Project description:The goal of this experiment was to explore the molecular network of glucose-TOR signaling in Arabidopsis seedling autotrophic transition stage. We used the whole-genome microarrays to detail the global program of gene expression mediated by glucose and TOR. Arabidopsis WT and estradiol inducible RNAi-tor seedlings were germinated in low light condition for 3 days to deplete seed nutrient supply. Fifteen mM glucose was adding back to reactivate the quiescent seedlings and the samples were harvested for RNA isolation. Transcript profiles were analyzed and compared between with and without glucose treatment to identify early glucose-TOR target genes. Each experimental condition was repeated three times and total 12 samples were generated.
Project description:To identify targets of the C2H2 zinc finger protein ZINC 34 FINGER OF ARABIDOPSIS THALIANA 14 (ZAT14, AT5G03510), we used an estradiol-inducible system for inducible expression of ZAT14, comparing gene expression in estradiol-treated plants and mock-treated 5-day old seedlings 8 hours after estradiol or mock treatment.
Project description:Arabidopsis SnRK1 is structurally and functionally related to the yeast Snf1 and mammalian AMP-activated kinases, which are activated in response to carbon/glucose limitation and stress conditions causing an imbalance of energy homeostasis increasing the AMP/ATP ratio. Mutations of the SNF4 activating subunit of trimeric Arabidopsis SnRK1 complexes are not transmitted through the male meiosis. Silencing of SNF4 by a β-estradiol-inducible artificial microRNA (amiR-SNF4) constructs was used to examine how inhibition of SnRK1 affects transcriptional regulation of different cellular pathways in dark and light grown seedlings. This study shows that amiR-SNF4 silencing of SnRK1 leads to coordinate transcriptional activation of salicylic acid and trehalose synthesis, oxidative/endoplasmic reticulum stress and pathogen defense responses by inducing simultaneous changes in numerous other essential hormonal and metabolic pathways in Arabidopsis. We used Affymetrix ATH1-121501 Genome Array to compare global transcript levels in wild type and β-estradiol-induced amiR-SNF4 mutant seedlings 5 days after germination in the dark or light.
Project description:Global transcriptome patterns were determined in XVE-14 and wild-type seedlings induced for 45 min b-estradiol in order to identify the genes early regulated by EBE transcription factor. We used microarrays to identify genes differentially expressed in EST-inducible EBE over-expression line #14 compared to wild-type plants, 45 min after 2µM EST induction. Three independent biological replications were performed. In order to identify potential direct/early target genes of EBE transcription factor, estradiol inducible overexpression system was used. Three week old Arabidopsis EBE-XVE (line 14) and WT seedlings were treated with ß-estradiol (2µM) for 45 min. RNA was isolated from shoot and subjected to hybridization on Affymetrix microarrays. Experiment was performed in 3 biological replications and genes differentially expressed between estradiol treated EBE-XVE and WT plants were identified as potential early targets of EBE.
Project description:To validate that the transcriptional program rapidly activated by PP242, WYE354, KU63794 and Torin2 was specifically associated with the complete inhibition of TOR kinase, we performed genome-wide transcript profiling using the estradiol-inducible tor-es mutant after reaching complete TOR protein silencing in Arabidopsis seedlings grown in the full-nutrient medium. The estradiol inducible tor-es mutant was treated without/with estradiol for 72 hours to complete inhibition of TOR kinase. And RNA-seq was performed for comparison the transcriptomes changes (http://gbnci.abcc.ncifcrf.gov/geo/gse_view.php?ID=58096).
Project description:To identify potenital target genes of the transcription factor KUODA1 (AT5G47390), we performed expression profiling using an estradiol inducible overexpression system. Estradiol-treatment allows for the nuclear translocation of the constitutively expressed chimeric transcription factor XVE and subsequent expression of KUA1 from the LexA promoter. Stable trangenic T3 seedlings were grown for 14 days and either treated with 15 M-BM-5M estradiol or ethanol (mock) for 4 hours. Two mock and two estradiol treated samples were used for RNA isolation and hybridization.
Project description:We designed an experiment to identify BAK1 interactors considering that it could be a coreceptor for the GOLVEN6 (GLV6) signaling peptide involved in lateral root organogenesis in Arabidopsis thaliana. A BAK1pro:BAK1-GFP/bak1-4 line crossed to an estradiol-inducible GLV6 overexpression line (BAK1pro:BAK1-GFP/bak1-4xiGLV6) was treated with mock or estradiol followed by affinity purification mass spectrometry (AP-MS) experiments using anti-GFP beads in microsomal-enriched fractions. Statistical analysis of estradiol-treated vs. mock seedlings did not reveal significant differences. Nevertheless, we remarked that regardless of the treatment, several LRR-RLKs co-eluted with BAK1-GFP, comprising known, as well as, unknown interactors. We confirmed interaction of BAK1 with these LRR-RLKs by BIFC and identified the PXC3 receptor as a BAK1 constitutive interactor, which was further verified by co-IP.
