Experimental data was collected by ROBuST member Peter Gould at the University of Liverpool. Sue Bird and Dana MacGregor performed the construction of promoters fused with the Luciferase gene at the University of York, whilst Peter Gould and Jack Young performed the inclusion into agrobacterium and the dipping of plants at the University of Liverpool.
2.2.1
– Seed Stock
Mutants used in the delayed fluorescence screen (Chapter 3) were obtained through the NASC database. Collaborators at York University developed constructs used for luciferase screens (Chapters 4 and 5). Additionally, members of the ROBuST consortium working at the University of Edinburgh crossed luciferase markers into circadian mutants. These were created as heterozygous lines and were homogenised using multiple self-crossing performed by technicians at the University of Liverpool.
2.2.2
– Seed Sterilisation
All seed underwent gas sterilisation in a fume hood prior to cold treatment. This was achieved by placing the open Eppendorf tubes containing the loose seed into a large desiccation jar. Five hundred ml of reverse osmosis water was used to dissolve two chlorine tablets (CLO-TABS, Arrow Solutions). This was atomised using 5ml of hydrochloric acid and the jar sealed. This was then left for 3 hours before seeds were removed from the desiccation jar and moved to a sterile flow hood for an additional 30 minutes to remove remaining chlorine traces. 0.15% agar was then added to the tubes and seed suspended within it (modified from (Desfeux et al. 2000)).
Materials and Methods
2.2.3
– Plant Growth Conditions
Seed was moved to a constant dark 4˚C room for 3 days to promote stratification prior to planting. Three hundred μL of Murashige and Skoog media (MS) was pipetted into 96 well microtitre plates. Around 8-12 seeds were placed into each well before a second, empty microtitre plate was placed inverted on top. The two plates were then taped together with microporous paper tape and placed in a 22˚C room grown under 12:12 light:dark cycles of 80 μmol/m2/s1 in a Sanyo MLR350 plant growth chamber. These plates were left
for 14 days for luciferase screens and 21 days for a delayed fluorescence screen.
2.2.4
– Circadian Screens
Circadian effects of genes were monitored using two different screens. When a LUC line existed, a luciferase screen was used to monitor the expression pattern of the gene (Southern et al. 2006). For mutants ordered from NASC, which had no inherent luciferase markers, a delayed fluorescence screen was used. Both screens were completed using the same apparatus (see Imaging System below). Similarly the raw output data was initially processed using the same methodology (see Image Analysis below).
2.2.4.1 – Imaging System
Circadian screens were carried out in Sanyo MIR-553 incubators (Sanyo Gallenkamp, UK) set to the desired experimental temperature. Image acquisition was done using a top mounted ORCA-II-BT 1024 16-bit camera (Hamamatsu Photonics, Japan) cooled to -80˚C (Southern et al., 2006). This setup allowed for the simultaneous imaging of six 96-well microtitre plates per cabinet. Lighting inside the cabinets (when needed) was supplied by red/blue light emitting diode (LED) arrays (MD Electronics, UK). These arrays were
Materials and Methods
controlled using WASABI imaging software (Hamamatsu Photonics, Japan) that also captured images from the cameras. Light intensity was set to a total of 40 μmol/m2/s, either split 20/20 for red/blue light or all 40 μmol/m2/s coming
from a single light source, depending on the light conditions desired (Gould et al. 2006).
2.2.4.2 – Luciferase Screening
A single column of 8 wells in a microtitre plate was assigned to each transgenic plant being screened. This allowed biological repeats of up to 12 genes to be processed per microtitre plate. These plates were grown for 14 days in 12:12 LD conditions at 22˚C. 5 mM D-luciferin (dissolved in 0.01% Triton X-100) was applied using a fine spray on the 13th day in a sterile flow hood. Plates were
moved to the imaging chamber at dawn on the 15th day after coming out of the
cold room, and images were taken every 2 hours for the next 7 days. During the first two days the plants experienced standard 12:12 LD cycles, before then entering 5 days of constant light (LL). Images were taken by turning off the lights, waiting 5 minutes to remove plant auto fluorescence, and then capturing light emission for 20 minutes. This led to a plant experiencing 1 hour 35 minutes of light every 2 hours during light conditions. Cabinets were set to remain at a constant temperature of either 12, 17, 22 or 27˚C.
2.2.4.3 – Delayed Fluorescence Screening
Microtitre plates were set up in the same way as luciferase screens (2.2.4.2) but were left to grow for 21 days before being moved to imaging chambers. Additionally, each plate had a wild type control on it. Plants were left in constant red/blue light conditions from the start, with images being taken every hour. Image capture occurred by turning off the lights and immediately recording photon emission for 1 minute before lights were turned back on (Gould et al. 2009). This was done using cabinets set to maintain a temperature of 12, 17 and 27˚C.
Materials and Methods
2.2.4.4 – Image Analysis
The RBF images produced during the screens were reloaded into WASABI and converted into TIFF files. These were then imported into Metamorph 6.0 image analysis software (Molecular Devices). Regions relating to each well were highlighted, as well as four additional background regions. The colour intensity of these regions were then measured for each time and outputted to an excel file. These files were then uploaded to Biodare (Moore et al. 2013) where they were background corrected, detrended, and normalised.
Regulation of the Arabidopsis Transcriptome by Temperature