Genome-wide survey of cold stress regulated alternative splicing in Arabidopsis thaliana
ABSTRACT: Alternative splicing plays a major role in expanding the potential informational content of eukaryotic genomes. It is an important post-transcriptional regulatory mechanism that can increase protein diversity and affect mRNA stability. Cold stress, which adversely affects plants growth and development, regulates the transcription and splicing of plants splicing factors. This affects the pre-mRNA processing of many genes. To identify cold regulated alternative splicing we applied Affymetrix Arabidopsis tiling arrays to survey the transcriptome under cold treatment conditions. Two-week old Arabidopsis seedlings grown on agar were subjected to 24 hours of cold (4°C) treatment under long day conditions. Control and cold-treated plants were harvested at the same time to ensure that observed differences would not be due to circadian clock effects on transcripts. Total RNA from four biological repeats were used for microarray hybridization.
Project description:St (common potato) is a freezing sensitive species unable to cold acclimate. The close wild relative Sc is freezing tolerant and able to cold acclimate. Here we compare the cold transcriptome of these two species with different levels of freezing tolerance. We also identify the putative CBF regulons by comparing the transcriptomes of wild type plants with that of 35S::AtCBF3 transgenic lines in both species. Plants were grown in 16:8 photoperiod. Eight hours after dawn, plants were either transfered to cold or kept in the warn. Wild type S. tuberosum and S. commersonii were grown at 2oC for 2h, 24h and 7 days. Wild type plants grown under warm temperatures for 2h was used as control for 2h cold samples; wild type warm grown plants for 24h were used as controls for 24h and 7 days cold samples. Under warm conditions, S. commersonii 35S::AtCBF3 lines were compared to S. commersonii wild type plants (same thing was done for S. tuberosum).
Project description:Stress acclimation is an effective mechanism that plants acquired for adaption to dynamic environmental conditions. After undergoing cold acclimation, plants become more tolerant to cold stress. In order to understand the mechanism of cold acclimation, we performed a systematic, comprehensive study of cold response and acclimation in Cassava (Manihot esculenta), a staple crop and major food source in the tropical regions of the world. We profiled mRNA genes and small-RNA species, using next generation sequencing, and performed an integrative analysis of the transcriptome and microRNAome of Cassava across the normal condition, a moderate cold stress at 14°C, a harsh stress at 4°C after cold acclimation at 14°C, and a cold shock from 24°C to 4°C. Two results from the analysis were striking. First, the moderate stress and cold shock, despite a difference of 10°C between the two, triggered comparable degrees of perturbation to the transcriptome; in contrary, further harsh stress after cold acclimation resulted in a much smaller degree of transcriptome variation. Second and more importantly, about two thirds of the up- or down-regulated genes after moderate stress reversed their expression to down- or up-regulation, respectively, under harsh stress after cold acclimation, resulting in a genome-wide rewiring of regulatory networks. MicroRNAs, which are key post-transcriptional gene regulators, were major players in this massive rewiring of genetic circuitry. Further, a function enrichment analysis of the perturbed genes revealed that cold acclimation helped the plant to develop immunity to further harsh stress by exclusively inducing genes with functions of nutrient reservoir; in contrast, many genes with functions of viral reproduction were induced by cold shock. Our study revealed, for the first time, the molecular basis of stress acclimation in plants, and shed lights on the role of microRNA gene regulation in cold response and acclimation in Euphorbia. Three organs/tissues (folded leaf, fully expanded leaf and roots) of Cassava cultivar SC124 harvested at 6h, 24h and 5d for three cold treatments of CA, CCA and CS, for gene expression profiling at the stages of initial response, secondary response, and functional adaption to cold stresses. Total RNA of each sample was isolated individually, and then pooled with an equal amount from each sample into one for profiling. As a result, four mRNA libraries and four small-RNA libraries, corresponding to the conditions of CA, CCA, CS and NC, were constructed.
Project description:Small noncoding RNA (sncRNA), including microRNAs (miRNAs) and endogenous small-interfering RNAs (endo-siRNAs) are key gene regulators in eukaryotes, playing critical roles in plant development and stress tolerance. Trans-acting siRNAs (ta-siRNAs), which are secondary siRNAs triggered by miRNAs, and siRNAs from natural antisense transcripts (nat-siRNAs) are two well-studied classes of endo-siRNAs. In order to understand sncRNAs’ roles in plant cold response and stress acclimation, we studied miRNAs and endo-siRNAs in Cassava (Manihot esculenta), a major source of food for the world populations in tropical regions. Combining Next-Generation sequencing and computational and experimental analyses, we profiled and characterized sncRNA species and mRNA genes from the plants that experienced severe and moderate cold stresses, that underwent further severe cold stress after cold acclimation at moderate stress, and that grew under the normal condition. We also included Castor bean (Ricinus communis) to understand conservation of sncRNAs. In addition to known miRNAs, we identified dozens of novel miRNAs as well as ta-siRNA-yielding and nat-siRNA-yielding loci in Cassava and Castor bean, respectively. Among the expressed sncRNAs, many sncRNAs were differentially expressed under cold stresses. Our study provided the results on gene regulation by sncRNAs in cold acclimation of Euphorbiaceous plants and the role of sncRNA-mediated pathways affected by cold stress and stress acclimation in Cassava. Examination of small RNA populations in Cassava cultivar SC124 under the normal condition (NC), gradual cold acclimation (CA), cold shock (CS) and stress acclimation Cold stress after cold acclimation (CCA).
