Coevolution of hormone metabolism and signaling networks expands plant adaptive plasticity
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ABSTRACT: Differential transcriptomic analysis of 10-day-old Arabidopsis seedlings treated with 50 μM ABA, PA, or DPA. 10-day-old Arabidopsis seedlings grown on MS medium was treated with 50 μM ABA, PA, or DPA. Total RNA was extracted from biological duplicates, Illumina RNAseq was performed on each sample.
Project description:The Arabidopsis seedlings treated with 2M t-zeatin (CTK) for 30 minutes and then total RNA was extracted from Arabidopsis seedlings using TRIzol reagent (Invitrogen) and treated with RNase-free DNase I. The poly(A) mRNA was isolated using oligo (dT) beads. First-strand cDNA was subsequently generated using random hexamer-primed reverse transcription, followed by synthesis of the second-strand cDNA using RNaseH and DNA polymerase I. Then, single-end and paired-end RNA-seq libraries were prepared following Illumina's protocols and sequenced on the Illumina GA II platform.
Project description:Purpose: The goal of this study is to compare the differently expressed genes in the wild type and the KDEL-tailed cysteine protease AtCEP1 knockout (atcep1) Arabidopsis using RNA-sequencing (RNA-seq). Methods: Arabidopsis buds mRNA profiles of anther development stages 5-6, 7-9, and 10-11 of the wild type (WT) and atcep1 mutant were generated by deep sequencing via Illumina HiSeqTM 2000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with the following steps: Remove reads with adaptor sequences; Remove reads in which the percentage of unknown bases (N) is greater than 10%; Remove low quality reads, in which the percentage of the low quality base (base with quality value ⤠5) is greater than 50%. The clean reads were mapped to the Arabidopsis reference genome and reference genes using SOAP aligner/SOAP2. No more than 2 mismatches were allowed in the alignment. The gene expression level was calculated using RPKM (Reads Per Kb per Million reads). Differential expression analysis between the wild type and the atcep1 mutant was performed using the DEGseq R package (1.12.0) based on normalized read counts. A corrected P value of < 0.005 and |log2Ratio| > 1 were set as the threshold for significantly differential expression. Results: We identified 872 genes showing significant differential expression, and in the atcep1 mutant, the upregulated genes significantly outnumbered the downregulated genes at the three time points. The GO analysis of the differently expressed genes showed that the expression of genes participating in anther tapetal secretory structure formation, pollen wall development, and tapetal cell wall generation, clearly changed. Arabidopsis buds mRNA profiles of development stages 5-6, 7-9, and 10-11 of the wild type (WT) and atcep1 muant were generated by deep sequencing via Illumina HiSeqTM 2000.
Project description:Glycolytic Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of glyceraldehyde 3-phospate to 1,3-bisphosphoglycerate by coupling with the reduction of NAD+ to NADH. We generated mutants of the Arabidopsis plastidial GAPDH isoforms (At1g79530, At1g16300; GAPCp1, GAPCp2). gapcp double mutants (gapcp1 gapcp2) display a drastic phenotype of arrested root development and sterility.Complex interactions occurring between ABA and sugar signal transduction pathways have been shown, but the molecular mechanisms connecting both pathways are not well understood. Since we found drastic carbohydrate changes in gapcp1 gapcp2, we studied their response to ABA. by performing a microarray analysis comparing gapcp1 gapcp2 and wild type seedlings after a long term treatment with ABA. 15-day old Arabidopsis seedlings (Col 0) and gapcp1gapcp2 double mutants were used for RNA extraction and hybridization on Affymetrix microarrays.
Project description:Identification of diacylglycerol pyrophosphate regulated genes in ABA signaling.<br><br> The specific plant phosphorylated form of phosphatidic acid (PA), diacylglycerol pyrophosphate (DGPP) was recently shown to be a second messenger in abscisic acid (ABA) signaling. The aim of the project is to identify among the set of ABA-regulated genes the ones also regulated by DGPP and/or PA.<br> Five ml of 3 days-old suspension cells was incubated with ABA or lipids for 3 h under the conditions of culture. Lipids were emulsified by sonication for 1 mi, four times, at 4°C, in one ml of culture medium then added to 4 ml suspension cells. Cells were filtrated under vacuum, frozen in liquid nitrogen and RNA extracted. Dioleoyl PA and dioctanoyl PA are from Sigma, dioleoyl DGPP and dioctanoyl DGPP are from Avanti Polar Lipids.