Project description:The experiment is aiming to analyze the effect of overexpression of the Arabidopsis At1g06160 AP2/ERF-domain transcription factor on gene expression and the involvement of the At1g06160-regulated genes in the jasmonic acid or jasmonic acid/ethylene signal transduction pathways. The experiment was performed with 12 hybridizations, using 24 samples of species [Arabidopsis thaliana], using 12 arrays of array design [Agilent Arabidopsis 3 Oligo Microarray [G4142A]], producing 12 raw data files and 0 transformed and/or normalized data files. In the first two hybridizations (#1 and #2), two independent transgenic Arabidopsis lines containing a construct carrying the Arabidopsis gene At1g06160 fused to an inducible promoter were used. This promoter is activated after the addition of the chemical estradiol to the plant medium. Addition of estradiol results in overexpression of At1g06160. For each independent line, total RNA from estradiol-treated and -untreated plants were collected and labeled with Cy3 and Cy5, respectively, and hybridization was performed for each At1g06160-inducible line. Two other hybridizations (#3 and #4) were performed using two independent transgenic Arabidopsis control lines containing a construct carrying the GUS gene fused to an inducible promoter. For each independent line, total RNA from estradiol-treated and -untreated plants were collected and labeled with Cy3 and Cy5, respectively and hybridization was performed for each GUS-inducible line. The last 8 hybridizations were performed using wild-type Arabidopsis plants. The aim was analyze the effect of the jasmonic acid hormone or the effect of a combination of the jasmonic acid and ethylene hormones on gene expression in wild-type. Hybridizations #5 and #6: wild-type Arabidopsis seedlings were treated with jasmonic acid (JA) or with the control DMSO and total RNA was collected after 8 hours. Total RNA from JA-treated and DMSO-treated plants were collected and labeled with Cy3 and Cy5, respectively, and hybridization was performed. A second hybridization was performed using independent biological samples that were treated and labeled similarly. Hybridizations #7 and #8: wild-type Arabidopsis seedlings were treated with jasmonic acid (JA) or with the control DMSO and total RNA was collected after 24 hours. Total RNA from JA-treated and DMSO-treated plants were collected and labeled with Cy3 and Cy5, respectively, and hybridization was performed. A second hybridization was performed using independent biological samples that were treated and labeled similarly. Hybridizations #9 and #10: wild-type Arabidopsis seedlings were treated with jasmonic acid (JA) and ethylene or with the control DMSO/Na-phosphate and total RNA was collected after 8 hours. Total RNA from JA/ethylene-treated and DMSO/Na-phosphate-treated plants were collected and labeled with Cy3 and Cy5, respectively, and hybridization was performed. A second hybridization was performed using independent biological samples that were treated and labeled similarly. Hybridizations #11 and #12: wild-type Arabidopsis seedlings were treated with jasmonic acid (JA) and ethylene or with the control DMSO/Na-phosphate and total RNA was collected after 24 hours. Total RNA from JA/ethylene-treated and DMSO/Na-phosphate-treated plants were collected and labeled with Cy3 and Cy5, respectively, and hybridization was performed. A second hybridization was performed using independent biological samples that were treated and labeled similarly.
Project description:The aim was to identify genes associated to the down-regulation TARGET OF RAPAMYCIN (TOR) pathway in Arabidopsis. For this purpose, three independent amiR-tor lines (amiR-tor9, amiR-tor17 and amiR-tor 20) and EV lines 3 and 6 days after EST- or non-induction were used for expression profile analysis using Affymetrix microarray. Two weeks-old transgenic seedlings were transferred to MS plate with 20µM estradiol to induce the overexpression of amiR-tor (consequently, repression of AtTOR levels) under the control of an estradiol-(EST) inducible promoter. Identically treated wild-type Col-0 and pER8 empty-vector (EV) transformed seedlings were served as controls. Seedlings were harvested after 3 and 6 (amiR-tor lines) days.
Project description:The goal of this experiment was to identify the downstream targets of the GOLVEN6 peptide signaling pathway in Arabidopsis thaliana, specifically during lateral root initiation. Using an estradiol inducible GLV6 overexpression construct in wildtype and rgi1rgi5 double mutant (mutant in receptors for the GLV6 peptide) backgrounds, in combination with gravistimulation induced lateral root formation, the RGI receptor dependent transcriptional effects of GLV6 overexpression were characterized. An estradiol inducible GLV6 overexpression line in a wildtype (iGLV6) and in an rgi1rgi5 double receptor mutant background (rgi1rgi5/iGLV6) were used. 4-day old seedlings of both lines were gravistimulated (vertically grown seedlings were turned counterclockwise by 90°) to induce lateral root initiation in the resulting root bends. 8h after gravistimulation, seedlings of both lines were treated with 2µM of estradiol to induce GLV6 overexpression, or DMSO as a mock treatment. 3h and 6h after treatment, root bends were dissected and collected for RNA-sequencing. This yielded a total of 8 samples per replicate; 3h mock treated iGLV6 (IM3), 3h estradiol treated iGLV6 (IE3), 3h mock treated rgi1rgi5/iGLV6 (RM3), 3h estradiol treated rgi1rgi5/iGLV6 (RE3), 6h mock treated iGLV6 (IM6), 6h estradiol treated iGLV6 (IE6), 6h mock treated rgi1rgi5/iGLV6 (RM6), 6h estradiol treated rgi1rgi5/iGLV6 (RE6). For each sample, 4 replicates were obtained. This setup enabled the comparison of the GLV6 induced transcriptional effects between wildtype and rgi1rgi5 mutants at 2 time points after treatment, in samples that are strongly enriched for lateral root initiation events.