Transcription profiling by array of Arabidopsis expressing DUO1 as a result of induction with estradiol
ABSTRACT: The experiment is designed to examine changes in gene expression upon ectopic expression of the germ line transcription factor DUO1 in Arabidopsis. Plants were transformed with the vector pMDC7DUO1, allowing induction of DUO1 by treatment with estradiol. T2 seedlings were selected on agar plates containing hygromycin for 12 days before seedlings were transferred to plates containing either 2 µM 17beta-estradiol (induced), or control plates containing DMSO (non-induced). Plants were grown for 6, 12 or 24 hours before harvesting. RNA was extracted using the RNAeasy kit from Qiagen. Each sample consists of 20 seedlings combined and all treatments have been conducted in triplicate. 18 samples were used in this experiment.
Project description:The experiment is designed to examine changes in gene expression upon ectopic expression of the germ line transcription factor DUO1 in Arabidopsis. Plants were transformed with the vector pMDC7DUO1, allowing induction of DUO1 by treatment with estradiol. T2 seedlings were selected on agar plates containing hygromycin for 12 days before seedlings were transferred to plates containing either 2 µM 17beta-estradiol (induced), or control plates containing DMSO (non-induced). Plants were grown for 6, 12 or 24 hours before harvesting. RNA was extracted using the RNAeasy kit from Qiagen. Each sample consists of 20 seedlings combined and all treatments have been conducted in triplicate. 18 samples were used in this experiment.
Project description:The aim of this investigation is to analyse the effect on nuclear gene expression of inhibition of chloroplast division. Transgenic Arabidopsis lines (Ler) were generated that contained a chemically inducible promoter (XVE) upstream of AtMinD1. AtMinD1 encodes a product involved in chloroplast division; overexpression of AtMinD1 leads to chloroplast division inhibition. 6 samples were used in this experiment. Three treatments were performed: Treatment 1: Empty vector control seedlings (transformed with PER-10 only) were sown onto lehle, sprayed at 13 days old with 2uM 17-B-estradiol (inducer) and harvested 24 hours later. Treatment 2: Seeds were sown on lehle containing inducer and sprayed every 24 hours with 2uM inducer until harvested at 14 days old. Treatment 3: Seeds were sown on lehle plates, sprayed at 13 days old with 2uM inducer and harvested 24 hours later. For all three treatments, seedlings were grown in constant light conditions, and tissue was harvested from 14-day-old seedlings (4 rosette leaves) and snap frozen in liquid nitrogen before RNA preparation.
Project description:ATM plays an important role in the response of plants to forms of DNA damage that cause DNA double-strand breaks. Arabidopsis ATM has been shown to be important for the transcriptional induction of several DNA repair genes including AtRAD51 (Garcia et al 2003 Plant Cell 15, 119-132). In our experiment we compare the transcript profiles of wild type and mutant plants exposed to 10Gy X-rays (dose rate ~0.8 Gy/min). Plants will be grown on 0.5*MS + 1%sucrose at 22degC under short (9h) day conditions and treated after 5h light. We will irradiate wild type (Col-0) and atatm-3 mutant (N589805) seedlings at growth stage 1.06 and harvest tissue 1h post irradiation. RNA will be isolated using the SV RNA isolation kit (Promega). Comparison of the transcript profiles will then allow AtATM-dependent X-ray inducible transcripts to be identified. 6 samples (3 wild type, 3 mutant) were used in this experiment.
Project description:AtIPT8/pga22 seedlings (gain-of-function mutant in Ws background; Sun et al. 2003, Plant Physiology 131, pp167-176) were grown on vertical plates for 7 days in LD. The seedlings were then incubated directly on the plate with medium containing 5 uM 17-beta-estradiol (for induction of the IPT8 gene) or 5 uM trans-zeatin for 12 and 24 h. 5 mm primary root tips were harvested from the seedlings and pooled for microarray analysis. Total RNA was isolated from the samples with the RNeasy Plant Mini Kit from Qiagen. 14 samples were used in this experiment.
Project description:The Arabidopsis mutant vtc2 is vitamin C-deficient and high light sensitive (Mueller-Moule et al., 2002. Plant Physiol 128: 970-977; Mueller-Moule et al., 2003. Plant Physiol 133: 748-760). Even though the VTC2 gene has been cloned (Jander et al., 2002. Plant Physiol 129: 440-450), its function is still unclear. The goal of this experiment is to learn more about the function of the VTC2 gene by determining differences in the transcriptomes of wild type and the vtc2-1 mutant. In order to differentiate between VTC2 gene-specific differences and vitamin C-induced differences, a second vitamin C-deficient mutant vtc1-1 (Conklin et al., 1996. PNAS 93: 9970-9974) is included in the experiment. The plants will be grown on agar plates in a growth chamber with a 16 hour light (about 40 umol photons m(-2)s(-1))/8 hour dark regime at 20C for 14 days to stage 1.04. Whole plants will be collected and total RNA will be isolated using the Qiagen Rneasy plant mini kit (Qiagen, Hilden, Germany). 9 samples were used in this experiment.
