Identification of ORS1 target genes using inducible overexpression system
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ABSTRACT: The aim was to identify early target genes of the senescence-associated transcription factor: ORS1. For this purpose we used DEX-inducible system and studied the expression profile 5h after treatment using Affymetrix microarray. Keywords: genetic modification Arabidopsis 46 day-old transgenic plants (Arabidopsis C24 background, inducible overexpression) were sprayed with 30µM DEX for 5h. As control mock (0.05% ethanol) treated ORS1-inducible over-expression plants were used. Whole rosettes were harvested 5h after induction and used for expression profiling using Affymetrix near-full genome ATH1 arrays. Experiment performed in two biological replications.
Project description:Arabidopsis plants expressing DEX-inducible version of the developmental regulator FIL were exposed to DEX or a solution lacking DEX and changes in gene expression assessed after 4h and 8h using microarray analysis.
Project description:The aim was to identify early target genes of the senescence-associated transcription factor: ORS1. For this purpose we used DEX-inducible system and studied the expression profile 5h after treatment using Affymetrix microarray. Keywords: genetic modification
Project description:The aim of this study is to identify early DELLA protein-responsive genes using a Dexamethasone (DEX)-inducible system. Two transgenic lines were used: one induces the expression of a dominant, gibberellin non-responsive DELLA protein (rga-delta17); the other is a control line that carries the same vector, but lacks the rga-delta17 transgene. By comparing the gene expression changes in the line that expresses the rga-delta17 protein in the presence or absence of DEX it is possible to identify putative targets of DELLA proteins. An empty vector transgenic line was included in this study to identify genes that might be regulated by the DEX inducible system that are not dependent on the DELLA protein. Experiment Overall Design: Seedlings of both transgenic lines were pretreated for 16 h with 2 uM GA4 to enhance gibberellin responses. Because DELLA proteins are strong signaling repressors, this pretreatment should maximize the effect of DELLA induction. Eight-day old seedlings were treated with 2 uM GA4 or a combination of 2 uM GA4 plus 10 uM DEX to induce the rga-delta17 transgene. Three biological replicas for the transgenic line that carries the DEX-inducible rga-delta17 transgene were generated at 2h and 4h. For the empty vector line, only 2 biological replicas were generated at 4h of treatment with 2 uM GA with or without 10 uM DEX.
Project description:MYB46 functions as a transcriptional switch that turns on the genes necessary for secondary wall biosynthesis. Elucidating the transcriptional regulatory network immediately downstream of MYB46 is crucial to our understanding of the molecular and biochemical processes involved in the biosynthesis and deposition of secondary walls in plants. The transcription factors identified here may include direct activators of secondary wall biosynthesis genes. The current study discovered novel hierarchical relationships among the transcription factors involved in the transcriptional regulation of secondary wall biosynthesis and generated several testable hypotheses. To gain insights into the MYB46-mediated transcriptional regulation, we first established an inducible secondary wall thickening system in Arabidopsis by expressing MYB46 under the control of dexamethasone inducible promoter. Then, we used ATH1 GeneChip microarray to obtain a series of time-course transcriptome profiles with regard to the induction of secondary wall development.
Project description:The development of trichomes (leaf hairs) from pluripotent epidermal cells in Arabidopsis provides a powerful system to investigate the regulatory motifs involved in plant cell differentiation. Genetic studies have revealed that a bHLH transcription factor, GL3, activates downstream genes required for trichome initiation by interacting with a R2R3-MYB protein, GL1. In order to investigate genome-wide regulatory functions of GL1 and GL3, we performed genome-wide expression analyses using GR inducible systems of GL1 and GL3. Transgenic plants carrying pGL1::GL1-GR and pGL3::GL3-GR were treated with mock or Dex for 4 and 24 hours.
Project description:In order to identify candidate target genes of the OBP1 (At3g50410) transcription factor we used dexamethasone inducible system (Lloyd et al, 1994). A single inducible over-expression line was compared to an empty vector control line 10h after DEX induction to identify candidate genes that were confirmed by quantitative RT-PCR.
Project description:Transgenic Arabidopsis seedlings RV86-5 (contains a dexamethasone-inducible ubiquitin variant that contains Arg instead of Lys at position 48; ̈Schlögelhofer et al., 2005) were surface-sterilized and sown on Uhelon 120T (Silk & Progress, Czech Republic) mesh placed on 1% (w/v) agar containing a half-strength Murashige and Skoog medium (pH 5.7), stratified at 4 °C for 3 d, and cultivated at 21 °C/19 °C day/night temperatures, with a 16 h photoperiod (90 μmol m−2 s−1 light intensity) for 14 d. On the fourteenth day (after the first 2 h of the day period), the Uhelon mesh with the seedlings was transferred onto a new solid medium supplemented with (i) 5×10−4% (v/v) DMSO (mock); (ii) 0.7 or 7.0 μM dexamethasone (DEX) and rinsed with DMSO or DEX-supplemented water for mock and DEX-treated seedlings, respectively, and incubated for 24 hours.
Project description:Global transcriptome patterns were performed using SHYG-IOE-5h (5h after Estradiol and Mock treatment) To identify genes more rapidly responding to elevated SHYG , we perfromed gene expression profiling (Affymetrix) upon 5h Est treatment of SHYG-IOE plants.
Project description:Plants typically contain two different types of cell walls: a primary wall that is being deposited around all growing cells, and a secondary wall that is produced in cells with specialized functions once they have ceased to grow. In Arabidopsis, VND7 is a transcription factor that is sufficient to activate secondary cell wall synthesis. To artificially turn on the secondary cell wall synthesis, VND7 was fused to the activation domain of the herpes virus VP16 protein and the glucocorticoid receptor (GR) domain. Thus, the transgenic plants harbouring the constructs can then be treated with dexamethasone (DEX), a glucocorticoid derivative, to induce the secondary cell wall formation. We used microarrays to investigate the global program of gene expression during the secondary cell wall development and identified up- and down-regulated genes associated with this process. Cultures were harvested at indicated time points after induction (1, 3, 6, 9, 12, 24, 30, 48 h) and at time point zero (0). Uninduced cultures (DMSO instead of DEX) served as controls for each time point, and thus, 48 microarray analyses for 8 time points (3 biological reps) for +/- induced VND7-VP16-GR cultures, and 3 arrays for time 0h. To ensure that the DEX did not influence the measurements, an empty vector construct (EV), i.e., without the VND7 gene, VP16-GR, was used at three time points, i.e., VP16-GR +/- DEX at 9h, 12h, and 24h after DEX induction.