time point: Day 70 daylight: Short day tissue: Lignified stems developmental stage: Plants were grown for the SD time course experiment. Lignified stems were harvested 70 days after the photoperiod was switched from LD to SD.
Growth protocol
Seedlings were received from CFS, Quebec. Trees are surplus individuals from starting association population being created by N. Isabel, LFC-CFS. The two-years-old plants were grown using a complete randomized block design in controlled-environment chambers at 20°C and 50-60% humidity, with full illumination for 16 hours and 8 hours of dark. At 6 to 8 weeks (46-58 days), part of the plant population was switched from long daylight photoperiod (16h) to short daylight photoperiod with 8 hours of light and 16 hours of dark for 18 to 25 weeks. A subset was subsequently transferred to low temperature at 2°C to 4°C for 3 to 4 weeks under short daylight photoperiod. During the long daylight photoperiod, plants were fertilized with 0.5 g/L of 20-20-20. After switching to short daylight photoperiod, 1.0 g/L of 15-30-15 fertilizations were applied for the first two weeks, 0.5 g/L of 20-20-20 from week 3 to week 8, and 0.5 g/L of 8-20-30 after week 8.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted according to a modified procedure of Chang et al. 1993, a simple and efficient method for isolating RNA from pine trees [Plant Molecular Biology Reporter, 11(2):113-116]. Ground tissue was treated with 750 µL CTAB + b-ME extraction buffer, and incubated in water bath at 65°C for 10 minutes, vortexed periodically. Each tube received 500 µl of chloroform:isoamyl alcohol (24:1 v/v), vortexed vigorously, and centrifuged at 14000 rpm for 15 minutes. The aqueous phase was transferred to new tubes, and purified with chloroform:isoamyl alcohol again. The volume of aqueous phase recovered after the second purification was determined, and mixed with an equal volume of LiCl-EDTA (7.5 M LiCl, 50mM EDTA) gently by inverting the tubes several times. These tubes were placed at -20°C freezer for one hour, and centrifuged for 15 minutes at 14000 rpm at 4ºC. Pellet from each tube was collected, and washed with 800 µL of 80% ethanol, followed by a brief vortex. Another 5 minutes of centrifugation at 14000 rpm at room temperature was applied to separate out the ethanol. After drying the pellet by removing the supernatant, the pellet was resuspended in sterile water. NanoDrop 1000 (Thermo Scientific, ON, Canada) and 2100 Bioanalyzer (Agilent, ON, Canada) were used to determine the relative quality and quantity of the extracted RNA.
Label
Alexa647, Alexa555
Label protocol
Two micrograms of total RNA was amplified with the amino allyl antisense RNA (aRNA) procedure (Superscript Indirect RNA Amplification System, Invitrogen). Five micrograms of aRNA was directly labeled using Alexa Fluor® 555 and/or 647 Dyes, (Invitrogen). Alexa fluor 647 conjugates virtually match those of the Cy5 dye, and spectra of the Alexa fluor 555 conjugates virtually match those of the Cy3 dye. Coupling efficiency was evaluated using the NanoDrop 1000 (Thermo Scientific).
time point: Day 0 daylight: Short day tissue: Lignified stems developmental stage: Plants were grown for the SD time course experiment. Lignified stems were harvested right after switching the photoperiod from LD to SD.
Growth protocol
Seedlings were received from CFS, Quebec. Trees are surplus individuals from starting association population being created by N. Isabel, LFC-CFS. The two-years-old plants were grown using a complete randomized block design in controlled-environment chambers at 20°C and 50-60% humidity, with full illumination for 16 hours and 8 hours of dark. At 6 to 8 weeks (46-58 days), part of the plant population was switched from long daylight photoperiod (16h) to short daylight photoperiod with 8 hours of light and 16 hours of dark for 18 to 25 weeks. A subset was subsequently transferred to low temperature at 2°C to 4°C for 3 to 4 weeks under short daylight photoperiod. During the long daylight photoperiod, plants were fertilized with 0.5 g/L of 20-20-20. After switching to short daylight photoperiod, 1.0 g/L of 15-30-15 fertilizations were applied for the first two weeks, 0.5 g/L of 20-20-20 from week 3 to week 8, and 0.5 g/L of 8-20-30 after week 8.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted according to a modified procedure of Chang et al. 1993, a simple and efficient method for isolating RNA from pine trees [Plant Molecular Biology Reporter, 11(2):113-116]. Ground tissue was treated with 750 µL CTAB + b-ME extraction buffer, and incubated in water bath at 65°C for 10 minutes, vortexed periodically. Each tube received 500 µl of chloroform:isoamyl alcohol (24:1 v/v), vortexed vigorously, and centrifuged at 14000 rpm for 15 minutes. The aqueous phase was transferred to new tubes, and purified with chloroform:isoamyl alcohol again. The volume of aqueous phase recovered after the second purification was determined, and mixed with an equal volume of LiCl-EDTA (7.5 M LiCl, 50mM EDTA) gently by inverting the tubes several times. These tubes were placed at -20°C freezer for one hour, and centrifuged for 15 minutes at 14000 rpm at 4ºC. Pellet from each tube was collected, and washed with 800 µL of 80% ethanol, followed by a brief vortex. Another 5 minutes of centrifugation at 14000 rpm at room temperature was applied to separate out the ethanol. After drying the pellet by removing the supernatant, the pellet was resuspended in sterile water. NanoDrop 1000 (Thermo Scientific, ON, Canada) and 2100 Bioanalyzer (Agilent, ON, Canada) were used to determine the relative quality and quantity of the extracted RNA.
