Analysis of GEK1 function that is required for ethanol tolerance in higher plants

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1.Phenotypic analysis using gek1 mutants.To investigate the effect of gek1 mutation on the metabolic pathway, we tried to detect the change in metabolites by muti-nuclear NMR measurement combined with ^<13>C, ^<15>N stable isotope labelling. The results showed that gek1 activated the amino acid metabolic pathway in the presence of ethanol more strongly than the wild type. Microarray analysis revealed that several stress inducible genes were upregulated more rapidly by ethanol treatment in the gekl mutant.2.Screening for gek1 suppressor mutants.Putative gek1 suppressor mutants, totally 34 lines, were isolated. We analyzed most of them and found they were adh mutants. We are going to analyze rest of them.3.Identification GEK1-interacting proteins.Three putative GEK1-interacting proteins were identified by yeast two-hybrid analysis. Two of them are Zn-finger proteins (#20,#24). The rest one is NFU2(#76) that is presumed to be involved in the formation of Fe-S cluster in chloroplasts. The subcellular localization and the analyses of disruption mutants (#24,#76) did not offer any clear link between these proteins and GEK1 so far.4.Recombinant GEK1 protein.A recombinant GEK1 protein expressed in E.coli was obtained. We investigated its activity to breakdown acetaldehyde in vitro. We tried several conditions, but failed to detect such activity. We obtained the recombinant protein of the Pyrococcus GEK1 homologus gene that is presumed to be more stable. We tried to detect the same activity but failed again. Therefore, we concluded that GEK1 does not have such activity. We also tried to see any activity to change the metabolite profiling. The extract from ^<13>C,^<15>N stable isotope labeled plants were reacted with the recombinant protein and measured by NMR. However, we could not detect any changes.