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Identifying targets of FLC at 27oC

ABSTRACT: FLOWERING LOCUS C (FLC) is a MADS box transcription factor that plays a well characterised role in repressing the vegetative to floral transition of Arabidopsis thaliana. FLC has also been shown to affect the Arabidopsis circadian clock, with mutant seedlings showing short circadian periods. In a previous study, we identified the temperature-dependent circadian period QTL PerCv5b near the FLC locus on the top arm of Chromosome 5. PerCv5b caused a significant period effect at 27oC but not at 12oC or 22oC. Temperature-dependent circadian period phenotypes and a known polymorphism in the Ler allele made FLC a strong candidate gene for PerCv5b. The period effect of FLC was enhanced by combination with alleles of FRIGIDA (FRI), a gene shown to up-regulate FLC's expression. We were interested in identifying how FLC affects the circadian clock, so we decided to identify its target genes. Greatest phenotypic difference was observed between fri; flc and FRI; FLC genotype seedlings at 27oC, so expression was compared between these lines (previously described in Michaels and Amasino1999 and 2001) on the Affymetrix ATH1 microarray. Seedlings were grown on media (MS 1.5% Agar containing 3% Sucrose) for 6 days under constant cool white fluorescent light (55-60 micro EINSTEINS) at 22oC then entrained for 4 days under (12h , 12h) light , dark cycles at 22oC. At dawn on the fourth day of entrainment they were transferred to constant light (25-30 micro EINSTEINS) at 27oC. Four samples were taken at 6 hour intervals starting 24h after the transfer to continuous conditions at the times 24h, 30h, 36h and 42h. Equal amounts of total RNA were pooled from the three time points to produce one sample per genotype. The pooling strategy was employed to reduce the effect of circadian regulation on genes expression. This was particularly important in our case as some interesting genes would likely be regulated by the circadian clock and may only show expression differences at particular phases that could easily be missed if using just one time point. Experimenter name = Kieron Edwards Experimenter phone = 0131 651 3326 Experimenter fax = 0131 650 5392 Experimenter department = Institute of Molecular Plant Sciences Experimenter institute = University of Edinbugh Experimenter address = Kings Buildings Experimenter address = Mayfield Road Experimenter address = Edinburgh Experimenter zip/postal_code = EH9 3JH Experimenter country = UK Keywords: genetic_modification_design

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE5528 | GEO | 2006/08/15

SECONDARY ACCESSION(S): PRJNA96007

REPOSITORIES: GEO

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Project description:FLOWERING LOCUS C (FLC) is a MADS box transcription factor that plays a well characterised role in repressing the vegetative to floral transition of Arabidopsis thaliana. FLC has also been shown to affect the Arabidopsis circadian clock, with mutant seedlings showing short circadian periods. In a previous study, we identified the temperature-dependent circadian period QTL PerCv5b near the FLC locus on the top arm of Chromosome 5. PerCv5b caused a significant period effect at 27oC but not at 12oC or 22oC. Temperature-dependent circadian period phenotypes and a known polymorphism in the Ler allele made FLC a strong candidate gene for PerCv5b. The period effect of FLC was enhanced by combination with alleles of FRIGIDA (FRI), a gene shown to up-regulate FLC's expression. We were interested in identifying how FLC affects the circadian clock, so we decided to identify its target genes. Greatest phenotypic difference was observed between fri; flc and FRI; FLC genotype seedlings at 27oC, so expression was compared between these lines (previously described in Michaels and Amasino1999 and 2001) on the Affymetrix ATH1 microarray. Seedlings were grown on media (MS 1.5% Agar containing 3% Sucrose) for 6 days under constant cool white fluorescent light (55-60 micro EINSTEINS) at 22oC then entrained for 4 days under (12h , 12h) light , dark cycles at 22oC. At dawn on the fourth day of entrainment they were transferred to constant light (25-30 micro EINSTEINS) at 27oC. Four samples were taken at 6 hour intervals starting 24h after the transfer to continuous conditions at the times 24h, 30h, 36h and 42h. Equal amounts of total RNA were pooled from the three time points to produce one sample per genotype. The pooling strategy was employed to reduce the effect of circadian regulation on genes expression. This was particularly important in our case as some interesting genes would likely be regulated by the circadian clock and may only show expression differences at particular phases that could easily be missed if using just one time point. Experimenter name = Kieron Edwards; Experimenter phone = 0131 651 3326; Experimenter fax = 0131 650 5392; Experimenter department = Institute of Molecular Plant Sciences; Experimenter institute = University of Edinbugh; Experimenter address = Kings Buildings; Experimenter address = Mayfield Road; Experimenter address = Edinburgh; Experimenter zip/postal_code = EH9 3JH; Experimenter country = UK Experiment Overall Design: 4 samples were used in this experiment

