Gene Expression
Nebulas
Gene Expression NebulasSummary: The circadian clock helps organisms to anticipate and coordinate gene regulatory responses to changes in environmental stimuli. Under growth limiting temperatures, time of day modulates the accumulation of polyadenylated mRNAs. In response to heat stress, plants will conserve energy and selectively translate mRNAs. How the clock and/or time of day regulates polyadenylated mRNAs bound by ribosomes in response to heat stress is unknown. In-depth analysis of Arabidopsis thaliana translating mRNAs found that time of day gates the response of approximately one-third of the circadian regulated heat responsive translatome. Specifically, time of day and heat stress interact to prioritize the pool of mRNAs in cue to be translated. For a subset of mRNAs, we observed a stronger gated response during the day, and preferentially before the peak of expression. We propose previously overlooked transcription factors (TFs) as regulatory nodes and show that the clock plays a role in the temperature response for select TFs. When the stress was removed, the redefined priorities for translation recovered within one-hour, though slower recovery was observed for abiotic stress regulators. Through hierarchical network connections between clock genes and prioritized TFs, our work provides a framework to target key nodes underlying heat stress tolerance throughout the day.
Overall Design: Analysis of the Arabidopsis transcriptome and translatome response to heat stress (37°C vs 22°C) at eight different times of day during a 24 h time course, and recovery post heat stress.
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| Growth Protocol: | Seeds were surface sterilized and were stratified at 4°C for three days in the dark. Seedlings were grown in square petri dishes on filter paper placed on Murashige and Skoog (MS) medium supplemented with 1.5 % (w/v) sucrose. Plates were randomly arranged in the growth chamber and seedlings were grown for 10 days in 12 h light (~130 μEm-2s-1) and 12 h dark (LD) cycles at constant 22°C temperature. On day 11, plants were transferred to constant light and light intensity was reduced to 80-100 μEm-2s-1 to limit oxidative stress. |
| Treatment Protocol: | Time of day is referred to as Zeitgeber Time (ZT), i.e. ZT48 and ZT60 corresponding to the beginning of the light and subjective night periods on day 13, respectively. On day 13, a 1 h heat stress (37°C) was applied to the seedlings (i.e. ZT47 to ZT48), every three hours (ZT48 - ZT69) on a 24 h time course, on different sets of plants (Fig. 1A). Control plants were maintained at 22°C (ZT48 - ZT72). To analyze the plant recovery, a 1 h heat stress was applied from ZT53 to ZT54. Stressed plants were transferred back to 22°C for recovery and control plants were maintained at 22°C. Seedlings were collected at ZT54, ZT55, ZT57 and ZT60, which correspond to 0 h, 1 h, 3 h and 6 h or recovery, respectively. |
| Extract Protocol: | To extract mRNAs associated with ribosomes, Translating Ribosome Affinity Purification (TRAP) was performed as previously described (Reynoso et al., 2015). Total and TRAP mRNAs were isolated using biotinylated oligo(dT) and streptavidin magnetic beads (New England Biolabs) as previously described (Townsley et al., 2015) |
| Library Construction Protocol: | Libraries were prepared as previously described (Townsley et al., 2015) with the modifications previously detailed (Blair et al., 2019). In the final enrichment step, indexed adapter enrichment primers were used (Townsley et al., 2015) and 12 cycles were performed to amplify the libraries |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | SINGLE |
| Library Strand: | Reverse; - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina NextSeq 500 |
| Strand-Specific: | Specific; Unspecific |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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