Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Primary stem development

ABSTRACT: Arabidopsis plants (ecotype Ler) were grown under short day conditions (8h cool white fluorescent light at 100 uE, 16h dark) at 20 Â°C and ambient humidity in PGW36 phytochambers (Controlled Environment Ltd., Winnipeg, MB, Canada). Plants were grown in Redi soil (Grace & Co, Ajax, ON, Canada) in 12 cm pots at a density of five plants per pot without fertilization for 8 weeks. Light conditions were then changed to long day (16h light, 8h dark), and plants were fertilized once with standard 10-15-10 plant fertilizer (150 ul in 300 ml water per pot) (Schultz, St. Louis, MO, USA). Twelve and 14 days after changing to long day conditions we harvested sections from stems totalling 4-6cm (â5cm stemâ) and 9-11cm (â10cm stemâ) in length, respectively. All flowers, siliques, cauline leaves, and secondary branches were discarded and the remaining bolting stems were cut with a razor blade to generate the following sections: 0-2cm and 2-4cm from 5cm stems; and 0-3cm, 3-5cm, 5-7cm, and 7-9cm from 10cm stems. For array hybridizations, labelled cDNA derived from each section was co-hybridized with cDNA from 0-2cm sections as a reference. Four replicate arrays (double dye-flip) were hybridized for each comparison. Keywords = fiber Keywords = lignin Keywords = stem developmet

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Brian Ellis

PROVIDER: E-GEOD-2000 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Global transcript profiling of primary stems from Arabidopsis thaliana identifies candidate genes for missing links in lignin biosynthesis and transcriptional regulators of fiber differentiation.

Ehlting Jürgen J Mattheus Nathalie N Aeschliman Dana S DS Li Eryang E Hamberger Britta B Cullis Ian F IF Zhuang Jun J Kaneda Minako M Mansfield Shawn D SD Samuels Lacey L Ritland Kermit K Ellis Brian E BE Bohlmann Jörg J Douglas Carl J CJ

The Plant journal : for cell and molecular biology 20050601 5

Different stages of vascular and interfascicular fiber differentiation can be identified along the axis of bolting stems in Arabidopsis. To gain insights into the metabolic, developmental, and regulatory events that control this pattern, we applied global transcript profiling employing an Arabidopsis full-genome longmer microarray. More than 5000 genes were differentially expressed, among which more than 3000 changed more than twofold, and were placed into eight expression clusters based on poly ...[more]

PMID: 15918878

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Project description:Arabidopsis thaliana plants (ecotype Landsberg erecta) were grown at 20ËC constant temperature, 8 hr/16 hr Light/Dark photoperiod at 50-60% ambient humidity, for 8 to 9 weeks. Short day conditions prevented the onset of flowering and the plants were thus maintained in growth stage 1 (leaf production) with 13 to 15 rosette leaves larger than 1mm (stage 1.13 to 1.14). Diamondback moth (DBM, Plutella xylostella) larvae were maintained on cabbage (Brassica oleracea) plants in a climate-controlled room at 25ÂºC, 12 hr photoperiod with 50%-60% relative humidity. Two days before exposing A. thaliana plants to herbivore treatment, plants were transferred to a climate-controlled room (22ÂºC, 50-60% humidity, 12 hr photoperiod). For insect treatment, seven Diamondback moth (DBM, Plutella xylostella) larvae (third to fifth instars) were placed on a group of four or five plants until time of harvest, for each time point separately. All rosette leaves were harvested at 1h, 4h, 12h, and 24h after onset of continuous herbivory and flash frozen in liquid nitrogen. Control plants were maintained under the same conditions and harvested in parallel. Keywords: time course, stress response, herbivory response For each time point (1h, 4h, 12h, and 24h) two biological replicates (DBM treatment) were performed. Control plants were harvested in parallel. Total RNA from each sample was used for two labelling reactions using dye-flips (Cy5 or Cy3) and labelled cDNA from treatment and control plants were co-hybridized. Thus, for each time-point four replicates were analyzed each comparing treatment with the corresponding control (rep1 and rep2 are dye-flips of biological replicate 1, and rep3 and rep4 are dye-flips of biological replicate 2). For background correction, we defined the mean of the lowest 10% of spot intensities from a particular subgrid as the background for that subgrid. This mean was subtracted from each spot in the subgrid. Signal intensities that did not exceed the background plus 3 standard deviations thereof were defined as not detectable and were excluded from further analyses. We normalized using loess curves thus generating log2 transformed expression ratios comparing DBM treatment with control. These ratios are given in the VALUE columns of each array.

