Genome-wide analysis of toc1-2 transcriptional networks
ABSTRACT: To gain insights into the mechanisms of TOC1 function in the Arabidopsis circadian clock we performed transcriptional profiling of Wild-Type (WT) and and TOC1 mutant plants (toc1-2) under constant light conditions for two days. Overall design: Comparisons of WT and toc1-2. Two biological replicates each per array. Two Arabidopsis Oligonucleotide Microarrays (two-color Cy3 and Cy5). synchronized under 12-hour light:12-hour dark (LD) cycles for 10 days followed by two days under constant light conditions. Samples were collected at circadian time 16 (CT16).
INSTRUMENT(S): Galbraith Laboratory Operon Long Oligonucleotide Microarray version 3.0 (ATv302.01.Z)
Project description:To gain insights into the mechanisms of TOC1 function in the Arabidopsis circadian clock we performed transcriptional profiling of Wild-Type (WT) and and TOC1 mutant plants (toc1-2) under constant light conditions for two days. Comparisons of WT and toc1-2. Two biological replicates each per array. Two Arabidopsis Oligonucleotide Microarrays (two-color Cy3 and Cy5). synchronized under 12-hour light:12-hour dark (LD) cycles for 10 days followed by two days under constant light conditions. Samples were collected at circadian time 16 (CT16).
Project description:To gain insights into the mechanisms of TOC1 function in the Arabidopsis circadian clock we performed transcriptional profiling of Wild-Type (WT) and and TOC1 overexpressor plants (TOC1-ox). Overall design: Comparisons of WT and TOC1-ox. Three biological replicates synchronized under 12-hour light:12-hour dark (LD) cycles for 12 days. Samples were collected at Zeitgeber Time 15 (ZT15).
Project description:To gain insights into the mechanisms of TOC1 function in the Arabidopsis circadian clock we performed transcriptional profiling of Wild-Type (WT) and and TOC1 overexpressor plants (TOC1-ox). Experiment Overall Design: Comparisons of WT and TOC1-ox. Three biological replicates synchronized under 12-hour light:12-hour dark (LD) cycles for 12 days. Samples were collected at Zeitgeber Time 15 (ZT15).
Project description:In higher plants, the circadian clock orchestrates fundamental processes such as light signaling and the transition to flowering. We isolated mutants of the circadian clock from an Arabidopsis thaliana mutagenized reporter line by screening for seedlings with long hypocotyl phenotypes and subsequently assaying for abnormal clock-regulated CAB2::LUC expression. This screen identified five mutant alleles of a clock gene, LUX ARRHYTHMO (LUX), that significantly affect amplitude and robustness of rhythms in both constant white light and dark conditions. In addition, the transition from vegetative to floral development is accelerated and hypocotyl elongation is accentuated in these mutants under light:dark cycles. We genetically mapped the mutations by bulk segregant analysis with high-density oligonucleotide array genotyping to a small putative Myb transcription factor related to other clock components and response regulators in Arabidopsis. The negative arm of the Arabidopsis circadian clock, CIRCADIAN CLOCK ASSOCIATED (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), is repressed in the lux mutants, whereas TIMING OF CAB2 EXPRESSION (TOC1) is activated. We demonstrate that CCA1 and LHY bind to the evening element motif in the LUX promoter, which strongly suggests that these proteins repress LUX expression, as they do TOC1. The data are also consistent with LUX being necessary for activation of CCA1 and LHY expression.
Project description:Most organisms anticipate daily environmental variations and orchestrate cellular functions thanks to a circadian clock which entrains robustly to the day/night cycle, despite fluctuations in light intensity due to weather or seasonal variations. Marine organisms are also subjected to fluctuations in light spectral composition as their depth varies, due to differential absorption of different wavelengths by sea water. Studying how light input pathways contribute to circadian clock robustness is therefore important. Ostreococcus tauri, a unicellular picoplanktonic marine green alga with low genomic complexity and simple cellular organization, has become a promising model organism for systems biology. Functional and modeling approaches have shown that a core circadian oscillator based on orthologs of Arabidopsis TOC1 and CCA1 clock genes accounts for most experimental data acquired under a wide range of conditions. Some evidence points at putative light input pathway(s) consisting of a two-component signaling system (TCS) controlled by the only two histidine kinases (HK) of O. tauri. LOV-HK is a blue light photoreceptor under circadian control, that is required for circadian clock function. An involvement of Rhodopsin-HK (Rhod-HK) is also conceivable since rhodopsin photoreceptors mediate blue to green light input in animal circadian clocks. Here, we probe the role of LOV-HK and Rhod-HK in mediating light input to the TOC1-CCA1 oscillator using a mathematical model incorporating the TCS hypothesis. This model agrees with clock gene expression time series representative of multiple environmental conditions in blue or green light, characterizing entrainment by light/dark cycles, free-running in constant light, and resetting. Experimental and theoretical results indicate that both blue and green light can reset O. tauri circadian clock. Moreover, our mathematical analysis suggests that Rhod-HK is a blue-green light receptor and drives the clock together with LOV-HK.