Project description:To investigate differences in plant responses to salt and ABA stimulus, differences in gene expression in Arabidopsis in response to salt and ABA were compared using an Agilent oligo microarray. Four-week-old Arabidopsis thaliana ecotype Columbia (Col-0) seedlings were treated with either 150 mM NaCl or 10 M-NM-<M ABA for 6 hours; unstressed seedlings (control sample) were collected in parallel to avoid the possible effects of circadian rhythms. The results revealed that 31 genes were up regulated by both NaCl and ABA stress, and 23 genes were down-regulated by these stressors. To provide further validation of our microarray experiment data, ten genes from this signature were quantified in the same RNA samples by quantitative real-time PCR. Differentially expression genes of Arabidopsis thaliana were measured under salt stressed, ABA stressed and normal condition for 6 hours, respectively. Three independent experiments were performed at each treatment using different plants for each experiment.
Project description:rs05-06_dgpp - dgpp - 2. The specific plant phosphorylated form of phosphatidic acid (PA), diacyglycerol pyrophosphate (DGPP), was recently shown to be a second messenger in abscisic acid (ABA) signaling. The aim of the project is to identify among the set of ABA-regulated genes the ones also regulated by DGPP and/or PA.
Project description:rs05-06_dgpp - dgpp - The specific plant phosphorylated form of phosphatidic acid (PA), diacyglycerol pyrophosphate (DGPP) was recently shown to be a second messenger in abscisic acid (ABA) signaling. The aim of the project is to identify among the set of ABA-regulated genes the ones also regulated by DGPP and/or PA. - Five ml of 3 days-old suspension cells was incubated with ABA or lipids for 3 h under the conditions of culture. Lipids were emulsified by sonication for 1 mi, four times, at 4°C, in one ml of culture medium then added to 4 ml suspension cells. Cells were filtrated under vacuum, frozen in liquid nitrogen and RNA extracted. Dioleoyl PA and dioctanoyl PA are from Sigma, dioleoyl DGPP and dioctanoyl DGPP are from Avanti Polar Lipids. Keywords: treated vs untreated comparison
Project description:rs05-06_dgpp - dgpp - The specific plant phosphorylated form of phosphatidic acid (PA), diacyglycerol pyrophosphate (DGPP) was recently shown to be a second messenger in abscisic acid (ABA) signaling. The aim of the project is to identify among the set of ABA-regulated genes the ones also regulated by DGPP and/or PA. - Five ml of 3 days-old suspension cells was incubated with ABA or lipids for 3 h under the conditions of culture. Lipids were emulsified by sonication for 1 mi, four times, at 4°C, in one ml of culture medium then added to 4 ml suspension cells. Cells were filtrated under vacuum, frozen in liquid nitrogen and RNA extracted. Dioleoyl PA and dioctanoyl PA are from Sigma, dioleoyl DGPP and dioctanoyl DGPP are from Avanti Polar Lipids. Keywords: treated vs untreated comparison 5 dye-swap - CATMA arrays
Project description:Dicamba is an auxin-like herbicide that can stimulate the production of ethylene and ABA biosynthesis. The subsequent stomatal closure and build-up of reactive oxygen species is hypothesized to contribute to plant death. In order to further understand the herbicide dicamba's mode of action at the molecular level, we used microarrays to dissect global gene expression in Arabidopsis seedlings 10 hrs after dicamba treatment. 4 day old Arabidopsis seedlings (a Columbia line containing a -800GSTF8::LUC reporter) were flooded with water or 7mM dicamba for 40 minutes before the treatment was poured off. Ten hours after the start of the treatment, whole seedlings were collected