biostudies-arrayexpressKarin KoehlOryza sativahttps://www.ebi.ac.uk/biostudies/studies/E-GEOD-7766We investigated the physiological and gene expression response of drought-tolerant (IR57311 and LC-93-4; subgroup indica) and drought-sensitive (Nipponbare and Taipai 309; subgroup japonica) rice (Oryza sativa) cultivars to 18 days of drought stress in climate chamber experiments. Rice plants were grown under water sufficient and water limiting conditions in three independent experiments in a controlled climate chamber with 12 h day length at 600 µE m-2 s-1; temperature was 26°C in the light and 22°C at night, with a relative humidity of 75% in the light and 70% at night. Leaf material for expression profiling analysis was harvested five hours after the beginning of the light period after 18 days of stress or control treatment. Normalization and statistical testing was performed using the R package limma (R 2.3.1, limma version 2.7.3; (Smyth, 2005)). The methods Robustspline for within array normalization and Quantile for between array normalization were applied. A linear model with the effects genotype, drought treatment, genotype x drought, dye was fitted to normalized data in limma that models the systemic variation in the data. Afterwards, for the comparisons of interest, moderated t-statistics that use an empirical Bayes method were calculated. Differentially expressed genes were identified using the decideTests function (method global, Benjamini & Hochberg fdr corrected p-value <0.05 in limma) Keywords: stress reponse Growth design. Three independent experiments were performed. The design was a split-plot design with five blocks per drought or control treatment in each experiment. Each treatment and cultivar was represented by five replicate pots with one plant per pot. Pots were randomized within the blocks. Block position was rotated daily. Hybridisation design: A total of 28 samples, each representing mRNA from four parallel plants, were hybridized to 14 arrays. The hybridization design was optimized for the estimation of the effects of condition and the condition x cultivar interaction taking a variance minimization approach. In a two-step procedure, a smaller design for one sensitive and one tolerant cultivar was enlarged to encompass all four cultivars by integrating eight additional arrays. The optimization was programmed using R [R Development Core Team 2006] and carried out on the 16-node Beowulf Linux-Cluster at the University of Potsdam. R-scripts are available upon request and from the Bioinformatics Team at the MPI of Molecular Plant Physiology (see the website for more information). Allocation of the three biological replicates of each of the combinations of treatment and cultivar was completed such that a balanced distribution with respect to the labeling was achieved. Each combination of condition and cultivar from all experiments has been used at least once.biostudies-arrayexpressGrowth Protocol - Seeds were pre-germinated in tab water at 28°C for ten days. Then, plantlets were transferred in 10 cm diameter pots filled with 540 g sand mixed with 8 g of Lewatit HD 50 (Lanxess, Langenfeld, Germany), an ion-exchange resin loaded with nutrient ions and 0.4 g Fetrilon Combi (Compo, Münster, Germany). Pots were transferred to a climate chamber with 12 h day length at 600 µE m-2 s-1; temperature was 26°C in the light and 22°C at night, with a relative humidity of 75% in the light and 70% at night. Pots were positioned in polypropylene boxes filled with water to the level of the substrate surface. Pot surfaces were covered with black, pinpricked polythene film (Aquafol, Reinmann, Emsdetten, Germany) to prevent growth of algae. Twenty-six days after sowing, water was removed from half of the boxes and plants were left to dry for four days, until the soil water content had reached the permanent wilting point (PWP) for 50% of the plants. Thereafter, the soil water content was kept constant to the fixed PWP value over a period of 14 days by weighing each pot at the end of the light period and adding the amount of water lost during the last 24 h. After a total of 19 days of drought stress, plants were harvested five