Project description:Tissue-specific functions of the circadian clock in Arabidopsis have recently been revealed. The vasculature clock shows distinctive gene expression profiles compared to the clock in other tissues under light-dark cycles. However, it has not yet been established whether the vasculature clock also shows unique gene expression patterns that correlate with temperature cycles, another important environmental cue. Here, we detected diel phase of TIMING OF CAB EXPRESSION 1 (TOC1) expression in the vasculature and whole leaf under long-day light-dark cycles and temperature cycles. We found that the vasculature clock had advanced TOC1 phase under light-dark cycles but not under temperature cycles, suggesting that the vasculature clock has lower sensitivity against temperature signals. Furthermore, the phase advancement of TOC1 was seen only under long-day condition but not under short-day condition. These results support our previous conclusion that the circadian clock in vasculature preferentially senses photoperiodic signals.
Project description:Although, the circadian clock is a universal biological system in plants and it orchestrates important role of plant production such as photosynthesis, floral induction and growth, there are few such studies on cultivated species. Lettuce is one major cultivated species for both open culture and plant factories and there is little information concerning its circadian clock system. In addition, most of the relevant genes have not been identified. In this study, we detected circadian oscillation in the lettuce transcriptome using time-course RNA sequencing (RNA-Seq) data. Constant light (LL) and light-dark (LD) conditions were used to detect circadian oscillation because the circadian clock has some basic properties: one is self-sustaining oscillation under constant light and another is entrainment to environmental cycles such as light and temperature. In the results, 215 contigs were detected as common oscillating contigs under both LL and LD conditions. The 215 common oscillating contigs included clock gene-like contigs CCA1 (CIRCADIAN CLOCK ASSOCIATED 1)-like, TOC1 (TIMING OF CAB EXPRESSION 1)-like and LHY (LATE ELONGATED HYPOCOTYL)-like, and their expression patterns were similar to those of Arabidopsis. Functional enrichment analysis by GO (gene ontology) Slim and GO Fat showed that the GO terms of response to light stimulus, response to stress, photosynthesis and circadian rhythms were enriched in the 215 common oscillating contigs and these terms were actually regulated by circadian clocks in plants. The 215 common oscillating contigs can be used to evaluate whether the gene expression pattern related to photosynthesis and optical response performs normally in lettuce.
Project description:Circadian clocks enable organisms to adapt to a 24 h diurnal cycle and anticipate rhythmic changes in the environment. The Arabidopsis central oscillator contains three genes encoding core clock components. CIRCADIAN CLOCK ASSOCIATED 1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY) and TIMING OF CAB EXPRESSION 1 (TOC1) reciprocally repress genes encoding each other and are critical for the generation of circadian rhythms controlling many clock outputs. A precise regulation of transcriptional events is, therefore, essential for proper circadian function. Here, we investigated histone 3 (H3) tail modifications of CCA1, LHY and TOC1 under various conditions. We found specific association of only H3K4Me3 and H3K9/14Ac with the translational start site of these three genes. These H3 marks were enriched at circadian time points of their increased transcription at different photoperiods and under free-running conditions, suggesting circadian regulation of H3 modifications. Analysis of clock-compromised CCA1-overexpressing lines provided evidence that light/dark photoperiods signal the establishment of these chromatin changes which are gated by the clock.
Project description:The circadian clock is a timekeeping system for regulation of numerous biological daily rhythms. One characteristic of the circadian clock is that period length remains relatively constant in spite of environmental fluctuations, such as temperature change. Here, using the curated collection of in-house small molecule chemical library (ITbM chemical library), we show that small molecule 3,4-dibromo-7-azaindole (B-AZ) lengthened the circadian period of Arabidopsis thaliana (Arabidopsis). B-AZ has not previously been reported to have any biological and biochemical activities. Target identification can elucidate the mode of action of small molecules, but we were unable to make a molecular probe of B-AZ for target identification. Instead, we performed other analysis, gene expression profiling that potentially reveals mode of action of molecules. Short-term treatment of B-AZ decreased the expression of four dawn- and morning-phased clock-associated genes, CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), PSEUDO-RESPONSE REGULATOR 9 (PRR9) and PRR7. Consistently, amounts of PRR5 and TIMING OF CAB EXPRESSION 1 (TOC1) proteins, transcriptional repressors of CCA1, LHY, PRR9 and PRR7 were increased upon B-AZ treatment. B-AZ inhibited Casein Kinase 1 family (CK1) that phosphorylates PRR5 and TOC1 for targeted degradation. A docking study and molecular dynamics simulation suggested that B-AZ interacts with the ATP-binding pocket of human CK1 delta, whose amino acid sequences are highly similar to those of Arabidopsis CK1. B-AZ-induced period-lengthening effect was attenuated in prr5 toc1 mutants. Collectively, this study provides a novel and simple structure CK1 inhibitor that modulates circadian clock via accumulation of PRR5 and TOC1.
Project description:Arabidopsis adapts to elevated temperature by promoting stem elongation and hyponastic growth through a temperature-responsive transcription factor PIF4. Here we show that the evening-expressed clock component TOC1 interacts with and inactivates PIF4, thereby suppressing thermoresponsive growth in the evening. We find that the expression of PIF4 target genes show circadian rhythms of thermosensitivity, with minimum responsiveness in the evening when TOC1 level is high. Loss of function of TOC1 and its close homologue PRR5 restores thermosensitivity in the evening, whereas TOC1 overexpression causes thermo insensitivity, demonstrating that TOC1 mediates the evening-specific inhibition of thermoresponses. We further show that PIF4 is required for thermoadaptation mediated by moderately elevated temperature. Our results demonstrate that the interaction between TOC1 and PIF4 mediates the circadian gating of thermoresponsive growth, which may serve to increase fitness by matching thermoresponsiveness with the day-night cycles of fluctuating temperature and light conditions.