| Property | Value |
|---|---|
| BioDare ID | 13227500410222 |
| Author | Edwards KD |
| Institution | University of Edinburgh |
| License | CC_BY |
Literature data from: 'FLOWERING LOCUS C mediates natural variation in the high-temperature response of the Arabidopsis circadian clock' by: Edwards KD.
Authors have analyzed FLC alleles in near-isogenic lines and induced mutants to eliminate other candidate genes. They showed
that FLC lengthened the circadian period specifically at 278C, contributing to temperature compensation of the circadian clock. Known upstream regulators of FLC expression in flowering time pathways similarly controlled its circadian effect. They sought to identify downstreamtargets of FLCregulation in the molecular mechanismof the circadian clock using genome-wide analysis to identify FLC-responsive genes and 3503 transcripts controlled by the circadian clock. A Bayesian clustering method based on Fourier coefficients allowed us to discriminate putative regulatory genes.Among rhythmic FLC-responsive genes, transcripts of the transcription factor LUX ARRHYTHMO (LUX) correlated in peak abundance with the circadian period in flc mutants. Mathematical modeling indicated that the modest change in peak LUX RNA abundance was sufficient to cause the period change due to FLC, providing a molecular target for the crosstalk between flowering time pathways and circadian regulation.
Identify downstream targets of FLCregulation in themolecular mechanism of the circadian clock using genome-wide analysis
NIL46 was produced by genotypic selection from a backcross of NIL187 (donated by M. Koornneef) to Ler. The fri; flc, FRI; FLC, fri; FLC, and FRI; flc genotypic combinations used for leaf movement and RNA time courses are in the Col background with combinations of either wildtype Col or flc-3 and FRI-SF2 alleles (Michaels and Amasino, 1999, 2001). The ld-1 and ld-1; flc-3 mutants have been described (Michaels and Amasino, 2001), as have the fld-3mutants (He et al., 2003). Wild-type Col-0 seedlings were used for the microarray circadian time-course experiment. Unless otherwise stated, seedlings were sterilized and grown as described (Edwards et al., 2005).
RT-PCR (Plant RNeasy kit and RNasefree DNase (Qiagen))
Literature data from: '' by: .
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,
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.
Growth on Agar 1.5% MS 1 3% Sucrose for 8.0 days (Growth1). "Eight-day-old Columbia (Col-0) seedlings grown under 12-h-light/12-h-dark cycles (LD 12:12)"
| Type | Duration (days) | Cycle (h) | Start | Duration | Spectrum | Source | Intensity |
|---|---|---|---|---|---|---|---|
| diurnal light | 8 | 24 | 0:00 | 12:00 | white | LED | 40 |
| Type | Duration (days) | Cycle (h) | Base (°C) | Warm (°C) | Warm start | Warm duration |
|---|---|---|---|---|---|---|
| constant temperature | 8 | 24 | 27 | -- | -- | -- |
Growth on Agar 1.5% MS 1 3% Sucrose for 2.0 days (Exp1). 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.
| Type | Duration (days) | Cycle (h) | Start | Duration | Spectrum | Source | Intensity |
|---|---|---|---|---|---|---|---|
| constant light | 2 | 24 | 0:00 | 24:00 | white | LED | 40 |
| Type | Duration (days) | Cycle (h) | Base (°C) | Warm (°C) | Warm start | Warm duration |
|---|---|---|---|---|---|---|
| constant temperature | 2 | 24 | 22 | -- | -- | -- |