Project description:Background All animals must be able to adapt to changes in nutrient quality and supply. One striking aspect of this adaptive response is the extension of lifespan when caloric intake is reduced. These animals are also more resistant to various stresses, including starvation. We have characterized the response of adult Drosophila males and females to starvation and sugar conditions using high density microarrays. Results We have organized the regulated genes into specific metabolic and physiologic pathways. Most of the highly regulated genes have a strong sex bias. In addition to expected sex specific changes, such as egg production in females, we found unexpected biases in sensory systems, amino acid and purine metabolism, and signaling pathways. Comparison of starvation to caloric restriction revealed numerous overlaps. Conclusions Our results support the notion that starvation response shares similarities to caloric restriction. They have relevance for sex specific differences in longevity and in response to nutrient conditions which alter lifespan. Nutrient Conditions and Fly handling Larvae were kept on apple juice agar plates with yeast, and the larvae were raised at 25Â°C. For the developmental control larvae were collected 39-41 h AEL, for the developmental time-points at 51-53 hours and every 12 hours until 99-101 hours. Adult flies were kept at 25Â°C in 50 ml PS-tubes flat bottom (Greiner bio-one, Frickenhausen, Germany) on standard fly food (16.5 g yeast, 81,5 g cornmeal, 8 g agar, 100 ml sugar beet sirup and 200 ml 10% Nipagin in EtOH). Flies were collected that had hatched in 24 hours and kept in groups of ~200 flies/tube. After 5 days we separated males and females and put them in groups of 80/28 ml PS-tube. We allowed for one day CO2 recovery on standard fly food before flies were moved to 1) tubes with yeast paste on PBS soaked filter paper (control) 2) tubes with only PBS soaked filter paper (starvation) or 3) tubes with filter paper soaked in PBS with 20% Sucrose (sugar). After 24 or 48 hours flies were shock frozen in liquid nitrogen and stored at - 80Â°C for further handling. Males and females were assayed separately. For females, we performed 24 and 48 starvation, and 24 and 48 hours sugar. For the males only 24 hour starvation was done, since most died upon 48 hour starvation; and 24 and 48 hour sugar. Briefly, flies were collected within a 24 hour period, allowed to stay together for 4 days, separated into males and females, and allowed one day recovery in normal fly food. Afterwards, the flies were placed into three conditions: fresh yeast plus PBS, PBS or PBS plus 20 % sucrose. These experiments essentially parallel the experimental set up of previous larval experiments (7). The starvation and PBS were then compared with yeast (control).