Project description:Fungal secondary metabolites can not only cause toxic effects in animals and humans, but also serve as virulence factors of the producing fungi for causing plant diseases.Thus, the severity of plant diseases associated with mycotoxins depend on the sensitivity towards the toxin. In previous experiments, we have evaluated the phytotoxic effect ofa mycotoxin on root growth of Arabidopsis wild-type and mutant seedlings. Mycotoxin treatment of a new conditional root expansion mutant partially restores the expansion phenotype (JE100; Werner et al., unpublished). AIM: This experiment aims to identify genes, in early and later phases after mycotoxin treatment in wild-type and mutant seedlings. EXPERIMENTAL PLAN: Eight Affymetrix chips are needed for this experiment. RNA preparation will be provided from wild-type, accession Columbia, and mutant seedlings after different time points of mycotoxin treatment. As control, separate seedlings will be treated with the same concentration of solvent (DMSO). Briefly, seeds will be sterilized, stratified for 48 hours and germinated on MS agar plates containing 4.5% sucrose at 22°C and 16h/8h light/dark cycles. 10 days after germination, seedlings will be transferred to liquid MS medium and shaken for another 3 days for acclimatization. Seedlings will be harvested after 2 and 24 hours of treatment with a single concentration (50 µM) of mycotoxin. To account for experimental variations (i.e. time needed for freezing the tissues, circadian clock,...), the experiment will be repeated three times and RNA samples will be pooled. EXPECTED RESULTS: The experiment should identify genes differentially expressed: 1) between wild-type and mutant seedlings, 2) upon mycotoxin treatment in wild-type, 3) upon mycotoxin treatment of mutant seedlings and 4) upon solvent treatment. The results will allow us to pinpoint the mode of action of this mycotoxin. They will also allow us to better understand the function of the mutated gene which affects the sensitivity towards the mycotoxin. Furthermore, we expect to identify the signaling pathway by which the plant responses towards the mycotoxinis triggered. Experimenter name = Ulrike Werner Experimenter phone = +43-1-36006-6371 Experimenter fax = +43-1-36006-6392 Experimenter department = Institute of Applied Genetics and Cell Biology Experimenter institute = BOKU Experimenter address = Center of Applied Genetics Experimenter address = University of Agricultural Sciences Vienna Experimenter address = Muthgasse 18 Experimenter address = Vienna Experimenter zip/postal_code = 1190 Experimenter country = Austria Keywords: genetic_modification_design; compound_treatment_design; time_series_design

Project description:Fungal secondary metabolites can not only cause toxic effects in animals and humans, but also serve as virulence factors of the producing fungi for causing plant diseases.Thus, the severity of plant diseases associated with mycotoxins depend on the sensitivity towards the toxin. In previous experiments, we have evaluated the phytotoxic effect ofa mycotoxin on root growth of Arabidopsis wild-type and mutant seedlings. Mycotoxin treatment of a new conditional root expansion mutant partially restores the expansion phenotype (JE100; Werner et al., unpublished). AIM: This experiment aims to identify genes, in early and later phases after mycotoxin treatment in wild-type and mutant seedlings. EXPERIMENTAL PLAN: Eight Affymetrix chips are needed for this experiment. RNA preparation will be provided from wild-type, accession Columbia, and mutant seedlings after different time points of mycotoxin treatment. As control, separate seedlings will be treated with the same concentration of solvent (DMSO). Briefly, seeds will be sterilized, stratified for 48 hours and germinated on MS agar plates containing 4.5% sucrose at 22Â°C and 16h/8h light/dark cycles. 10 days after germination, seedlings will be transferred to liquid MS medium and shaken for another 3 days for acclimatization. Seedlings will be harvested after 2 and 24 hours of treatment with a single concentration (50 ÂµM) of mycotoxin. To account for experimental variations (i.e. time needed for freezing the tissues, circadian clock,...), the experiment will be repeated three times and RNA samples will be pooled. EXPECTED RESULTS: The experiment should identify genes differentially expressed:; 1) between wild-type and mutant seedlings,; 2) upon mycotoxin treatment in wild-type,; 3) upon mycotoxin treatment of mutant seedlings and; 4) upon solvent treatment. The results will allow us to pinpoint the mode of action of this mycotoxin. They will also allow us to better understand the function of the mutated gene which affects the sensitivity towards the mycotoxin. Furthermore, we expect to identify the signaling pathway by which the plant responses towards the mycotoxinis triggered. Experimenter name = Ulrike Werner; Experimenter phone = +43-1-36006-6371; Experimenter fax = +43-1-36006-6392; Experimenter department = Institute of Applied Genetics and Cell Biology; Experimenter institute = BOKU; Experimenter address = Center of Applied Genetics; Experimenter address = University of Agricultural Sciences Vienna; Experimenter address = Muthgasse 18; Experimenter address = Vienna; Experimenter zip/postal_code = 1190; Experimenter country = Austria Experiment Overall Design: 8 samples were used in this experiment

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Identifying targets of FLC at 27oC

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