Project description:Oxidative and Cytokinin treatment of Arabidopsis wildtype, crf6 mutant, and CRF6 overexpressing seedlings Arabidopsis seedlings were treated with oxidative stress or cytokinin to determine transcripts altered in each genotype background
Project description:According to the well-documented scenario with regard to the cytokinin-mediated phosphorelay signal transduction in Arabidopsis thaliana, certain members of the type-B ARR family are crucially implicated in the regulatory networks that are primarily propagated by the cytokinin-receptors (AHKs) in response to cytokinin. Nevertheless, clarification of the biological impact of these type-B ARR transcription factors is at a very early stage. Here we focused on a pair of highly homologous ARR10 and ARR12 genes by constructing an arr10 and arr12 double-null mutant. The mutant alleles used in this study were arr10-5 and arr12-1. arr10-5 is the SALK_098604 T-DNA insertion line, whose mutation was determined to be located in the fifth exon of the ARR10 coding sequence. arr12-1 is the SALK_054752 T-DNA insertion line, whose mutation was determined to be located in the third exon of the ARR12 coding sequence. The resulting mutant showed remarkable phenotypes with special reference to the cytokinin-action in roots (e.g., inhibition of root elongation, green callus formation from explants). Furthermore, we demonstrated that ARR10 and ARR12 are involved in the AHK-dependent signaling pathway that modulates the differentiation of root-vascular tissues (i.e., protoxylem-specification), suggesting that ARR10 and ARR12 are the prominent players that act redundantly in the AHK-dependent cytokinin signaling in roots. Keeping this in mind, we then collected the root-specific and combinatorial DNA microarray datasets with regard to the cytokinin-responsible genes by employing both the wild-type and arr10 arr12 double-mutant plants. In this study, wild-type and the arr10 arr12 mutant grown vertically on MS agar plates for 2 weeks were treated with 20 microM of the cytokinin trans-zeatin (TZ) or 0.02% DMSO (solvent for trans-zeatin solution) for 1h. These treated plant samples were divided into three portions, from which RNA samples were prepared separately from roots of seedlings with use of RNeasy Plant Mini Kit (Qiagen, Valencia, CA, U.S.A.). The quality of RNAs prepared was analyzed by Bioanalyzer 2100 (Agilent Technologies). These RNA samples were processed as recommended by the Affymetrix instruction (Affymetrix GeneChip Expression Analysis Technical Manual, Affymetrix). These datasets will provide us with bases for understanding the early response to cytokinin on roots of seedlings in Arabidopsis thaliana.
Project description:According to the well-documented scenario with regard to the cytokinin-mediated phosphorelay signal transduction in Arabidopsis thaliana, certain members of the type-B ARR family are crucially implicated in the regulatory networks that are primarily propagated by the cytokinin-receptors (AHKs) in response to cytokinin. Nevertheless, clarification of the biological impact of these type-B ARR transcription factors is at a very early stage. Here we focused on a pair of highly homologous ARR10 and ARR12 genes by constructing an arr10 and arr12 double-null mutant. The mutant alleles used in this study were arr10-5 and arr12-1. arr10-5 is the SALK_098604 T-DNA insertion line, whose mutation was determined to be located in the fifth exon of the ARR10 coding sequence. arr12-1 is the SALK_054752 T-DNA insertion line, whose mutation was determined to be located in the third exon of the ARR12 coding sequence. The resulting mutant showed remarkable phenotypes with special reference to the cytokinin-action in roots (e.g., inhibition of root elongation, green callus formation from explants). Furthermore, we demonstrated that ARR10 and ARR12 are involved in the AHK-dependent signaling pathway that modulates the differentiation of root-vascular tissues (i.e., protoxylem-specification), suggesting that ARR10 and ARR12 are the prominent players that act redundantly in the AHK-dependent cytokinin signaling in roots. Keeping this in mind, we then collected the root-specific and combinatorial DNA microarray datasets with regard to the cytokinin-responsible genes by employing both the wild-type and arr10 arr12 double-mutant plants. In this study, wild-type and the arr10 arr12 mutant grown vertically on MS agar plates for 2 weeks were treated with 20 microM of the cytokinin trans-zeatin (TZ) or 0.02% DMSO (solvent for trans-zeatin solution) for 1h. These treated plant samples were divided into three portions, from which RNA samples were prepared separately from roots of seedlings with use of RNeasy Plant Mini Kit (Qiagen, Valencia, CA, U.S.A.). The quality of RNAs prepared was analyzed by Bioanalyzer 2100 (Agilent Technologies). These RNA samples were processed as recommended by the Affymetrix instruction (Affymetrix GeneChip Expression Analysis Technical Manual, Affymetrix). These datasets will provide us with bases for understanding the early response to cytokinin on roots of seedlings in Arabidopsis thaliana. 12 samples were used in this experiment.
