Transcriptional profile (mRNA and sRNA) of Clostridium acetobutylicum to metabolite stress, butanol and butyrate
ABSTRACT: The transcription profile of C. acetobutylicum to two major metabolite stress, butanol and butyric acid, was comprehensively investigated at three different concentrations of each metabolite and at four different time points (15, 30, 60 and 75 min post stress). All experiments were performed in 3 parallel biological replicates and the RNA extraction was perfomed in a manner to retain the small RNAs and hence, investigate their role and expression under stress. Genome-wide small RNA and mRNA profiling was done by Illumina TruSeq sample preparation followed by high-throughput sequencing with Illumina HiSeq 2000 platform. Please note that all the samples were analyzed for both mRNAs and smallRNAs. We used the miRNeasy Kit from Qiagen to ensure the extraction of the low molecular weight small RNA during the extraction of the total RNA and hence, mRNAs and sRNAs were analyzed together in the total RNA-seq.
Project description:Bacillus subtilis is exposed to a wide range of transitory stress and starvation conditions. Here we investigate the expression changes observed in the B. subtilis wild type strain 168 and its isogenic sigB mutant(BSM29) with respect to each stress condition tested. Gene expression was queried for the stress conditions: ethanol-, butanol-, osmotic- and oxidative stress, heat shock, low temperature growth, glucose as well as oxygen limitation. For butanol-, ethanol-, osmotic-, and oxidative stress as well as heat shock : time points (0min, 5min, 10min, 15min and 20min) ; for glucose limitation and oxygen limitation : time points (0min, 15min, 30min, 45min, 60min or 90min) and for low temperature growth, samples for recording of expression values were taken during mid-exponential growth at OD540 0.9 and 1.0.
Project description:Purpose: Brassica. juncea is vulnerable to abiotic stresses at specific stages in its life cycle. However, till date no attempts have been made to elucidate the genome-wide changes in the transcriptome of B. juncea subjected to either high temperature or drought stress. Hence, to gain global insights into genes, transcription factors and kinases regulated by these stresses and to provide basic information on coding transcripts that are associated with traits of agronomic importance, we utilized a combinatorial approach of next generation sequencing and de novo assembly to discover B. juncea transcriptome associated with high temperature and drought. Results: We constructed and sequenced three transcriptome libraries namely Brassica control (BC), Brassica high temperature stress (BHS) and Brassica drought stress (BDS) from control, high temperature treated and drought treated seedlings of Brassica juncea. More than 180 million purity filtered reads were generated which were processed through quality parameters and high quality reads were assembled de-novo using SOAPde-novo assembler. A total of 77750 unique transcripts were identified out of which 69,245 (89%) were annotated with high confidence. We established a subset of 19110 transcripts, which were differentially regulated by either high temperature and/or drought stress. Furthermore, 886 and 2834 transcripts that code for transcription factors and kinases, respectively, were also identified. Investigation of identified transcription factors revealed that 92 responded to high temperature, 72 exhibited alterations in expression during drought stress, and 60 were commonly associated with both the stresses. Similarly, 217, 259 and 193 kinases were responsive to high temperature, drought or both stresses, respectively. Maximum number of up-regulated transcription factors in high temperature and drought stress belonged to heat shock factors (HSFs) and dehydration responsive element-binding (DREB) families respectively. We also identified 239 metabolic pathways, which were perturbed during high temperature and drought treatments. Analysis of gene ontologies associated with differentially regulated genes forecasted their involvement in diverse biological processes. Conclusions: Our study provides first comprehensive discovery of B. juncea transcriptome under high temperature and drought stress conditions. Transcriptome resources generated in this study will enhance our understanding on the molecular mechanisms involved in defining the response of B. juncea against two important abiotic stresses. Furthermore this information would benefit designing of efficient crop improvement strategies for tolerance against conditions of high temperature regimes and water scarcity. Total three RNA-Seq libraries were prepared and sequenced independently [B. juncea control (BC), B. juncea high temperature stressed (BHS) and B. juncea drought stressed (BDS) on Illumina GAIIx sequencer].
