Project description:The scope of the project is to find DNA binding sites of the DNA binding protein AtDEK3. For the experiments AtDEK3 overexpressor (OE) plants have been obtained. The AtDEK3 was tagged with a CFP-tag. With ChIP the protein was pulled down and eluted DNA was send for sequencing. Gene ontology (GO) term classification of genes enriched for AtDEK3 binding indicated that AtDEK3 target genes are involved in diverse biological processes.
Project description:We characterized the global response of plants carrying a mitochondrial dysfunction induced by the expression of the unedited form of the ATP synthase 9 subunit. The u-ATP9 transgene driven by A9 and Apetala3 promoters induce mitochondrial dysfunction revealed by a decrease in both oxygen uptake and ATP levels, with an increase in ROS and a concomitant oxidative stress response. The transcriptome analysis of young Arabidopsis flowers, validated by RT-PCR and enzymatic or functional tests, show dramatic changes in u-ATP9 plants. Both lines present a modification in the expression of several genes involved in carbon, lipid and cell wall metabolism, suggesting that an important metabolic readjustment occurs in plants with a mitochondrial dysfunction. Interestingly, transcript levels involved in mitochondrial biogenesis, protein synthesis, and degradation are affected. Moreover, several mRNA levels for transcription factors and DNA binding proteins were also changed. Some of them are involved in stress and hormone response, suggesting that several signaling pathways overlap. Indeed, the transcriptome data reveal that the mitochondrial dysfunction dramatically alters genes involved in signaling pathways, including those involved in ethylene, absicic acid and auxin signal transduction. Our data suggest that the mitochondrial dysfunction model used in this rapport may be useful to uncover the retrograde signaling mechanism between the nucleus and mitochondria in plant cells.
Project description:Genomic integrity requires faithful chromosome duplication. Origins of replication are the genomic sites where DNA replication initiates in every cell cycle. There are multiple origins scattered throughout the eukaryotic genome whose genome-wide identification has been a hard challenge, especially in multicellular organisms. Thus, very little is known on the distinctive features of origins in terms of DNA sequence and chromatin context at a genomic scale. As part of a project for profiling replication origins in Arabidopsis thaliana, we have performed ChIP-chip analysis of the binding of ORC1 and CDC6, two proteins involved in initiation of DNA replication. Here, we provide the data of the ORC1-bound and CDC6-bound genomic sites using as control genomic DNA.
Project description:The scope of the project is to find DNA binding sites of the DNA binding protein AtDEK3. For the experiments AtDEK3 overexpressor (OE) plants have been obtained. The AtDEK3 was tagged with a CFP-tag. With ChIP the protein was pulled down and eluted DNA was send for sequencing. Gene ontology (GO) term classification of genes enriched for AtDEK3 binding indicated that AtDEK3 target genes are involved in diverse biological processes. ChIP Seq of three indenpendendt AtDEK3 OE and their respective inputs were used.
Project description:As one of the largest protein family in landplants pentatricopeptide repeat (PPR) proteins offer a wide research array. They are mostly involved in the RNA processing steps in Chloroplasts and Mitochondria. Recently it has been shown, that they are not only involved in the RNA processing steps, but are also part of the mitoribosome (rPPRs) as regulators of translation and ribosome stabilizers. This experiment focuses on a non-previously reported rPPR protein. The RNA-Seq data are needed for a ribosome profiling experiment that aimed to analyze DWEORG1's impact on mitochondrial translation. Arabidopsis mutants are compared to the wild type using duplicates for the RNA-Seq experiment. RNASEQ (determining the transcript abundance) and RIBOSEQ ( for detecting ribosome occupancy on each transcript) of wild type and mutants were conducted to determine the translational efficiency of mitochondrial transcripts in the mutant and wild type. The plants were grown in the Greenhouse under long day conditions. Flowers were harvested simultaneously for all genotypes, snap-frozen in liquid nitrogen and stored at -80°C until use.Total RNA was prepared from a mix of flower buds and open flowers.For total RNA extraction from flowers Trizol was used. Total RNA were treated with Dnase and depleted of rRNAs using the RiboMinusTM Plant Kit for RNA-Seq (invitrogen), following the manufacturer’s instructions.The sequencing libraries of two biological repeats per genotype were prepared using the NEXTflex Rapid Directional RNA-Seq Kit (Bioo Scientific) following the manufacturer's instructions. For sequencing Illumina NextSeq 500 75 bp single reads were used. The reads were than mapped to the Col-0 mitochondrial genome of Arabidopsis thaliana acccession JF729201.
