| Property | Value |
|---|---|
| BioDare ID | 13395774721132 |
| Author | Benedicte Wenden |
| Institution | University of Edinburgh |
| License | CC_BY |
Literature data from: 'Light inputs shape the Arabidopsis circadian system' by: Benedicte Wenden.
Experiments performed on phyA and Col-0, 22 March to 26 Aug 2009, and on elf4, 8 March through November 2010. Published in Wenden et al. Plant Journal 2011. Curated by Andrew Millar. Comparable LUC data from this paper are also available in BioDare.
Testing clock gene dynamics in FR-grown plants under FR-D cycles, cFR and DD. Col-0 is compared to the phyA-211 mutant (phyA was the candidate photoreceptor) in this study, and to the elf4-101 mutant (ELF4 was a candidate target of the photoreceptor input) in a separate upload.
qPCR data represent biological duplicates. The data uploaded are means, and the data from each replicate is attached in a separate XL file.
Note that the same WT data form the control for both phyA and elf4 mutants, but they were recalibrated for ease of plotting so the values from the two mutants cannot be directly compared. The two comparisons are uploaded as separate experiments.
Approximately 75–100 seedlings were harvested in RNAlater (Sigma, http://www.sigmaaldrich.com/) and total RNA was extracted using a Plant RNeasy kit (Qiagen, http://www.qiagen.com/) with on-column DNase digestion. The cDNA samples for real-time PCR applications were reverse transcribed from 1 lg of RNA using the SuperScript VILO cDNA Synthesis kit (Invitrogen, http:// www.invitrogen.com/), and the cDNA product was diluted 1:10 in RNase-free water.
RT-PCR (Roche LightCycler 480)
The qPCR was set up with a liquid handling robot (TECAN freedom Evo, http://www.tecan.com/) and cDNAs for each sample were quantified in triplicate using the LightCycler 480 Real- Time PCR System (Roche, http//http://www.roche.com/). Primers used are described in the Supporting Information.
Growth on Agar 2% MS 1 3% Sucrose for 4.0 days (FR-D cycles). The seeds were stratified at 4°C for 96h in darkness. 3h of 80 μmol m-2 s-1 of fluorescent white light at 22°C were applied to allow germination. After 21h of darkness at 22°C, seeds were entrained under (12 hr/12 hr) light/dark cycles of 2 μmol m−2 sec−1 FR (Figure 8 of the TPJ paper). In Figure 8, the FR LED source was filtered with two layers of Italian Blue gel (Roscolux; Roscolab Ltd, Sydenham UK), resulting in a λmax of 760nm.
Half the samples were entrained to inverted LD cycles, so that harvesting of day and night samples fell within a 12h interval on each of the six days of sampling.
| Type | Duration (days) | Cycle (h) | Start | Duration | Spectrum | Source | Intensity |
|---|---|---|---|---|---|---|---|
| diurnal light | 4 | 24 | 0:00 | 12:00 | far red | LED | 2 |
| Type | Duration (days) | Cycle (h) | Base (°C) | Warm (°C) | Warm start | Warm duration |
|---|---|---|---|---|---|---|
| constant temperature | 4 | 24 | 22 | -- | -- | -- |
Growth on Agar 2% MS 1 3% Sucrose for 6.0 days (1 x FR-D - 3 x cFR - 2 x DD). Half the samples were entrained to inverted LD cycles, so that harvesting of day and night samples fell within a 12h interval on each of the six days of sampling.
| Type | Duration (days) | Cycle (h) | Start | Duration | Spectrum | Source | Intensity |
|---|---|---|---|---|---|---|---|
| diurnal light | 1 | 24 | 0:00 | 12:00 | far red | LED | 2 |
| constant light | 3 | 24 | 0:00 | 24:00 | far red | LED | 2 |
| constant dark | 2 | 24 | -- | -- | far red | LED | 2 |
| Type | Duration (days) | Cycle (h) | Base (°C) | Warm (°C) | Warm start | Warm duration |
|---|---|---|---|---|---|---|
| constant temperature | 6 | 24 | 22 | -- | -- | -- |