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We characterize the time for coffee ( tic ) mutant that disrupts circadian gating, photoperiodism, and multiple circadian rhythms, with differential effects among rhythms. TIC is distinct in physiological functions and genetic map position from other rhythm mutants and their homologous loci. Detailed rhythm analysis shows that the chlorophyll a/b-binding protein gene expression rhythm requires TIC function in the mid to late subjective night, when human activity may require coffee, in contrast to the function of EARLY-FLOWERING3 ( ELF3 ) in the late day to early night. tic mutants misexpress genes that are thought to be critical for circadian timing, consistent with our functional analysis. Thus, we identify\nTIC as a regulator of the clock gene circuit. In contrast to tic and elf3 single mutants, tic elf3 double mutants are completely arrhythmic. 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Even the robust circadian clock of plants cannot function with defects at two different phases.", "pubmed_id": "14555691", "provenance": { "pub_title": "The TIME FOR COFFEE gene maintains the amplitude and timing of Arabidopsis circadian clocks", "first_author": "Hall A", "group": "Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom", "pub_date": "2003-10-10" }, "growth_condition": { "name": "Growth1", "medium": "Agar 1.5% MS 1 3% Sucrose", "stratification": "3 days at 4C", "duration": "8", "comment": "\"Eight-day-old Columbia (Col-0) seedlings grown under 12-h-light/12-h-dark cycles (LD 12:12)\"", "temperature_conditions": { "constant_temp": { "cycle_length": "24", "base_temp": "27" } }, "light_conditions": { "light_channel": { "spectrum": "red", "intensity": "40", "source": "LED", "diurnal_light": { "cycle_length": "24", "start_time": "0", "duration": "12" } } } }, "experimental_condition": { "name": "Exp1", "medium": "Agar 1.5% MS 1 3% Sucrose", "duration": "2", "comment": "transferred to constant light at 228C and harvested at 13 time points, covering two circadian cycles in 4-h intervals, starting 26 h after the last dark–light transition.", "temperature_conditions": { "constant_temp": { "cycle_length": "24", "base_temp": "22" } }, "light_conditions": { "light_channel": { "spectrum": "white", "intensity": "40", "source": "LED", "constant_light": { "cycle_length": "24" } } } }, "measurement": { "technique": "RT-PCR", "equipment": "Plant RNeasy kit and RNasefree DNase (Qiagen)", "description": "Approximately 100 seedlings were ground under liquid nitrogen per time point, and total RNA was extracted using a Plant RNeasy kit and RNasefree DNase (Qiagen) according to the manufacturer’s instructions. cDNA samples for real-time PCR applications were reverse transcribed from 1 mg of RNA using the RevertAid first-strand cDNA synthesis kit (Fermentas, Helena Biosciences) according to the manufacturer’s instructions, and the cDNA product was diluted 1:5 in RNase-free water. Transcript abundance of TOC1, CCA1, LHY, GI, EPR1, and LUX were assessed by quantitative real-time PCR in either an ABI PRISM 7700 (Applied Biosystems) or Bio-Rad iCycler IQ using ABI SYBR Green PCR Master Mix (Applied Biosystems) in 15-mL reactions. Transcript levels were normalized to ACT2 using a cDNA dilution series for each primer set in each experiment. Each RNA sample was assayed in triplicate. Primers for GI and ACT2 have been described previously (Locke et al., 2005b). Primer sequences to assess other transcripts are shown below: TOC1 forward, 59-ATCTTCGCAGAATCCCTGTGATA-39; TOC1 reverse,\n 59-GCACCTAGCTTCAAGCACTTTACA-39; CCA1 forward, 59-CTGTGTCTGACGAGGGTCGAA- 39; CCA1 reverse, 59-ATATGTAAAACTTTGCGGCAATACCT- 39; LHY forward, 59-CAACAGCAACAACAATGCAACTAC-39 LHY reverse, 59-AGAGAGCCTGAAACGCTATACGA-39; EPR1 forward, 