hours after the beginning of the light period. Samples for expression profiling were harvested from the middle section of the blades of fully expanded green leaves and immediately frozen in liquid nitrogenSample Processing - see growth protocolGrowth Protocol - Seeds were pre-germinated in tab water at 28°C for ten days. Then, plantlets were transferred in 10 cm diameter pots filled with 540 g sand mixed with 8 g of Lewatit HD 50 (Lanxess, Langenfeld, Germany), an ion-exchange resin loaded with nutrient ions and 0.4 g Fetrilon Combi (Compo, Münster, Germany). Pots were transferred to a climate chamber with 12 h day length at 600 µE m-2 s-1; temperature was 26°C in the light and 22°C at night, with a relative humidity of 75% in the light and 70% at night. Pots were positioned in polypropylene boxes filled with water to the level of the substrate surface. Pot surfaces were covered with black, pinpricked polythene film (Aquafol, Reinmann, Emsdetten, Germany) to prevent growth of algae. Forty-five days after sowing, plants were harvested five hours after the beginning of the light period. Samples for expression profiling were harvested from the middle section of the blades of fully expanded green leaves and immediately frozen in liquid nitrogen.Labeling - For labeling 1 mg cDNA (in 15 µl buffer (10 mM Tris, pH 8.5)) were mixed with 5 µl of the fluorescent dye (Alexa Fluor 532 and 647 (Qiagen, Hilden, Germany)) and incubated for 30 - 45 min at 85 °C in the dark, followed by adding of 5 µl Stop Solution and an incubation for 10 min at room temperature.Labeling - For labeling 1 mg cDNA (in 15 µl buffer (10 mM Tris, pH 8.5)) were mixed with 5 µl of the fluorescent dye (Alexa Fluor 532 and 647 (Qiagen, Hilden, Germany)) and incubated for 30 - 45 min at 85 °C in the dark, followed by adding of 5 µl Stop Solution and an incubation for 10 min at room temperature.)Nucleic Acid Extraction - PolyA+-RNA was extracted with magnetic beads (Dynabeads oligo (dT)25, Dynal, Oslo, Norway) following the manual instruction. In brief, 100mg leaf material was homogenized for 1.5 min at 28 Hz in a ball mill (Retsch) and 1.5 ml Lysis buffer (100 mM Tris, 500 mM LiCl, 10 mM EDTA, 1 % LiDS, 5 mM DTT, pH 8) was added. The solution was mixed and centrifugated at 4°C for 10min at 14000 rpm. 125µl beads were washed twice with 200µl Lysis buffer and were added to the supernatant. After gentle mixing for 5 min beads were washed twice with 1 ml washing buffer A (10 mM Tris, 150 mM LiCl, 1 mM EDTA, 0.1 % LiDS, 0.05% Tween, pH 8), beads were transferred to a new reaction vial and beads were washed twice with washing buffer B (10 mM Tris, 150 mM LiCl, 1 mM EDTA, 0.05% Tween, pH 8). PolyA+-RNA was extracted two times with 10 µl 10 mM Tris pH 7.5 heating 2 min to 95oC. For DNase treatment 20 µl RNA, 4 µl 5x Superscript III Puffer, 0.5 µl RNaseOUT (40 U, Invitrogen), 1 µl DNase (Roche) were incubated for 15 min at room temperature. After adding 1 µl 110 mM EDTA reaction was stopped by incubating for 10 min at 70°C. mRNA from four plants originating from the same experiment, condition and cultivar was pooled. cDNA was synthesized using SuperScript III (Invitrogen, Carlsbad, CA, USA) and purified by precipitation, using Bioline Sure Clean (Bioline, Luckenwalde, Germany).Hybridization - Prehybridization was done following the manufactures instructions: blocking of slide for 20 min at 42°C with Block Solution (2X SSC, 0.05% SDS, 0.25% NaBH4) followed by washing of slide with 1X SSC, 0.2X SSC and rinsing with Nanopure water. For hybridization labelled DNA was dissolved in 130µl hybridization buffer (0.1 % SDS, 25 % Formamide, 5x SSC, 10 mg/ml BSA, 40 mM Na3PO4 (pH 6.8)) and applied to the pre-warmed slide in the hybridization station Hybarray12 (Perkin Elmer, Wellesley, MA, USA). The Initial hybridization temperature was 45°C. The hybridization temperature was stepwise reduced every hour by 1K to reach final hybridization temperature of 41°C. After 10-12 hours of hybridization at 41°C, slides were washed with 2X SSC, 0.2% SDS at 42°C, 2X SSC and 0.2X SSC (all 4 cycles with 20s flow and 40s hold) and very shortly in Nanopure water, all at room temperature. Slides were dried