Project description:ZmDREB2A is a DREB2-type transcription factor cloned from maize, whose transcript was upregulated by drought, high salt, low temperature and heat stresses. The ZmDREB2A gene possesses two kinds of transcription forms by alternative splicing. Only the functional form was studied to be highly induced by stresses. Transgenic plants overexpressing ZmDREB2A (35S:ZmDREB2A) showed dwarfism and enhanced drought stress tolerance. Microarray analysis of two independent transgenic plants revealed that in addition to genes encoding LEA proteins, some genes related to heat shock and detoxification were also upregulated. Experiments on termotolerance tests of these transgenic plants showed overexpressing ZmDREB2A gene also improved plant tolerance to heat stress.
Project description:Introns are removed by the spliceosome, a large complex composed of five ribonucleoprotein subcomplexes (U snRNP). In metazoans, the U1 snRNP, which binds to 5’ splice sites, also fulfills regulatory roles in splice site selection and possesses non-splicing related functions. Here, we show that an Arabidopsis U1 snRNP subunit, LUC7, affects constitutive and alternative splicing. Interestingly, LUC7 specifically promotes splicing of a subset of terminal introns. Splicing of LUC7-dependent terminal introns is a prerequisite for nuclear export and can be modulated by stress. Globally, intron retention under stress conditions occurs preferentially among first and terminal introns, uncovering an unknown bias for splicing regulation in Arabidopsis. Taken together, our study reveals that the Arabidopsis U1 snRNP is important for alternative splicing and removal of terminal introns and it suggests that Arabidopsis terminal introns fine-tune gene expression under stress conditions. Overall design: Poly A libraries from wild type plants (WT Col-0) and a triple mutant (luc7 a-2, b-1, rl-1) were performed in triplicates
Project description:Many biological processes involve post-transcriptional regulation of gene expression by alternative pre-mRNA splicing. Here, we show that an alternative splicing factor SRSF6 affects tissue homeostasis of the skin. In this dataset, we study effects on gene expression and alternative splicing upon SRSF6 overexpression (+doxycycline) in mouse skin using inducible R26-rtTA+/-, ColA1-TREtight-SRSF6+/- transgenic mice 4 mixed-background strain samples (2 SRSF6-induced skin samples and 2 uninduced skin control samples)
Project description:Temperature reduction is a common environmental stress for plants. Land plants need to cope with cold stress on the basis of complex transcriptional and metabolic changes. The transcriptional responses and signaling networks that contribute to cold acclimation of seed plants have been analyzed previously. Here, we present the whole-genome transcriptomic cold stress response of the model moss species Physcomitrella patens as the representative of an early diverged lineage of haploid-dominant and poikilohydric land plants On the basis of time-series microarray experiments we characterized transcriptomic changes related to early stress signaling and the initiation of cold-acclimatory mechanisms, and as secondary effects, of dehydration and oxidative stress.
Project description:Anthurium andraeanum is one of the most popular tropical flowers. In temperate and cold zones, a much greater risk of cold stress occurs in the supply of Anthurium plants. Unlike the freeze-tolerant model plants, Anthurium plants are particularly sensitive to low temperatures. Improvement of chilling tolerance in Anthurium may significantly increase its production and extend its shelf-life. To date, no previous genomic information has been reported in Anthurium plants.
Project description:ABSTRACT: Exposure to abiotic stresses triggers global changes in the expression of thousands of eukaryotic genes at the transcriptional 70 and post-transcriptional levels. Small RNA (smRNA) pathways and splicing both function as crucial mechanisms regulating stress-responsive gene expression. However, examples of smRNAs regulating gene expression remain largely limited to effects on mRNA stability, translation, and epigenetic regulation. Also, our understanding of the networks controlling plant gene expression in response to environmental changes, and examples of these regulatory pathways intersecting, remains limited. Here, to investigate the role of smRNAs in stress responses we examined smRNA transcriptomes of Brachypodium distachyon plants subjected to various abiotic stresses. We found that exposure to different abiotic stresses specifically induced a group 75 of novel, endogenous small interfering RNAs (stress-induced, UTR-derived siRNAs, or sutr-siRNAs) that originate from the 3′ UTRs of a subset of coding genes. Our bioinformatics analyses predicted that sutr-siRNAs have potential regulatory functions and that over 90% of sutr-siRNAs target intronic regions of many mRNAs in trans. Importantly, a subgroup of these sutr- siRNAs target the important intron regulatory regions, such as branch point sequences, that could affect splicing. Our study indicates that in Brachypodium, sutr-siRNAs may affect splicing by masking or changing accessibility of specific cis-elements 80 through base-pairing interactions to mediate gene expression in response to stresses. We hypothesize that this mode of regulation of gene expression may also serve as a general mechanism for regulation of gene expression in plants and potentially in other eukaryotes. Analysis of smRNA populations in Brachypodium plants challenged by abiotic stresses: To profile the populations of smRNAs in the model monocot Brachypodium distachyon and examine their regulation in response to abiotic stresses, we conducted high-throughput sequencing of small RNAs from plants exposed to four different abiotic stress conditions, cold, heat (air), heat (water immersion), and salt, in the wild type Brachypodium cultivar Bd21. For our experiments we used information from the literature to select two time-points for stress durations, short and long, which differed for each stress: cold (6 and 24 hours), heat-air (1 and 3 hours), heat-water (1 and 3 hours), and salt (48 hours). We generated small RNA libraries for Illumina sequencing (GAII) from the leaves of Brachypodium plants subjected to stresses and selected smRNAs between 15 and 40 nt in length, which we mapped to the Brachypodium genome.