Project description:The severity of impact of drought on crops is contingent on the developmental stage of the plant, with the most sensitive stage being the reproductive stage. Hence, gene expression profiling has been used to understanding drought response and resistance mechanism in rice. Here we present drought transcriptomes of rice in three developmental stages and gain insights into the processes and regulatory mechanisms involved in common and stage specific drought responses. Total RNA was isolated from the rice seedlings, vegetative (V4) and reproductive (R4) tissues of both control and stress treated plants for hybridization on Affymetrix microarrays. Two independent replicates for seedling and reproductive stages, and three replicates for vegetative stages were generated, for both control and stress samples. For drought treatments, plants were gradually subjected to field drought conditions in order to reach 50% field capacity (FC) by regulating water supply, whereas control plants were maintained at 100% FC.
Project description:Purpose: Water stress limits plant survival and production in many parts of the world. Identification of genes and alleles responding to water stress conditions is important in breeding plants better adapted to drought. Currently there are no studies examining the transcriptome wide gene and allelic expression patterns under water stress conditions. We used RNA sequencing (RNA-seq) to identify the candidate genes and alleles and to explore the evolutionary signatures of selection Methods: We studied the effect of water stress on gene expression in Eucalyptus camaldulensis seedlings derived from three natural populations. We used reference-guided transcriptome mapping to study gene expression. Reads mapping to predicted transcripts were analysed with edgeR pacakge to study differntial expression of genes under control and water-stress conditions. SNPs within the genes were obtained using pileup file generated with samtools and the SNP frequencies were analysed with VarScan package. SNPs were further characterised as sysnonymous and non-synonymous using popoolation2 pacakge. Results: Several genes showed differential expression between control and stress conditions. Gene ontology (GO) enrichment tests revealed up-regulation of 140 stress-related gene categories and down-regulation of 35 metabolic and cell wall organisation gene categories. More than 190,000 single nucleotide polymorphisms (SNPs) were detected and 2737 of these showed differential allelic expression. Allelic expression of 52% of these variants was correlated with total gene expression. Signatures of selection patterns were studied by estimating the proportion of nonsynonymous to synonymous substitution rates (Ka/Ks). The average Ka/Ks ratio among the 13,719 genes was 0.39 indicating that most of the genes are under purifying selection. Among the positively selected genes (Ka/Ks > 1.5) apoptosis and cell death categories were enriched. Of the positively selected genes, ninety genes showed differential expression and 27 SNPs from 17 positively selected genes showed differential allelic expression between treatments. Conclusions: Correlation of allelic expression of several SNPs with total gene expression indicates that these variants may be the cis-acting variants or in linkage disequilibrium with such variants. Enrichment of apoptosis and cell death gene categories among the positively selected genes reveals the past selection pressures experienced by the populations used in this study. Leaf mRNA of 12 samples were used to study the gene expression under control and water-stress conditions. Three samples under water-stress conditions and three samples under control conditions were used as biological replicates for differenctial gene expression between the treatments. High-throughput sequence reads from mRNA samples were generated by deep sequencing with Illumina GAIIx.