in a centrifuge.Growth Protocol - Seeds were pre-germinated in tab water at 28°C for ten days. Then, plantlets were transferred in 10 cm diameter pots filled with 540 g sand mixed with 8 g of Lewatit HD 50 (Lanxess, Langenfeld, Germany), an ion-exchange resin loaded with nutrient ions and 0.4 g Fetrilon Combi (Compo, Münster, Germany). Pots were transferred to a climate chamber with 12 h day length at 600 µE m-2 s-1; temperature was 26°C in the light and 22°C at night, with a relative humidity of 75% in the light and 70% at night. Pots were positioned in polypropylene boxes filled with water to the level of the substrate surface. Pot surfaces were covered with black, pinpricked polythene film (Aquafol, Reinmann, Emsdetten, Germany) to prevent growth of algae. Twenty-six days after sowing, water was removed from half of the boxes and plants were left to dry for four days, until the soil water content had reached the permanent wilting point (PWP) for 50% of the plants. Thereafter, the soil water content was kept constant to the fixed PWP value over a period of 14 days by weighing each pot at the end of the light period and adding the amount of water lost during the last 24 h. After a total of 19 days of drought stress, plants were harvested five hours after the beginning of the light period. Samples for expression profiling were harvested from the middle section of the blades of fully expanded green leaves and immediately frozen in liquid nitrogen.MIAME ScoreRaw DataOrganizationAssays and DataProcessed DataMAGE-TAB FilesArray DesignsImage Adquisition - Microarrays were scanned with a FLA-8000 laser scanner (Fuji, Tokyo, Japan) at maximum intensity. Software version was FLA-8000 V 1.12.Feature Extraction - The software GeneSpotter 2.4.3 (Microdiscovery, Berlin, Germany) was used to fit the grid position of the spots and to calculate spot intensities. Spots for which intensity was affected by dust or dirt were flagged manually (user tag column = 1). Background correction was done based on the expression signal of 218 spots representing a hygromycin resistance gene that is not in the genome of the investigated cultivars. Any spot with an expression below the mean plus threefold standard deviation of the intensity of the hygromycin gene spots of the respective array and dye was labelled as below background (tag column = 1). Normalization was performed using the R package limma (R 2.3.1, limma version 2.7.3; (Smyth, 2005)). The methods Robustspline for within array normalization and Quantile for between array normalization were applied.Assay Data Transformation - ID_REF = <br>Internal_ID = Primary ID for internal use<br>VALUE = M value of normalized data; M=(log2(red intensity/green intensity)<br>Value_A = A value of normalized data; A=(0.5*log2(red intensity*green intensity)<br>Median_red_norm = Normalized expression value of median red intensity<br>Median_green_norm = Normalized expression value of median green intensity<br>BG_Mean_Red = mean value of red channel local background intensity<br>BG_Mean_Green = mean value of green channel local background intensity<br>used = 1: spot was used for statistical analysis; 0: spot was classified as below background or dirt or dust affected on more than 8 arrays of experimentMetabolomicsUnknownTranscriptomicsGenomicsProteomicsUnderstanding the molecular basis of plant performance under water-limiting conditions will help to breed crop plants with a lower water demand. We investigated the physiological and gene expression response of drought-tolerant (IR57311 and LC-93-4) and drought-sensitive (Nipponbare and Taipei 309) rice (Oryza sativa L.) cultivars to 18 days of drought stress in climate chamber experiments. Drought stressed plants grew significantly slower than the controls. Gene expression profiles were measured in leaf samples with the 20 K NSF oligonucleotide microarray. A linear model was fitted to the data to identify genes that were significantly regulated under drought stress. In all drought stressed cultivars, 245 genes were significantly repressed and 413 genes induced. Genes differing in their expression pattern under drought stress between tolerant and sensitive cultivars