Project description:Effect of the cytokinin BA on wt and arr1,10,12 mutant seedlings The type B Arabidopsis Response Regulators (ARRs) of Arabidopsis thaliana are transcription factors that act as positive regulators in the two-component cytokinin signaling pathway. We employed a mutant-based approach to perform a detailed characterization of the roles of ARR1, ARR10, and ARR12 in plant growth and development. The most pronounced phenotype was found in the arr1-3 arr10-5 arr12-1 triple loss-of-function mutant, which showed almost complete insensitivity to high levels of exogenously applied cytokinins. The triple mutant exhibited reduced stature due to decreased cell division in the shoot, enhanced seed size, increased sensitivity to light, altered chlorophyll and anthocyanin concentrations, and an aborted primary root with protoxylem but no metaxylem. Microarray analysis revealed that expression of the majority of cytokinin-regulated genes requires the function of ARR1, ARR10, and ARR12. Characterization of double mutants revealed differing contributions of the type B ARRs to mutant phenotypes. Our results support a model in which cytokinin regulates a wide array of downstream responses through the action of a multistep phosphorelay that culminates in transcriptional regulation by ARR1, ARR10, and ARR12. This data was originally made available through ArrayExpress under the accession number E-MEXP-1573.
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:Differentially regulated genes in rosette leaves and roots of hydroponically grown Arabidopsis thaliana Col-0 and nrt1.5-5 mutant plants were identified by microarray analyses.
Project description:Here we use bisulfite conversion of rRNA depleted RNA combined with high-throughput Illumina sequencing (RBS-seq) to identify single-nucleotide resolution of m5C sites transcriptome-wide in Arabidopsis thaliana roots. m5C sites were analyzed in wild type (WT) and an Arabidopsis T-DNA KO mutant for the RNA methyltransferase TRM4B.
Project description:Identification of genes regulated by apical auxin and basal cytokinin treatment of the nodal stem in cauline buds of Arabidopsis thaliana
Project description:Muraro2014 - Vascular patterning in Arabidopsis roots
Using a multicellular model, maintanence of vascular patterning in Arabidopsis roots has been studied. The model that is provided here is the single-cell version of the model. The two-cell and multicellular models described in the paper can be downloaded as python scripts (follow the curation tab to get these files).
This model is described in the article:
Integration of hormonal signaling networks and mobile microRNAs is required for vascular patterning in Arabidopsis roots.
Muraro D, Mellor N, Pound MP, Help H, Lucas M, Chopard J, Byrne HM, Godin C, Hodgman TC, King JR, Pridmore TP, Helariutta Y, Bennett MJ, Bishopp A.
Proc Natl Acad Sci U S A. 2014 Jan 14;111(2):857-62.
Abstract:
As multicellular organisms grow, positional information is continually needed to regulate the pattern in which cells are arranged. In the Arabidopsis root, most cell types are organized in a radially symmetric pattern; however, a symmetry-breaking event generates bisymmetric auxin and cytokinin signaling domains in the stele. Bidirectional cross-talk between the stele and the surrounding tissues involving a mobile transcription factor, SHORT ROOT (SHR), and mobile microRNA species also determines vascular pattern, but it is currently unclear how these signals integrate. We use a multicellular model to determine a minimal set of components necessary for maintaining a stable vascular pattern. Simulations perturbing the signaling network show that, in addition to the mutually inhibitory interaction between auxin and cytokinin, signaling through SHR, microRNA165/6, and PHABULOSA is required to maintain a stable bisymmetric pattern. We have verified this prediction by observing loss of bisymmetry in shr mutants. The model reveals the importance of several features of the network, namely the mutual degradation of microRNA165/6 and PHABULOSA and the existence of an additional negative regulator of cytokinin signaling. These components form a plausible mechanism capable of patterning vascular tissues in the absence of positional inputs provided by the transport of hormones from the shoot.
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Project description:De novo shoot organogenesis (DNSO) is a commonly used pathway for plant biotechnology, and is a hormonally regulated process, where auxin and cytokinin coordinates suites of genes encoding transcription factors, general transcription factors, and RNA metabolism machinery genes. Here we report that silencing Arabidopsis thaliana CTD phosphatase-like 4 (CPL4RNAi), which increases phosphorylation level of RNA polymerase II (pol II) CTD, altered lateral root development and DNSO efficiency of the host plants, suggesting an importance of precise control of pol II activities during DNSO. Under standard condition, roots of CPL4RNAi lines produced no or few lateral roots. When induced by high concentration of auxin, CPL4RNAi lines failed to produce focused auxin maxima at the meristem of lateral root primordia, and produced fasciated lateral roots. By contrast, root explants of CPL4RNAi lines were highly competent for DNSO. Efficient DNSO of CPL4RNAi lines were observed even under 10 times less cytokinin required for wild type explants. Transcriptome analysis showed CPL4RNAi but not wild type explants expressed high levels of shoot meristem related genes during priming by high auxin/cytokinin ratio, and subsequent shoot induction with cytokinin. These results indicate that CPL4 functions as a repressor of the early stage of DNSO, during acquisition of competency by high auxin/cytokinin ratio, perhaps via regulation of pol II activities.
Project description:This model is from the article:
The influence of cytokinin-auxin cross-regulation on cell-fate determination in Arabidopsis thaliana root development
Muraro D, Byrne H, King J, Voss U, Kieber J, Bennett M.
J Theor Biol.2011 Aug 21;283(1):152-67.
PMID: 21640126,
Abstract:
Root growth and development in Arabidopsis thaliana are sustained by a specialised zone termed the meristem, which contains a population of dividing and differentiating cells that are functionally analogous to a stem cell niche in animals. The hormones auxin and cytokinin control meristem size antagonistically. Local accumulation of auxin promotes cell division and the initiation of a lateral root primordium. By contrast, high cytokinin concentrations disrupt the regular pattern of divisions that characterises lateral root development, and promote differentiation. The way in which the hormones interact is controlled by a genetic regulatory network. In this paper, we propose a deterministic mathematical model to describe this network and present model simulations that reproduce the experimentally observed effects of cytokinin on the expression of auxin regulated genes. We show how auxin response genes and auxin efflux transporters may be affected by the presence of cytokinin. We also analyse and compare the responses of the hormones auxin and cytokinin to changes in their supply with the responses obtained by genetic mutations of SHY2, which encodes a protein that plays a key role in balancing cytokinin and auxin regulation of meristem size. We show that although shy2 mutations can qualitatively reproduce the effect of varying auxin and cytokinin supply on their response genes, some elements of the network respond differently to changes in hormonal supply and to genetic mutations, implying a different, general response of the network. We conclude that an analysis based on the ratio between these two hormones may be misleading and that a mathematical model can serve as a useful tool for stimulate further experimental work by predicting the response of the network to changes in hormone levels and to other genetic mutations.