Project description:Clostridium acetobutylicum is a Gram positive, endospore forming firmicute that has been known as the model organims for ABE (acetone-butanol-ethanol) fermentation. With its ability to consume a wide variety of substrates, C. acetobutylicum carries out a biphasic ABE fermentation, which consists of the acidogenic growth phase with the formation of butyric acid and acetic acid, followed by the solventogenic stationary phase with the formation of acetone, butanol and ethanol, characterised by the reassimilation of acids. The production butanol is of renewed ineterest both as a potential biofuel and bulk chemical production. Both butanol and butyric acid posses toxic characteristic and here, we focus on understanding and modeling the stress response of C. acetobutylicum to one of the two important toxic metabolites: butyric acid. C. acetobutylicum cultures were grown, three biological replicates, to mid-exponential phase and then stressed with four levels of butyric acid (0 mM - No stress; 30 mM - Low stress; 40 mM - Medium stress; & 50 mM - High stress). Butyric acid was pH adjusted with 10M KOH to match the pH of the cultures, prior to addition. Samples were collected following the stress at 0, 15, 30, 45, 60 and 75 min, post stress. These sampling times, which are of the order of the doubling time of these cells, were meant to capture largely the direct and immediate impact of these stresses on gene expression. The RNA extracted from two biological replicates were used for microarray hybridization foloowing cDNA generation and labelling using Agilent 44K arrays, while the third was used for q-RT-PCR validation. For each stress level, 6 time points with 2 biological replicates and dye swaps (Cy3/Cy5) were prepared for comparison. The hybridization was perfomed against an equal amount of oppositely labeled cDNA from common reference pool prepared using equal amounts of labeled cDNA from all four stress levels.
Project description:In the current study we did microarray of upland rice cultivar Nagina22 for drought stress at reproductive stage (panicle initiation) and analyzed drought stress responsive genes. We have taken flag leaf for our study as it is most essential organ for photosynthesis in rice. Normal watering Vs Drought Stress Flag leaf of Control (Three biological replicates) plant of Nagina22: C1, C2, C3 Flag leaf of drought stressed (Three biological replicates) plant of Nagina 22: S1, S2, S3
Project description:Stress is a powerful modulator of neuroendocrine, behavioral and immunological functions. After 4.5 days of repeated combined acoustic and restraint stress as a murine model of chronic psychological stress severe metabolic dysregulations became detectable in female BALB/c mice. Stress-induced alterations of metabolic processes that were found in a hepatic mRNA expression profiling were verified by in vivo analyses. Repeatedly stressed mice developed a hypermetabolic syndrome with severe loss of lean body mass, hyperglycemia, dyslipidemia, increased amino acid turn-over, and acidosis. This was associated with hypercortisolism, hyperleptinemia, insulin resistance, and hypothyroidism. In contrast, after a single acute stress exposure changes in expression of metabolic genes were much less pronounced and predominantly confined to gluconeogenesis, probably indicating that metabolic disturbances might be initiated already early but will only manifest in repeatedly stressed mice .Thus, in our murine model, repeated stress caused severe metabolic dysregulations leading to a drastic reduction of the individual’s energy reserves. Under such circumstances stress may further reduce the ability to cope with new stressors such as infection or cancer. Endocrinology Epub ahead of print, March 11, 2008; doi:10.1210/en.2008-0038; This SuperSeries is composed of the following subset Series:; GSE11123: Mouse liver gene expression after a single acute 2h exposure to combined acoustic and restraint stress vs. control; GSE11125: Mouse liver gene expression after 4.5 days of repeated combined acoustic and restraint stress vs. control Experiment Overall Design: Refer to individual Series