Project description:Our aim is to identify circadian transcripts that are co-regulated with [Ca2+]cyt, with the eventual goal of identifying genetic regulators and targets for circadian oscillations of [Ca2+]cyt. We have identified two conditions in which [Ca2+]cyt behaves differently to other circadian outputs. 1. Treatment of plants with nicotinamide, a metabolic inhibitor of ADPR cyclase, abolishes the circadian oscillations of [Ca 2+]cyt. However, leaf movement, CCA1, LHY, TOC1 and CAB transcript abundance and CAB promoter activity are all rhythmic albeit with a longer period (Dodd et al., 2007). 2. The toc1-1 mutant, which shortens the circadian period of all other rhythms tested, has no effect on the period of [Ca2+]cyt oscillations (Xu et al., 2007). We will measure the circadian regulation of transcript abundance in wild type (C24), toc1-1 and nicotinamide (C24)-treated plants. Method: Wild type (C24) and toc1-1 seeds were sown on 1/2MS 0.8% agar plates and imbibed at 4 C for 48 hours. Seedlings were grown in LD cycles of 12hL:12hD at 19 C for 11 days to entrain the oscillator. Following transfer to LL at dawn on the 12th day, 50% of the wild type seedlings were dosed with 50 mM nicotinamide every 2 hours over the entire course of the experiment. Wild type, toc1-1 and nicotinamide-treated seedlings (approx. 100 for each sample, excluding roots) were harvested at 4 hour intervals from 49 to 93 hours in LL (12 time points covering the entire third and fourth circadian cycles). Two independent replicates of the whole experiment will be hybridised. 72 samples were used in this experiment.
Project description:When Arabidopsis seedlings are grown in the dark, their shoot meristem remains in a repressed state and does not develop leaves. The tight control of leaf development by light provides the opportunity to analyse a fundamental plant developmental process in its very early stages. We have observed that activation of photoreceptors in dark-grown seedlings leads to a rapid and dramatic increase in mitotic activity in and around the shoot meristem. Cotyledons also exhibit an induction of cell cycle activity by light, but this consists of endoreduplication and occurs later. Many other processes take place upon induction by light, including the development of the photosynthetic apparatus and the synthesis of sunscreens, but these processes are expected to take place in both the leaf primordia and the cotyledons. We have used this information to carry out a whole transcriptome analysis of shoot meristem activation and early stages of leaf development by light. For this we dissected the shoot apical regions from seedlings grown in the dark or 1, 2, 6, 24, 48 or 72 h after transfer to light. We also dissected cotyledons from equivalent seedlings grown in the dark or 1 or 6 h after transfer to light. From these samples we obtained RNA used for hybridisations against the ATH1 GenChip. 16 samples were used in this experiment.
Project description:We used N-(1-naphthyl) phthalamic acid (NPA)-induced vascular overgrowth in Arabidopsis leaves to look for differential up-regulation of genes in NPA-treated tissues that may be involved in vascular differentiation. Arabidopsis thaliana Col-0 plants were grown for approximately 2 weeks on solid ATS medium (1) containing a final concentration of 10 um NPA (dissolved in DMSO) or an equivalent volume of DMSO (control). At this stage plants had approximately 6 rosette leaves. RNA was prepared from entire shoot tissues of control (DMSO) or NPA-treated plants.(1) Lincoln et al., 1990. Plant Cell 2: 1071-1080. 2 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:Reference: De Vos and Jander, 2009 - Plant, Cell, and Environment Aim: Identification of genes responding to aphid saliva. Background: During feeding on phloem sap aphids repeatedly salivate into the sieve element. It is thought that compounds in aphid saliva play a role in sustainable feeding. These compounds may include proteins and small molecules, which can function as virulence factors. Growth conditions Plants: Seeds of wild-type Arabidopsis thaliana (Col-0) were obtained from the were kept in 0.1% Phytagar (Invitrogen, Carlsbad, CA) for 24 h at 4°C prior to planting on Cornell mix with Osmocoat fertilizer. Plants were grown in Conviron growth chambers in 20- x 40-cm nursery flats at a photosynthetic photon flux density of 200 mmol m-2 s-1 and a 16-h photoperiod. The temperature in the chambers was 23°C and the relative humidity was 50%. Plants were grown for 3 weeks and used in experiments before flowering. Aphids: All experiments were conducted with a tobacco-adapted red lineage of M. persicae. Aphids were raised on cabbage (Brassica oleracea) with a 16-h day (150 mmol m-2 s-1 at 24°C) and an 8-h night (19°C) at 50% relative humidity. Experimental set-up/treatment: Fifty aphids were allowed to feed from 50 µL artificial diet, containing sucrose and amino acids (Kim and Jander, 2007) between two layers of Parafilm. After 24 h, artificial diet from 20 aphids and control (0 aphids) diet cups was collected and infiltrated into leaves of intact Arabidopsis plants using a 1-mL syringe without the needle. Plants for control diet and aphid saliva containing diet were grown in the same pot to allow for a paired comparison. Eighteen leaves (3 leaves from 6 plants) treated with control and aphid saliva containing diet were harvested and immediately frozen in liquid nitrogen. This experiment was repeated 3 times to function as independent biological replicates. RNA extraction + processing: RNA was extracted using the Qiagen Plant RNeasy kit. RNA quality and quantity was assessed with an Agilent BioAnalyser 2100. Samples were processed by the Cornell Microarray facility. Whole genome gene expression profiling was done using Affymetrix ATH1 GeneChips. Data analysis: Raw data from the microarrays was normalized at probe-level using gcRMA algorithm. The detection calls (present, marginal, absent) for each probe set was obtained using the GCOS system. Significance of gene expression was determined using the LIMMA (Smyth, 2004) program and raw p values of multiple tests were corrected using False Discovery Rate (FDR). 6 samples were used in this experiment