Project description:We characterized the global response of plants carrying a mitochondrial dysfunction induced by the expression of the unedited form of the ATP synthase 9 subunit. The u-ATP9 transgene driven by A9 and Apetala3 promoters induce mitochondrial dysfunction revealed by a decrease in both oxygen uptake and ATP levels, with an increase in ROS and a concomitant oxidative stress response. The transcriptome analysis of young Arabidopsis flowers, validated by RT-PCR and enzymatic or functional tests, show dramatic changes in u-ATP9 plants. Both lines present a modification in the expression of several genes involved in carbon, lipid and cell wall metabolism, suggesting that an important metabolic readjustment occurs in plants with a mitochondrial dysfunction. Interestingly, transcript levels involved in mitochondrial biogenesis, protein synthesis, and degradation are affected. Moreover, several mRNA levels for transcription factors and DNA binding proteins were also changed. Some of them are involved in stress and hormone response, suggesting that several signaling pathways overlap. Indeed, the transcriptome data reveal that the mitochondrial dysfunction dramatically alters genes involved in signaling pathways, including those involved in ethylene, absicic acid and auxin signal transduction. Our data suggest that the mitochondrial dysfunction model used in this rapport may be useful to uncover the retrograde signaling mechanism between the nucleus and mitochondria in plant cells. Arabidopsis oligonucleotide microarrays fabricated by the University of Arizona contain 26,000 oligonucleotides (http://www.ag.arizona.edu/microarray/). RNA was isolated from 6-week-old flowers from u-ATP9 and wt plants. The experimental (mutant) and reference (wild type) RNA samples were reverse-transcribed and directly labeled with either Cy5-dUTP or Cy3-dUTP fluorescent dye (GE Healthcare), using random hexamer primers (Invitrogen). Excess nucleotides and primers were removed using QIAquick PCR Purification Kit (Qiagen). Labeled samples were mixed and then hybridized to microarray for 15 h at 60°C. The slides were washed at room temperature in three wash steps: 2 x SSC, 0.5% SDS; 0.5 x SSC; and 0.05 x SSC for 5 min each with gentle shaking. The slides were scanned with a GenePix 4000B Scanner (Axon Instruments Inc., Union City, CA). Normalization between the Cy3 and Cy5 fluorescent dye emission channels was achieved by adjusting the levels of both image intensities. The experiments were repeated four times with samples from different experiments, as biological replicates. In dye swapping experiments, the RNA samples from different experiments were reciprocally labeled, both as a biological and technical repetition for comparing the reproducibility of the experiments. Hybridization intensities for each microarray element were measured using ScanAlyze 4.24 (available at http://genome-www4.stanford.edu/MicroArray/SMD/restech.html). The two channels were normalized in log space using the z-score normalization on a 95% trimmed data set. We removed unreliable spots according to the following criteria: spots flagged as having false intensity caused by dust or background on the array were removed; and spots for which intensity was less than three fold above background were also eliminated. Data from multiple experiments were normalized (Bolstad, 2003) and signals from spots from different experiments were statistically analyzed using Significance Analysis of Microarrays using the one class response (SAM, http://www-stat.stanford.edu/~tibs/SAM/), cut at a false discovery rate < 10% (Tusher, 2001).
Project description:The goal of this project is to compare the primary metabolite profile in different tissue types of the model plant Arabidopsis thaliana. Specifically, plants were grown hydroponically under the long-day (16hr light/day) condition at 21C. Tissue samples, including leaves, inflorescences, and roots were harvest 4 1/2 weeks post sowing. Untargeted primary metabolites profiling was carried out using GCTOF.
Project description:Stresses that target mitochondrial function lead to altered transcriptional responses for 100-1000s of genes genome wide, and are signalled via retrograde signalling pathways within the cell. rao1 mutants contain a mutation in a gene encoding a Cyclin-Dependant Kinase E;1 and cannot induce stress responsive genes (such as the mitochondrial alternative oxidase 1a) in response to mitochondrial dysfunction. We sought to define the global gene network regulated through RAO1 function in response to mitochondrial stress (mimicked through treatment of plants with antimycin A - a specific inhibitor of complex III in the mitochondrial electron transfer chain). We have defined global stress responses that are positively and negatively mediated by RAO1 function, as well as global stress responses to antimycin A treatment that are regulated independently of RAO1. We used Affymetrix microarray to characterise global gene expression profiles for mutant plants with compromised mitochondrial retrograde signalling under stress (rao1 mutants), to define the genome wide transcriptional network regulated through RAO1 function.