were identified by the genotype x environment (G x E) interaction term. More genes were significantly drought regulated in the sensitive than in the tolerant cultivars. Localizing all expressed genes on the rice genome map, we checked which genes with a significant G x E interaction co-localized with published quantitative trait loci regions for drought tolerance. These genes are more likely to be important for drought tolerance in an agricultural environment. To identify the metabolic processes with a significant G x E effect, we adapted the analysis software MapMan for rice. We found a drought stress induced shift toward senescence related degradation processes that was more pronounced in the sensitive than in the tolerant cultivars. In spite of higher growth rates and water use, more photosynthesis related genes were down-regulated in the tolerant than in the sensitive cultivars.transcription profiling by arrayOryza sativaExpression profiling of rice cultivars differing in their tolerance to long-term drought stress.Karin KoehlEllen ZutherDirk HinchaPhuc DoDegenkolbe T, Do PT, Zuther E, Repsilber D, Walther D, Hincha DK, Köhl KIThomas DegenkolbeDirk RepsilberDirk WaltherfalseModerate long-term drought stress in riceWe investigated the physiological and gene expression response of drought-tolerant (IR57311 and LC-93-4; subgroup indica) and drought-sensitive (Nipponbare and Taipai 309; subgroup japonica) rice (Oryza sativa) cultivars to 18 days of drought stress in climate chamber experiments. Rice plants were grown under water sufficient and water limiting conditions in three independent experiments in a controlled climate chamber with 12 h day length at 600 µE m-2 s-1; temperature was 26°C in the light and 22°C at night, with a relative humidity of 75% in the light and 70% at night. Leaf material for expression profiling analysis was harvested five hours after the beginning of the light period after 18 days of stress or control treatment. Normalization and statistical testing was performed using the R package limma (R 2.3.1, limma version 2.7.3; (Smyth, 2005)). The methods Robustspline for within array normalization and Quantile for between array normalization were applied. A linear model with the effects genotype, drought treatment, genotype x drought, dye was fitted to normalized data in limma that models the systemic variation in the data. Afterwards, for the comparisons of interest, moderated t-statistics that use an empirical Bayes method were calculated. Differentially expressed genes were identified using the decideTests function (method global, Benjamini & Hochberg fdr corrected p-value <0.05 in limma) Keywords: stress reponse Growth design. Three independent experiments were performed. The design was a split-plot design with five blocks per drought or control treatment in each experiment. Each treatment and cultivar was represented by five replicate pots with one plant per pot. Pots were randomized within the blocks. Block position was rotated daily. Hybridisation design: A total of 28 samples, each representing mRNA from four parallel plants, were hybridized to 14 arrays. The hybridization design was optimized for the estimation of the effects of condition and the condition x cultivar interaction taking a variance minimization approach. In a two-step procedure, a smaller design for one sensitive and one tolerant cultivar was enlarged to encompass all four cultivars by integrating eight additional arrays. The optimization was programmed using R [R Development Core Team 2006] and carried out on the 16-node Beowulf Linux-Cluster at the University of Potsdam. R-scripts are available upon request and from the Bioinformatics Team at the MPI of Molecular Plant Physiology (see the website for more information). Allocation of the three biological replicates of each of the combinations of treatment and cultivar was completed such that a balanced distribution with respect to the labeling was achieved. Each combination of condition and cultivar from all experiments has been used at least once.2008-10-29T00:00:00Z2023-09-18T03:41:56.528Z2022-02-03T13:34:07.717ZE-GEOD-7766GSE776618931976EFO_000276810.1007/s11103-008-9412-7