Project description:The plant hormone cytokinin regulates diverse aspects of plant growth and development, likely through context-dependent transcriptional regulation that relies on a dynamic interplay between regulatory proteins and chromatin. We employed the Assay for Transposase Accessible Chromatin (ATAC-Seq) to profile changes in the chromatin landscape of Arabidopsis roots and shoots in response to cytokinin. Our results reveal differentially accessible chromatin regions indicative of dynamic regulation in response to cytokinin. These changes in chromatin occur preferentially upstream of genes whose expression changes in response to cytokinin and largely overlap with binding sites for the type-B ARRs, transcription factors that mediate the primary response to cytokinin. Further, the type-B ARRs were found to be necessary for the changes in chromatin state in response to cytokinin. Lastly, we find context-dependent responses by comparing root and shoot profiles. These datasets provide new insights into the transcriptional regulatory mechanisms involved in the cytokinin response and how cytokinin mediates its pleiotropic effects.
Project description:Above ground tissue of 10 day old Arabidopsis seedlings of Col wild-type, 35S-ARR7, arr7, 35S-ARR15 was treated with Cytokinin (benzyladenine), Auxin (indole-3-acetic acid) or both.
Project description:In Arabidopsis thaliana, the immediate early response of plants to cytokinin is formulated as the multistep AHK-AHP-ARR phosphorelay signaling circuitry, which is initiated by the cytokinin-receptor histidine protein kinases. In the hope of finding components (or genes) that function downstream of the cytokinin-mediated His-Asp phosphorelay signaling circuitry, we carried out genome-wide microarray analyses. To this end, we focused on a pair of highly homologous ARR10 and ARR12 genes by constructing an arr10 arr12 double null mutant. The mutant alleles used in this study were arr10-5 and arr12-1. arr10-5 is the SALK_098604 T-DNA insertion line, whose mutation was determined to be located in the fifth exon of the ARR10 coding sequence. Arr12-1 is the SALK_054752 T-DNA insertion line, whose mutation was determined to be located in the third exon of the ARR12 coding sequence. The resulting mutant exhibits a characteristic phenotype with regard to the cytokinin-mediated His-Asp phosphorelay. Here we, therefore, compared response to cytokinin in wild type with that in arr10 arr12 double mutant. In this study, wild type and the arr10 arr12 double mutant grown vertically on MS agar plates for 2 weeks were treated with 20uM t-zeatin or 0.02% DMSO (solvent for t-zetion solution) for 1h. These treated plant samples were divided into three portions, from which RNA samples were prepared separately from aerial parts of seedlings with use of RNeasy Plant Mini Kit (Qiagen, Valencia, CA, U.S.A.). The Quality of RNAs prepared was analyzed by Bioanalyzer 2100 (Agilent Technologies). These RNA samples were processed as recommended by the Affymetrix instruction (Affymetrix GeneChip Expression Analysis Technical Manual, Affymetrix). These dataset will provide us with bases for understanding the early response to cytokinin on aerial parts of seedlings in Arabidopsis thaliana.