Project description:Salt stress is a primary cause of crop losses worldwide, and it has been the subject of intense investigation to unravel the complex mechanisms responsible for salinity tolerance. MicroRNA is implicated in many developmental processes and in responses to various abiotic stresses, playing pivotal roles in plant adaptation. Deep sequencing technology was chosen to determine the small RNA transcriptome of Saccharum sp cultivars grown on saline conditions. We constructed four small RNAs libraries prepared from plants grown on hydroponic culture submitted to 170mM NaCl and harvested after 1h, 6hs and 24hs. Each library was sequenced individually and together generated more than 50 million short reads. Ninety-eight conserved miRNAs and 33 miRNAs* were identified by bioinformatics. Several of the microRNA showed considerable differences of expression in the four libraries. To confirm the results of the bioinformatics-based analysis, we studied the expression of the 10 most abundant miRNAs and 1 miRNA* in plants treated with 170mM and with a severe treatment of 340mM NaCl. The results showed that 11 selected miRNAs had higher expression in samples treated with severe salt treatment compared to the mild one. We also investigated the regulation of the same miRNAs in shoots of four cultivars grown on soil treated with 170mM NaCl. Cultivars could be grouped according to miRNAs expression in response to salt stress. Furthermore, the majority of the predicted target genes had an inverse regulation with their correspondent microRNAs. The targets encode a wide range of proteins, including transcription factors, metabolic enzymes and genes involved in hormone signaling pathways of, probably assisting the plants to develop tolerance. Our work provides insights into the regulatory functions of miRNAs, thereby expanding our knowledge on potential salt-stressed regulated genes. Screenning of sRNA transcriptome of sugarcane plants infected with Acidovorax avenae subsp avenae after seven days
Project description:Stress is a powerful modulator of neuroendocrine, behavioral and immunological functions. After 4.5 days of repeated combined acoustic and restraint stress as a murine model of chronic psychological stress severe metabolic dysregulations became detectable in female BALB/c mice. Stress-induced alterations of metabolic processes that were found in a hepatic mRNA expression profiling were verified by in vivo analyses. Repeatedly stressed mice developed a hypermetabolic syndrome with severe loss of lean body mass, hyperglycemia, dyslipidemia, increased amino acid turn-over, and acidosis. This was associated with hypercortisolism, hyperleptinemia, insulin resistance, and hypothyroidism. In contrast, after a single acute stress exposure changes in expression of metabolic genes were much less pronounced and predominantly confined to gluconeogenesis, probably indicating that metabolic disturbances might be initiated already early but will only manifest in repeatedly stressed mice .Thus, in our murine model, repeated stress caused severe metabolic dysregulations leading to a drastic reduction of the individual’s energy reserves. Under such circumstances stress may further reduce the ability to cope with new stressors such as infection or cancer. Endocrinology Epub ahead of print, March 11, 2008; doi:10.1210/en.2008-0038 Experiment Overall Design: Two biological experiments of repeated combined acoustic and restraint stress were performed, which consist each of a repeatedly stressed group and an untreated control group. Liver RNA expression profiles were analyzed in technical duplicates using pools of 8 or 9 individual RNAs of each group.
Project description:Identification of all expressed transcripts in a sequenced genome is essential both for genome analysis and for realization of the goals of systems biology. We used the transcriptional profiling technologies like ‘massively parallel signature sequencing (MPSS)’ and ‘Sequencing by Synthesis’ (SBS) to develop a comprehensive expression atlas of rice (Oryza sativa cv Nipponbare). Illumina’s SBS technology can generate large amounts of sequence data in a short time at low cost compared to traditional Sanger sequencing based methods. Using the MPSS technology, we previously analyzed the transcriptomes of 72 rice tissues. To validate the sequencing results from MPSS technology, we employed SBS technology and constructed SBS libraries from 32 rice tissues (47 libraries including replications). For SBS library construction, we used the same mRNA samples and same restriction enzyme (DpnII) that were used for the construction of the MPSS libraries. These libraries include six abiotic-stress libraries, eight pathogen-infected libraries, five insect-damaged libraries, three developing seed libraries, and 10 untreated rice tissue libraries. This study was carried out with the following objectives; a) Identification and quantification of expressed genes in rice at all developmental stages of plant growth, response to biotic and abiotic stresses, and developing seeds; b) Compare SBS signatures with rice genomic sequence to identify novel transcripts; c) To validate the transcriptional data obtained through MPSS technology; and To create query and analysis tools to facilitate public use of and access to rice MPSS and SBS data and to display abundance and chromosomal locations of rice MPSS and SBS signatures. The SBS data will be available at http://mpss.udel.edu/rice_sbs/. 32 rice tissues (47 libraries including replications)