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 Experiment Overall Design: 12 samples were used in this experiment

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: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:In Arabidopsis thaliana, each member of a large family of AS2/LOB (ASYMMETRIC LEAVES 2 / LATERAL ORGAN BOUNDARIES) genes encodes a highly homologous plant-specific protein. A mutational lesion of the representative AS2 gene results in the development of anomalous asymmetric leaves, implying that these family members might commonly play some roles in plant development. Here, we found that ectopic and over-expression of ASL9 (ASYMMETRIC LEAVES 2 LIKE 9) in transgenic plants displayed a markedly anomalous architecture during development of adult plants. The expression pattern of ASL9 was quite characteristic in that it was expressed at the restricted bases of lateral organs formed from vegetative, inflorescence, and floral meristems. It was also demonstrated that, among the AS2/LOB family members, ASL9 is uniquely distinct from others in that this one is exclusively, markedly, and specifically regulated by the plant hormone cytokinin in a manner dependent on the His-Asp phosphorelay signal transduction. To gain further insight into the function of ASL9, we carried out transcriptome analysis of transgenic lines that ectopically overexpress ASL9 (ASL9-ox). Wild-type and ASL9-ox plants were grown on agar plates containing MS salt and 1% sucrose under continuous fluorescent light for 2 weeks. For each strain, these 2-week-old 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). The resultant data set will be of help to discuss and hypothesize that ASL9 might be implicated in the regulation of differentiation of lateral organs with a linkage to the actions of cytokinin that is primarily propagated through the His-Asp phosphorelay signal transduction.

Project description:In Arabidopsis thaliana, each member of a large family of AS2/LOB (ASYMMETRIC LEAVES 2 / LATERAL ORGAN BOUNDARIES) genes encodes a highly homologous plant-specific protein. A mutational lesion of the representative AS2 gene results in the development of anomalous asymmetric leaves, implying that these family members might commonly play some roles in plant development. Here, we found that ectopic and over-expression of ASL9 (ASYMMETRIC LEAVES 2 LIKE 9) in transgenic plants displayed a markedly anomalous architecture during development of adult plants. The expression pattern of ASL9 was quite characteristic in that it was expressed at the restricted bases of lateral organs formed from vegetative, inflorescence, and floral meristems. It was also demonstrated that, among the AS2/LOB family members, ASL9 is uniquely distinct from others in that this one is exclusively, markedly, and specifically regulated by the plant hormone cytokinin in a manner dependent on the His-Asp phosphorelay signal transduction. To gain further insight into the function of ASL9, we carried out transcriptome analysis of transgenic lines that ectopically overexpress ASL9 (ASL9-ox). Wild-type and ASL9-ox plants were grown on agar plates containing MS salt and 1% sucrose under continuous fluorescent light for 2 weeks. For each strain, these 2-week-old 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). The resultant data set will be of help to discuss and hypothesize that ASL9 might be implicated in the regulation of differentiation of lateral organs with a linkage to the actions of cytokinin that is primarily propagated through the His-Asp phosphorelay signal transduction. 6 samples were used in this experiment.

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Cytokinin treatment on aerial parts of seedlings

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