Label
Alexa555, Alexa647
Label protocol
Two micrograms of total RNA was amplified with the amino allyl antisense RNA (aRNA) procedure (Superscript Indirect RNA Amplification System, Invitrogen). Five micrograms of aRNA was directly labeled using Alexa Fluor® 555 and/or 647 Dyes, (Invitrogen). Alexa fluor 647 conjugates virtually match those of the Cy5 dye, and spectra of the Alexa fluor 555 conjugates virtually match those of the Cy3 dye. Coupling efficiency was evaluated using the NanoDrop 1000 (Thermo Scientific).
Hybridization protocol
Microarray slides were incubated for 2 hours at 65°C in pre-hybridization buffer (5 × SSC, 0.1% sodium dodecyl sulphate (SDS), 0.2 mg ml−1 bovine serum albumin (BSA), and 0.1 mg ml−1 herring sperm DNA) to prevent nonspecific binding. Slides were washed twice with 0.1 × SSC, and twice with water at room temperature, and were then transferred to a water bath at 85°C for 90 seconds before drying by centrifugation for 2 minutes at 480 g in a bench top centrifuge (Eppendorf 5804). Alexa Fluor® 555 and Alexa Fluor® 647 labeled aRNA were denatured at 95°C for 2 minutes, cooled on ice and centrifuged. 51 µl of hybridization buffer (50% formamide, 5 × SSC, 0.1% SDS and 0.1 mg ml−1 herring sperm DNA) was preheated to 46°C, and added to the labeled aRNAs (referred as aRNA probes). The aRNA probes were kept at 46°C until applying to each slide. Cover-slip (Erie Scientific Company, Portsmouth, NH, USA) was placed on each slide over the subarrays, and aRNA probes were added to one edge of the cover-slip, which allowed the probe to wick underneath it. The slides were put into the hybridization chambers (Corning, New York, NY, USA), with a drop of water being added to dedicated wells to maintain humidity, and incubated for 18 hours in a hybridization oven at 45°C. Cover-slips were then removed by floating the slides in a Coplin jar (Bel-Art Products Inc, Pequannock, NJ, USA) containing 2 × SSC and 0.5% SDS preheated to 45°C. The slides were incubated in the hybridization oven at 45°C for three periods of 15 minutes, with shaking every few minutes: once in 2 × SSC, 0.5% SDS, and twice in 0.5 × SSC, 0.1% SDS. The slides were then washed at room temperature twice with 0.1 × SSC for 1 minute and twice with water for 20 seconds before drying by centrifugation for 2 minutes at 480 g in Eppendorf 5804.
Scan protocol
Microarray images were obtained using a GenePix 4000B microarray scanner and analyzed with Genepix Pro 6.0 (Axon Instruments). Adaptive circles were used to indicate spots (spot diameter resize feature, 80 to 150%; composite pixel intensity, 300). After manual elimination of bad spots (highly irregular spot shape, dust, or salt particles), the median background intensity was subtracted from the mean spot intensity applying the local method (default option).
Description
4 biological replicates were used, and raw data file 'HA013-017.gpr' and 'HA013-015.gpr' were dye-swap [i.e. ch1 labeled with Alexa555 and ch2 with Alexa647].
Data processing
Within-array data normalization was first applied using print-tip loess detrending procedure, followed by between-array scale normalization to enforce the data having the same median-absolute-deviation across arrays from the same experiment. Taking the normalized data, linear models were fitted using duplicate correlation for each sequence. To reduce the false discovery rate, a nonspecific filtering was applied by removing invalid and low intensity sequences prior statistical analysis. A moderated t-statistic, empirical Bayes statistic, was used to obtain p-values, and these p-values were further adjusted by Benjamini-Hochberg procedure. A list of statistically significant differentially expressed (DE) sequences was selected by an adjusted p-value cut-off at 0.01, and this list was further filtered to include sequences with fold change greater than 1.5 or smaller than 0.67. The VALUEs in the data table represent the averaged values of 4 biological replicates.
