozone induction was not affected. This shows that ozone-induced gene expression occurs via at least two different signalling mechanisms and suggests an additional ethylene independent signalling pathway for ozone-induced expression of genes involved in phytoalexin biosynthesis.

GSF -Na tional Re search Cen ter for En vi ron ment and Health, In sti tute of Bio chem i cal Plant Pa thol ogy, Re cent stud ies showed, that ozone-in duced gene ex pres sion oc curs via at least two dif fer ent sig nal ling mech a nisms that are eth yl ene-de pend ent (ß-1,3-glucanases) and eth yl ene-in de pend ent in de pend ent (stilbene synthase). To iden tify trans-act ing fac tors in volved in ozone-in duced gene ex pres sion we an alyzed a 150 bp PCR frag ment of an ozone-re spon sive pro moter seg ment of the grape vine resveratrol synthase gene (Vst1) in com bi na tion of a cDNA li brary pre pared from ozone-treated to bacco plants, us ing the yeast one-hy brid screen ing sys tem. Two cDNA clones that en code WRKY bind ing pro teins were iso lated by this screen ing tech nique. The open read ing frame of NtWRKY10 and NtWRKY11 showed an iden tity of 93.5 % and the de duced amino acid se quence an iden tity of 89.3 %. Ac cord ing to the WRKY do main clas si fi ca tion in Arabidopsis, both pro teins be long to sub group II. Com par i son with known to bacco WRKY pro teins in di cate that WRKY10 and WRKY11 be long to a new class of to bacco WRKY tran scription fac tors. Elec tro pho retic mo bil ity shift as says (EMSA) of yeast ex tracts, con tain ing the WRKY fu sion pro tein showed a weak bind ing to the ra dio ac tively la belled 150 bp ozone-respon sive Vst1 frag ment. These re sults are con sis tent with an in volve ment of WRKY pro teins in ozone-in duced phytoalexin gene ex pres sion.List of ab bre vi a tions: Vst1 -resveratrol synthase gene; EMSA -elec tro pho retic mo bil ity shift as say In tro duc tionStrato spheric ozone pro tects life from det ri men tal ul tra vi o let-B ra di a tion, but tro po spheric ozone is a se ri ous world-wide pol lut ant (Krupa 2000). The phytotoxic ef fects of ozone were rec og nized as early as 1950, and sub se quent stud ies have shown the dam ag ing ef fects of ozone at the phys i o log i cal, bio chem i cal and ge netic level (Runeckles and Chevo ne 1992, Kangasjärvi et al. 1994, Sandermann 1996).The spe cific mech a nisms by which ozone causes changes in plant gene ex pres sion are not known, but it is gen er ally ac cepted that ozone ini ti ates an ox i da tive burst and an ac tive pro duc tion of re ac tive ox y gen in ter me di ates (Schraudner et al. 1998, Pellinen et al. 1999, Rao and Da vis 1999. Sev eral signal mol e cules like eth yl ene, jasmonic acid and sal icylic acid, have been hy poth e sized to act as a second or third mes sen ger for ozone-in duced gene expres sion (Mehlhorn and

Ab stractSe lected cDNA clones of Arabidopsis thaliana, iso lated pre viously by sup pres sion subtractive hy bridi sa tion, were used to dif fer en ti ate be tween abiotic stress fac tors. Changes in ex pression pat terns of 79 genes were ex am ined by ar ray anal y sis in Arabidopsis thaliana af ter fu mi ga tion with ozone and af ter short-or long-term mer cu ric-ion ex po sure. Sub stan tial changes in the abun dance of 42 tran scripts were re corded in response to the treat ments, and 6 tran script clus ters were observed. The abun dance of 37 mRNAs was in creased more then 1.5-fold, whereas that of 5 mRNAs was re duced. The abundances of 5, 6 and 9 mRNAs were spe cif i cally in creased by short-term mer cury ap pli ca tion, ozone fu mi ga tion, and long-term mer cu ric-ion ex po sure, re spec tively. The tran scription of the other 5 tran scripts was in duced by both ozone and short-term mer cu ric-ion treat ment. The abun dance of 10 differ ent mRNAs was in creased by the dif fer ent mer cu ric-ion appli ca tions. Two tran scripts were in duced by ozone fu mi ga tion, as well as long-term, mer cury treat ment. Fi nally, 5 tran scripts were re pressed by ozone ex po sure, and 3 out of them by short-term mer cu ric-ion treat ment. These re sults show that the ar ray tech nique can be used to ana lyse the ex pres sion pat tern in Arabidopsis thaliana un der ozone and mer cu ric-ion stress. Searches against the Arabidopsis da ta base fur ther more provide a clas si fi ca tion of most genes. In ad di tion pos si ble cis-acting reg u la tory el e ments were iden ti fied by an in silico approach us ing the MIPS Arabidopsis thaliana da ta base.