Project description:Phytochromes are red/far red photosensors regulating numerous developmental programs in plants. Among them phytochrome A (phyA) is essential to enable seedling de-etiolation in continuous far-red (FR) light a condition mimicking the environment under a dense canopy. The ecological relevance of this response is demonstrated by the high mortality rate of phyA mutants germinating in deep vegetational shade. phyA signaling involves a direct interaction of the photoreceptor with members of the bHLH transcription factor family, PIF1 and PIF3 (Phytochrome Interacting Factor). Here we investigated the involvement of PIF4 and PIF5 in phyA signaling and found that they redundantly control de-etiolation in FR light. The pif4pif5 double mutant is hypersensitive to low fluence rates of FR light. This phenotype is dependent on FR light perception by phyA but does not rely on alterations of the phyA level. Our microarrays analysis shows that PIF4 and PIF5 are part of an inhibitory mechanism repressing the expression of some light-responsive genes in the dark and are also needed for full expression of several growth-related genes in the light. Unlike PIF1 and PIF3, PIF4 and PIF5 are not degraded in response to FR light indicating that they are light-regulated by a different mechanism. Our genetic analysis suggests that this is achieved through the sequestration of these PIFs by the closely related bHLH transcription factor HFR1 (long Hypocotyl in FR light). Experiment Overall Design: he pif4pif5 double mutant were compared to wild-type plants when kept in the dark or subjected to 1 or 24 hours of 0.5 or 5 µmol/m2/s far-red light respectively.
Project description:The mRNA expression profiles of the WT and b''ab''b were analyzed in 1 hour 8 μmol/m2s red-light exposure and darkness after 4-day dark-grown. The gene expression changes was analyzed and compared between dark and red-light treated samples.
Project description:Light initiates the seedling deetiolation transition by promoting major changes in gene expression mainly regulated by phytochrome (phy) photoreceptors. During the initial dark-to-light transition, phy photoactivation induces rapid changes in gene expression that eventually lead to the photomorphogenic development. Recent reports indicate that this process is achieved by phy-induced degradation of Phy-Interacting bHLH transcription Factors (PIFs) PIF1, PIF3 PIF4 and PIF5, which are partly redundant constitutive repressors of photomorphogenesis that accumulate in darkness. In order to test whether light/phy-regulated gene expression occurs through these PIFs, we have performed whole-genome expression analysis in the pif1pif3pif4pif5 quadruple mutant (pifq). Wild-type and pifq mutant seeds were plated on GM medium without sucrose at room temperature. During this procedure the seeds were routinely exposed to white light (WL) for a total of 1.5 hours after imbibition. Seeds were then stratified for 5 days at 4ºC in darkness, induced to germinate with a 5-min red pulse (Rp) (46 μmol/m2/s) and then incubated in the dark for 3h at 21°C before exposure to a terminal 5-min far red pulse (FRp) (58 μmol/m2/s) to suppress pseudo-dark effects. Seeds were then placed in either dark (D) or constant red light (Rc) (6.7 μmol/ m2/s) at 21°C for 45h (2d-old seedlings). Alternatively, 2d-old dark-grown seedlings were treated with 1h of red light (R1) (7.5 μmol/m2/s). Seed samples were harvested after stratification (5d stratified seeds).
Project description:Purpose: The goals of this study are to compare the transcriptome profiling and alternative splicing (AS) profiling between Col-0 wild type and SFPS knockout mutant (sfps-2) through RNA-seq to determine the molecular mechanisms of how splicing factor SFPS regulates photomorphogenesis in Arabidopsis. Results: Using an optimized data analysis workflow, we mapped about 100 million sequence reads per sample to the Arabidopsis genome (TAIR10) and identified 1495 differentially expressed genes between Col-0 and mutant dark samples; 1361 differentially expressed genes between Col-0 and mutant red light treated samples; 4291 differentially expressed genes between Col-0 dark and red light treated samples; and 4479 differentially expressed genes between mutant dark and red light treated samples. Except for gene expression, we also discovered 788 differentially spliced bins between Col-0 and mutant dark samples; 827 differentially spliced bins between Col-0 and mutant red light treated samples; 610 differentially spliced bins between Col-0 dark and red light treated samples; and 405 differentially spliced bins between mutant dark and red light treated samples. Altered splicing of 9 genes was confirmed with qRT-PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Conclusions: Our study represents the first detailed analysis of SFPS mutant transcriptomes, with biologic replicates, generated by RNA-seq technology. Our results show that SFPS regulates photomorphogenesis in Arabidopisis through regulating the splicing activity of light signaling genes, which helps us.