Project description:Mice used in this study were 129/sv males, 8-15 weeks of age, which were housed individually and received water ad libitum. Normal fed animals were fed before and during the 48h experiment with standard food ad libitum. Sugar fed animals were fed before the experiment with standard food ad libitum and during the 48h experiment with 50% (w/v) sugar solution (40% glucose, 10% sucrose). Mice were killed by CO2 asphyxiation. The livers were snap-frozen in liquid nitrogen and stored at -80 degrees C. Total RNA was prepared using the Nucleospin RNA L kit (Macherey-Nagel, Düren, Germany), with an additional step in which the final eluate was used for a subsequent elution step. PolyA RNA was extracted with the Ambion (Ambion Inc, Austin, USA) Purist kit according to the manufacturer's protocols. Equal amounts of polyA RNA were pooled to generate pools of all animals. Pool1, normal = 3 animals (N1, N2, N3), pool2, normal = 3 animals (N4, N5, N6), pool3, = 4 animals (N7, N8, N9, N10), pool1, sugar = 3 animals (Su1, Su2, Su3), pool2, sugar = 3 animals (Su4, Su5, Su6), pool3, sugar = 4 animals (Su7, Su8, Su9, Su10). Labeled cDNA was synthesized from 1.5 µg polyA RNA using the Amersham (Amersham Europe, Freiburg, Germany) direct cDNA labeling kit. Removal of the unincorporated dyes and concentration of the target were done with Microcon 30 (Millipore, Bedford, MA) spin columns according to the manufacturer's instructions. The concentrated probes were hybridized to the microarray in 1x dig easy hyb buffer (Hoffman-la Roche, Basel, CH) overnight at 42 degrees C. After hybridization the microarrays are washed 5’ in 2x SSC, 0.1% SDS at 42 degrees C; 10’ in 0.1x SSC, 0.1% SDS at RT; 4 times 1’ in 0.1x SSC at RT and shortly dipped into 0.01x SSC. Drying is performed in centrifuge on a microtiterplate holder at 130 rcf for 7’. Slides are stored in dark until they are scanned. Arrays were scanned using the dual laser scanner Axon 4000B and the corresponding software Genepix 4 (Axon Inc., Union City, CA, USA). Both channels (532 nm for Cy3 and 635 nm Cy5) were scanned in parallel and stored as 16 bit tiff files. The absolute intensity values span the range from 0 to 65,535. The scans were performed with a resolution of 10 µm. From each spot with a mean diameter of 100 µm, 100 data pixels were recorded. Individual local background area around the spots were defined, which included approximately 400 pixels and excludes neighboring spots. For each channel, the raw data was calculated as the median intensity of all foreground pixels with respect to all background pixels. Each array was scanned three times (low, medium and high scan) with different signal amplification factors (voltage settings of the photo multiplier tubes), but with the same laser power. The channels for Cy3 and Cy5 were balanced in each scan for approximately the same intensity profile. In the low scan no spot was saturated, in the high scan the signal amplification for Cy5 was set to approximately 80% of maximum and the Cy3 amplification was adjusted to this. The settings used in the medium scan lie between the low and the high scan. This method for scanning has several advantages. In the low scan where no spot is in saturation, it is possible to calculate the real ratio for genes with high expression levels; however, those with a low expression level are most likely not recognized. In order not to lose these a high scan is made; in this case the information on the saturated spots is lost so the two scans complement each other. The medium scan produces additional values for subsequent calculations. By scanning the arrays three times errors which occur while recording and which might increase the error factor in the normalization are averaged. The data processing was automated in a Visual Basic/Excel (Microsoft) program, which integrates the following steps. Raw data are derived from the result files generated by scanning and picture analysis software Genepix. The spot diameter is to lie in the range from 80 to 120 µm otherwise these spots are not included in analysis. From the pixel to pixel ratios between the foreground values of both colors, the standard deviation (SD) is calculated. Those spots which show a ratio SD of higher than 3 are excluded from analysis due to inconsistency. The foreground signal of each spot is corrected by subtracting the corresponding local background. Resulting values which are lower than the background are replaced by the background value. This defines the minimum measurement threshold as the background and avoids calculations of completely wrong ratios, e.g., if the background corrected value for one channel is close to zero. Spots are marked as saturated in one channel if more than 10% of the foreground pixels are at the highest absolute value. From the unsaturated data points of all three scans, linear regression between the low and medium, and between low and high scans, are calculated for both colors. Taking these regressions, the values of saturated spots are replaced by estimated ones which reflect the real intensity. The generated data set of each scan is normalized afterwards based on the intensity-dependent methods as described by Yang et al (Yang YH, Dudoit S, Luu P, Lin DM, Peng V, Ngai J, and Speed TP. Normalization for cDNA microarray data: robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. 30: e15, 2002). The ratios are calculated as log2 transformed values: M=log2 (Cy5/Cy3). Number describing the spot intensity A=log2sqrt (Cy5*Cy3) is calculated according to Yang et al. A within pin-tip group loess fit to the MA-plot was made. The loess scatter plot smoother performs robust locally fits using a tricube function to weight the values relative to the median point in the intensity interval. As interval size 20% was chosen, which corresponds to 180 data points in a pin-tip group containing 30 by 30 spots. After combining the normalized data from all blocks the data sets of all scans are additionally normalized using a global approach. The normalization was performed so that the sum of all log transformed ratios (M) is 0. All statistical normalizations are based on the assumption that (1) the majority of genes are not changed in their expression and (2) that the overall up and down regulations statistically compensate each other in sum. Taking this into account, the standard deviation in the ratios of the stable genes (80% of the most unchanged genes) could be seen as the measurement noise of the array experiments. To be able to compare the different scans and different normalized microarrays, the SD of the stable genes ratios was adjusted to a medium estimated value of 0.2, which is dependent on the used array system. This value does not influence the subsequent statistical analysis because the normal distribution of the data is not changed. For each microarray the normalized and adjusted log ratios of the three scans are averaged. Keywords = microarray analysis Keywords = nutrient response Keywords = caloric restriction Keywords = metabolic signaling Keywords = aging/longevity This SuperSeries is composed of the following subset Series: GSE851: liver, 48h sugar GSE852: liver, 48h starved GSE853: liver, 24h starved Refer to individual Series