Project description:In Arabidopsis thaliana, the immediate early response of plants to cytokinin is formulated as the multistep AHK-AHP-ARR phosphorelay signaling circuitry, which is initiated by the cytokinin-receptor histidine protein kinases. In the hope of finding components (or genes) that function downstream of the cytokinin-mediated His-Asp phosphorelay signaling circuitry, we carried out genome-wide microarray analyses. To this end, we focused on a pair of highly homologous ARR10 and ARR12 genes by constructing an arr10 arr12 double null mutant. The mutant alleles used in this study were arr10-5 and arr12-1. arr10-5 is the SALK_098604 T-DNA insertion line, whose mutation was determined to be located in the fifth exon of the ARR10 coding sequence. Arr12-1 is the SALK_054752 T-DNA insertion line, whose mutation was determined to be located in the third exon of the ARR12 coding sequence. The resulting mutant exhibits a characteristic phenotype with regard to the cytokinin-mediated His-Asp phosphorelay. Here we, therefore, compared response to cytokinin in wild type with that in arr10 arr12 double mutant. In this study, wild type and the arr10 arr12 double mutant grown vertically on MS agar plates for 2 weeks were treated with 20uM t-zeatin or 0.02% DMSO (solvent for t-zetion solution) for 1h. These treated plant samples were divided into three portions, from which RNA samples were prepared separately from aerial parts of seedlings with use of RNeasy Plant Mini Kit (Qiagen, Valencia, CA, U.S.A.). The Quality of RNAs prepared was analyzed by Bioanalyzer 2100 (Agilent Technologies). These RNA samples were processed as recommended by the Affymetrix instruction (Affymetrix GeneChip Expression Analysis Technical Manual, Affymetrix). These dataset will provide us with bases for understanding the early response to cytokinin on aerial parts of seedlings in Arabidopsis thaliana. Experimenter name: Hitoshi SAKAKIBARA Experimenter phone: 81455039576 Experimenter fax: 81455039609 Experimenter address: Biodynamics Research Team Experimenter address: RIKEN Plant Science Center Experimenter address: 1-7-22 Suehiro, Tsurumi Experimenter address: Yokohama Experimenter zip/postal_code: 230-0045 Experimenter country: JAPAN Keywords: compound_treatment_design;
Project description:In Arabidopsis thaliana, cytokinin responsive B-type ARR transcription factors and HD-ZIP III transcription factors such as REVOLUTA (REV), act cooperatively as master regulators of shoot regeneration. To identify the downstream targets of ARR-HD-ZIP III transcriptional complex, we used an inducible line of REV, 35S::FLAG-GR-rREV, in which FLAG-tagged miR165/6-non-targetable form of REV (rREV)-GR fusion protein was expressed from 35S promoter. DEX treatment induced activation of REV by translocation of FLAG-GR-rREV fusion protein from cytoplasm to the nucleus. We treated 35S::FLAG-GR-rREV seedlings with 6-benzylaminopurine (6-BA, a cytokinin), dexamethasone (DEX), or 6-BA+DEX for 2 hours. Total RNAs were extracted and subjected to Agilent Arabidopsis Gene Expression Microarray analyses. The differentially expressed genes (>1.5-fold, p<0.05) were identified.
Project description:5 to 7 days old Arabidopsis thaliana seedlings of wildtype (Col-0) and the cytokinin deficient transgenic line 35S:CKX1 (Werner et al. Plant Cell 15, 2532) at growth stage 1.00 (Boyes scale) grown in liquid medium were induced witn 5 µM 6-Benzylaminopurine for different periods of time in order to find cytokinin-related changes in gene expression.
Project description:6 days old Arabidopsis thaliana of Col-0 wild-type and 35S:ARR1-SRDX transgenic seedlings grown in liquid culture (1/2 MS, 1 g/L sucrose, 0.5 g/L MES, pH 5.7) were induced with either 5 µM 6-Benzyladenine (a cytokinin analog) for 15 or 120 min, or mock-treated 120 min as a control. These samples were subjected to microarray analysis.