59-CCAAGATGGCTCAGGAAGCT-39; EPR1 reverse, 59-AAGGATGTGCCGGTTTTCTCT- 39; LUX forward, 59-GACGATGATTCTGATGATAAGG-39; LUX reverse, 59-CAGTTTATGCACATCATATGGG-39." }, "sample_preparation": { "method": "n/a" }, "samples": { "sample": [ { "sample_id": "1", "sample_type": "seedling", "origin": "seedling", "growth_stage": "seedling", "genetic_info": { "species": "Arabidopsis thaliana", "background": "C24", "genotype": "Wild Type" }, "marker": "CCA1", "growth_cond_name": "Growth1", "experiment_cond_name": "Exp1" }, { "sample_id": "2", "sample_type": "seedling", "origin": "seedling", "growth_stage": "seedling", "genetic_info": { "species": "Arabidopsis thaliana", "background": "C24", "genotype": "tic-1" }, "marker": "CCA1", "growth_cond_name": "Growth1", "experiment_cond_name": "Exp1" }, { "sample_id": "3", "sample_type": "seedling", "origin": "seedling", "growth_stage": "seedling", "genetic_info": { "species": "Arabidopsis thaliana", "background": "C24", "genotype": "elf3" }, "marker": "CCA1", "growth_cond_name": "Growth1", "experiment_cond_name": "Exp1" }, { "sample_id": "4", "sample_type": "seedling", "origin": "seedling", "growth_stage": "seedling", "genetic_info": { "species": "Arabidopsis thaliana", "background": "C24", "genotype": "tic;elf3" }, "marker": "CCA1", "growth_cond_name": "Growth1", "experiment_cond_name": "Exp1" }, { "sample_id": "5", "sample_type": "seedling", "origin": "seedling", "growth_stage": "seedling", "genetic_info": { "species": "Arabidopsis thaliana", "background": "C24", "genotype": "Wild Type" }, "marker": "TOC1", "growth_cond_name": "Growth1", "experiment_cond_name": "Exp1" }, { "sample_id": "6", "sample_type": "seedling", "origin": "seedling", "growth_stage": "seedling", "genetic_info": { "species": "Arabidopsis thaliana", "background": "C24", "genotype": "tic-1" }, "marker": "TOC1", "growth_cond_name": "Growth1", "experiment_cond_name": "Exp1" }, { "sample_id": "7", "sample_type": "seedling", "origin": "seedling", "growth_stage": "seedling", "genetic_info": { "species": "Arabidopsis thaliana", "background": "C24", "genotype": "elf3" }, "marker": "TOC1", "growth_cond_name": "Growth1", "experiment_cond_name": "Exp1" }, { "sample_id": "8", "sample_type": "seedling", "origin": "seedling", "growth_stage": "seedling", "genetic_info": { "species": "Arabidopsis thaliana", "background": "C24", "genotype": "tic;elf3" }, "marker": "TOC1", "growth_cond_name": "Growth1", "experiment_cond_name": "Exp1" }, { "sample_id": "9", "sample_type": "seedling", "origin": "seedling", "growth_stage": "seedling", "genetic_info": { "species": "Arabidopsis thaliana", "background": "C24", "genotype": "Wild Type" }, "marker": "ELF3", "growth_cond_name": "Growth1", "experiment_cond_name": "Exp1" }, { "sample_id": "10", "sample_type": "seedling", "origin": "seedling", "growth_stage": "seedling", "genetic_info": { "species": "Arabidopsis thaliana", "background": "C24", "genotype": "tic-1" }, "marker": "ELF3", "growth_cond_name": "Growth1", "experiment_cond_name": "Exp1" }, { "sample_id": "11", "sample_type": "seedling", "origin": "seedling", "growth_stage": "seedling", "genetic_info": { "species": "Arabidopsis thaliana", "background": "C24", "genotype": "elf3" }, "marker": "ELF3", "growth_cond_name": "Growth1", "experiment_cond_name": "Exp1" }, { "sample_id": "12", "sample_type": "seedling", "origin": "seedling", "growth_stage": "seedling", "genetic_info": { "species": "Arabidopsis thaliana", "background": "C24", "genotype": "tic;elf3" }, "marker": "ELF3", "growth_cond_name": "Growth1", "experiment_cond_name": "Exp1" } ] } }, "biblio": { "principal": "Millar AJ", "institution": "University of Edinburgh", "author": [ "Hall A", "Bastow RM", "Davis SJ", "Hanano S", "McWatters HG", "Hibberd V", "Doyle MR", "Sung S", "Halliday KJ", "Amasino RM" ] } }