Project description:Background Insulin signaling pathway is conserved from worms to humans. In Drosophila, three insulin-like peptides (dilps) are expressed in the insulin producing cells (IPCs) in the brain. In order to identify target genes of insulin signaling, the IPCs of female flies were destroyed during development by expressing apoptosis inducing factors reaper and head involution defective under control of the promoter region of dilp3. These insulin-deficient flies were analysed using high density microarrays. Results From the genes that appeared to be regulated in the absence of IPCs, the strongest difference was found for a gene involved in carbohydrate metabolism. Sequence analysis revealed that it is an alpha-glucosidase, which is able to break down disaccharides and/or glycogen. Conclusions We could identify an alpha-glucosidase, involved in carbohydrate metabolism, to be strongly affected in the context of decreased insulin signaling. The striking feature of this regulation was not only that it was the highest regulated, but it was the only gene regulated to this degree. Nutrient Conditions and Fly handling Experimental and control female flies were collected after hatching, then kept on fly maintenance food and separated from males after 11 days. After one day of recovery from carbon dioxide treatment, females were put in cages with PBS agar plates and supplied with yeast paste as only food source for 48 hours. Flies were shock frozen in liquid nitrogen and stored at - 80Â°C for further handling. Keywords: IPC knockout, 14 day old female flies, 48h yeast feeding Insulin producing cell deficient flies were compared to control flies. Two independent biological repeats were performed. From the two repeats, 5 chips were hybridised, including dye swap Experimental and control female flies were collected after hatching, then kept on fly maintenance food and separated from males after 11 days. After one day of recovery from carbon dioxide treatment, females were put in cages with PBS agar plates and supplied with yeast paste as only food source for 48 hours. The microarray was scanned 3 times: low scan (suffix _L in data table) with a low amplification / intensity to avoid saturated spots for proper data analysis, high scan (suffix _H in data table) with a high amplification / intensity to detect also weak fluorescent spots which are missed in the low scan and medium scan which lies between high and low intensity and gives additional data points for subsequent calculations. Both copies and all hybridisation repeats were used for normalisation, VALUE therefore has the same value for these copies in all relevant hybridisations. For additional information see publication and web link. Keywords = insulin, aging, longevity, nutrient dependance Lot batch = FP7

Project description:The human gastric pathogen Helicobacter pylori is extremely well adapted to the highly acidic conditions encountered in the stomach. The pronounced acid resistance of H. pylori relies mainly on the ammonia-producing enzyme urease, however, urease-independent mechanisms are likely to contribute to acid adaptation. Acid-responsive gene regulation is mediated at least in part by the ArsRS two-component system consisting of the essential OmpR-like response regulator ArsR and the non-essential cognate histidine kinase ArsS whose autophosphorylation is triggered in response to low pH. In this study by global transcriptional profiling of an ArsS-deficient H. pylori mutant grown at pH 5.0 we define the ArsR~P- dependent regulon consisting of 110 genes including the urease gene cluster, the genes encoding the aliphatic amidases AmiE and AmiF and the rocF gene encoding arginase. Transcriptome analyses were performed using a whole-genome microarray containing 1649 PCR products generated with specific primer pairs derived from the genome sequences of H. pylori 26695 (Tomb et al., 1997. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388:539-547) and J99 (Alm et al., 1999. Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature 397:176-180) which were spotted in duplicate. Microarrays were produced as described by Gressmann et al. (Gressmann et al., 2005. Gain and loss of multiple genes during the evolution of Helicobacter pylori. PLoS Genet 1(4):e43). To determine genes which are differentially expressed in the ArsS-deficient mutant G27/HP165::km at pH 5.0, cDNA was prepared from RNA extracted from H. pylori G27 and G27/HP165::km after exposing the bacteria for one hour to acidic pH. A total of eight RNA samples from two independent RNA preparations from strain G27 and G27/HP165::km, respectively, was used for cDNA labelling und hybridisation. Dye reversal colour swaps were performed as follows: One cDNA sample was generated using Cy3-dCTP and the other using Cy5-dCTP resulting in four labelled cDNAs per colour swap. Cy5-dCTP and Cy3-dCTP labelled cDNAs were combined and hybridized to the H. pylori microarray. The slides were scanned using ScanArray HT and analysed by using the ScanArray express software (Perkin Elmer). Spots were flagged and eliminated from analysis when the signal to background ratio was less then three or in obvious instances of high background or stray fluorescent signals. Median intensities of spots were background corrected and differences in dye bias were normalized by using the LOWESS algorithm (Yang et al., 2002. Normalization for cDNA microarray data: a robuste composite method addressing single and multiple slide systematic variation. Nucleic Acid Res. 30:e15). The signal ratios as measure of differential expression between the red and green channels were obtained from processed signal intensities. Ratios were further analysed with Microsoft Excel (Microsoft) and SAM software for statistic significance (Tusher et al., 2001. Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl. Acad. Sci. USA 98:5116-5121). To determine the significance of differential expression RNA was isolated from the H. pylori G27 wild-type grown in BHI broth (pH 5.0), and 20 Âµg of this RNA were labelled either with Cy3-dCTP or with Cy5-dCTP. The two cDNA probes generated were hybridized onto the same slide, and the data were analysed as mentioned above. Signal ratios < 0.5 and > 2.0 were analyzed further.

Dataset Information

Primary stem development

Publications

Global transcript profiling of primary stems from Arabidopsis thaliana identifies candidate genes for missing links in lignin biosynthesis and transcriptional regulators of fiber differentiation.

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