Transcription profiling by array of Arabidopsis knocked out for At5g10140 grown at 27degC
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 Experiment Overall Design: 4 samples were used in this experiment
Project description:This experiment was annotated by TAIR (http://arabidopsis.org).FRI FLC time series Experimenter name = Markus Schmid Experimenter phone = ++49-7071-601-1413 Experimenter fax = ++49-7071-601-1412 Experimenter department = Detlef Weigel Laboratory Germany Experimenter institute = Max-Planck-Institute for Developmental Biology Experimenter address = Dept. of Molecular Biology Experimenter address = Spemannstr. 37-39 Experimenter address = Tübingen Experimenter zip/postal_code = 72076 Experimenter country = Germnay Keywords: time_series_design; strain_or_line_design; innate_behavior_design Overall design: 36 samples were used in this experiment
Project description:This experiment was annotated by TAIR (http://arabidopsis.org).FRI FLC time series; Experimenter name = Markus Schmid; Experimenter phone = ++49-7071-601-1413; Experimenter fax = ++49-7071-601-1412; Experimenter department = Detlef Weigel Laboratory Germany; Experimenter institute = Max-Planck-Institute for Developmental Biology; Experimenter address = Dept. of Molecular Biology; Experimenter address = Spemannstr. 37-39; Experimenter address = Tübingen; Experimenter zip/postal_code = 72076; Experimenter country = Germnay Experiment Overall Design: 36 samples were used in this experiment
Project description:Our aim is to study the circadian expression of genes to aid in our attempt of modelling the Arabidopsis circadian clock. Circadian microarray data have previously been published for plants after white light (WL)-dark cycles, using the 8k chip (Harmer et al. 2000). We intend to repeat this experiment using the 26k chips and are coordinating with Dr. Harmer, who is pursuing complementary experiments in UC Davis. Plants will be transferred to continuous WL after entrainment to 12h:12h light dark cycles. RNAs will be harvested every 4 hours over two days, with the same accession and sampling intervals used previously by Harmer et al. The two days of sampling provide internal replication. Our experience shows that this is the most economical design: it is easier to identify rhythms over a two-day timecourse than in two replicates of a single day. Hence: 13 RNA samples on 13 chips in total. METHOD: Seed was sown on MS agar plates with 3% sucrose, imbibed at 4 C for 96 hours. Seed was then entrained for 7 days at 22C, in cycles of 12 hours white light, 12 hours darkness. After 7 days they were transferred to constant white light at 22 C: this is time 0h. Tissue harvested at the time points shown after time 0. Experimenter name = Kieron Edwards; Experimenter phone = 024 7652 8374; Experimenter fax = 024 7652 3701; Experimenter department = Department of Biological Sciences; Experimenter institute = University of Warwick; Experimenter address = Department of Biological Sciences; Experimenter address = University of Warwick; Experimenter address = Gibbet Hill Road; Experimenter address = Coventry; Experimenter zip/postal_code = CV4 7AL; Experimenter country = UK Experiment Overall Design: 13 samples were used in this experiment
Project description:Arabiposis plants with conbinations of different FRIGIDA (FRI) and FLOWERING LOCUS C (FLC) alleles grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D). Genotypes:; Columbia wild type (Col-0): fri FLC; Columbia with introgressed FRI from Sf-2: FRI FLC; Columbia with introgressed FRI and deleted FLC (flc-3): FRI flc; Columbia with deleted FLC (flc-3): fri flc; Time points:; 0, 2, and 4 days after shift to long days
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 Experiment Overall Design: 8 samples were used in this experiment
Project description:Most higher organisms, including plants and animals, have developed a time-keeping mechanism that allows them to anticipate daily fluctuations of environmental parameters such as light and temperature. This circadian clock efficiently coordinates plant growth and metabolism with respect to time-of-day by producing self-sustained rhythms of gene expression with an approximately 24-hour period. The importance of these rhythms has in fact been demonstrated in both phytoplankton and higher plants: organisms that have an internal clock period matched to the external environment possess a competitive advantage over those that do not. We used microarrays to identify circadian-regulated genes of Arabidopsis thaliana to elucidate how the clock provides an adaptive advantage by understanding how the clock regulates outputs and determining which pathways and processes may be under circadian control. Keywords: time course Overall design: Groups of Arabidopsis seedlings were grown in light/dark cycles for 7 d before, transferred to constant light, and after 24 h in constant light 12 samples were harvested at 4-h intervals over the next 44 h for RNA extraction and hybridization on Affymetrix microarrays.
Project description:Plants of three different genotypes (FRI FLC, FRI flc and fri flc) were induced to flowering by shifting from short day conditions to long day conditions. FRI=FRIGIDA, FLC=FLOWERING LOCUS C.
Project description:Most higher organisms, including plants and animals, have developed a time-keeping mechanism that allows them to anticipate daily fluctuations of environmental parameters such as light and temperature. This circadian clock efficiently coordinates plant growth and metabolism with respect to time-of-day by producing self-sustained rhythms of gene expression with an approximately 24-hour period. The importance of these rhythms has in fact been demonstrated in both phytoplankton and higher plants: organisms that have an internal clock period matched to the external environment possess a competitive advantage over those that do not. We used microarrays to identify circadian-regulated genes of Arabidopsis thaliana to elucidate how the clock provides an adaptive advantage by understanding how the clock regulates outputs and determining which pathways and processes may be under circadian control. Experiment Overall Design: Groups of Arabidopsis seedlings were grown in light/dark cycles for 7 d before, transferred to constant light, and after 24 h in constant light 12 samples were harvested at 4-h intervals over the next 44 h for RNA extraction and hybridization on Affymetrix microarrays.
Project description:The Arabidopsis transcription factor WRKY27 was found to be involved in plant defense towards Ralstonia solanacearum GMI1000. To identify target genes of WRKY27, we introduced a functional tagged version of WRKY27 into two independent Arabidopsis wrky27 knockout lines (ecotype Columbia) under the control of the estrogen receptor-based chemical-inducible system. 18 days old plants grown on soil (Metro Mix 200) at 22oC under a 10/14 h light/dark cycle were treated for 6h with 10 microM beta-estradiol after which RNA was immediately isolated. The two independent knockout lines transformed with the empty vectors (pMD::XVE-SALK and pMD::XVE-ETL) served as controls and were grown under the same conditions and treated identically as the experimental plants. Experimenter name = Shahid Mukhtar; Experimenter phone = +49-221-5062-310; Experimenter fax = +49-221-5062-353; Experimenter address = Max.Planck-Institute for Plant Breeding; Experimenter address = Dept. Plant Microbe Interactions; Experimenter address = Carl-von-Linne Weg 10; Experimenter address = Koeln; Experimenter zip/postal_code = 50829; Experimenter country = Germany Experiment Overall Design: 4 samples were used in this experiment
Project description:Circadian clocks coordinate time-of-day specific metabolic and physiological processes to maximize performance and fitness. In addition to light, which is considered the strongest time cue to entrain animal circadian clocks, metabolic input has emerged as an important signal for clock modulation and entrainment, especially in peripheral clocks. Circadian clock proteins have been to be substrates of O-GlcNAcylation, a nutrient sensitive post-translational modification (PTM), and the interplay between clock protein O-GlcNAcylation and other PTMs, like phosphorylation, is expected to facilitate the regulation of circadian physiology by metabolic signals. Here, we used mass spectrometry proteomics to identify PTMs on PERIOD, the key biochemical timer of the Drosophila clock, over the circadian cycle.