Arabidopsis seeds were sterilized and sown on 1/2 X Murashige and Skoog-agar plates (hereafter, referred to as MS-agar plates). The sterilized seeds were incubated at 4 °C for 3 days for cold stratification. They were then allowed to germinate and grow in a temperature-controlled culture room set at 22 °C with relative humidity of 55% under short days (8-h light and 16-h dark). White light illumination (120 μmol photons m−2 s−1) was provided by fluorescent FLR40D/A tubes (Osram, Seoul, Korea). The seedlings were then transferred to various temperature conditions. Seven-day-old seedlings were photographed, and hypocotyl lengths were measured using the ImageJ software40 (link). Fifteen plants were analyzed for each measurement, unless otherwise specified.
Flr40d a tubes
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The FLR40D/A tubes are a type of fluorescent lighting products manufactured by Osram. They are designed to provide illumination for various applications. The key function of these tubes is to generate light through the process of fluorescence when an electric current is passed through them.
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7 protocols using flr40d a tubes
1
Arabidopsis Hypocotyl Length Measurement
2
Genetic regulation of Arabidopsis growth and development
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All Arabidopsis thaliana lines used were in the Col-0 background. Arabidopsis plants were grown in a controlled culture room set at 23°C with relative humidity of 55% under LDs with white light illumination (120 μM photons m−2s−1) provided by fluorescent FLR40D/A tubes (Osram, Seoul, Korea). The idd8-3, akin10-1, and akin11-1 mutants have been described previously [20 (link),26 (link),45 (link)].
To generate 35S:MYC-IDD8 transgenic plant, a full-size IDD8 cDNA (At5g44160) was fused in-frame to the 3′ end of the MYC-coding sequence under the control of the CaMV 35S promoter in the myc-pBA vector [46 (link)]. The expression construct was transformed into Col-0 plants. To generate transgenic plants overexpressing AKIN10 and AKIN11 genes (At3g01090 and At3g29160, respectively), full-size cDNAs were subcloned under the control of the CaMV 35S promoter into the binary pB2GW7 vector [47 (link)]. Agrobacterium-mediated transformation was performed according to a modified floral-dip method [48 (link)].
For dark treatments, 10-day-old plants grown on 1/2 X Murashige and Skoog-agar plates (MS-agar plates) were covered with aluminum foil and incubated at 23°C for 2 days in complete darkness. For DCMU treatments, 10-day-old plants grown on MS-agar plates were transfer to MS liquid culture containing 50 μM DCMU for 2 days under constant light conditions.
To generate 35S:MYC-IDD8 transgenic plant, a full-size IDD8 cDNA (At5g44160) was fused in-frame to the 3′ end of the MYC-coding sequence under the control of the CaMV 35S promoter in the myc-pBA vector [46 (link)]. The expression construct was transformed into Col-0 plants. To generate transgenic plants overexpressing AKIN10 and AKIN11 genes (At3g01090 and At3g29160, respectively), full-size cDNAs were subcloned under the control of the CaMV 35S promoter into the binary pB2GW7 vector [47 (link)]. Agrobacterium-mediated transformation was performed according to a modified floral-dip method [48 (link)].
For dark treatments, 10-day-old plants grown on 1/2 X Murashige and Skoog-agar plates (MS-agar plates) were covered with aluminum foil and incubated at 23°C for 2 days in complete darkness. For DCMU treatments, 10-day-old plants grown on MS-agar plates were transfer to MS liquid culture containing 50 μM DCMU for 2 days under constant light conditions.
3
Arabidopsis Growth Conditions for RNA and Protein Analysis
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Sterilized Arabidopsis seeds were cold-stratified for 3 days and were allowed to germinate on half-strength Murashige and Skoog agar (hereafter referred to as MS-agar) plates under either LDs (16-h light and 8-h dark) or SDs (8-h light and 16-h dark) with white light mmol m À2 s À1 ) provided by fluorescent FLR40D/A tubes (Osram, Seoul, Korea) in a controlled growth chamber set at 23 C. For phenotypic assays, 3-day-old seedlings were further grown at either 23 C or 28 C under either LDs or SDs for 4 additional days. To prepare total RNA and protein samples, we transferred 5-day-old seedlings to either 23 C or 28 C for the indicated time periods before harvesting whole seedlings.
4
Brachypodium and Arabidopsis Growth Conditions
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Brachypodium distachyon ecotype Bd21-3, which is a community standard diploid inbred line, was used in all experiments. Brachypodium plants were grown in a controlled growth chamber with relative humidity of 60% under long day conditions (20-h light/4-h dark). Growth conditions were 24°C during the day and 18°C at night with white light illumination provided by FLR40D/A fluorescent tubes (150 μmol photons/m2s, Osram, Seoul, Korea).
The Arabidopsis ecotype Columbia (Col-0) was used for BdCBF gene transformation. Arabidopsis plants were grown in a controlled culture room at 22°C with relative humidity of 55% under long day conditions (16-h light/8-h dark) with white light illumination provided by fluorescent FLR40D/A tubes (120 μmol photons/m2s, Osram).
The Arabidopsis ecotype Columbia (Col-0) was used for BdCBF gene transformation. Arabidopsis plants were grown in a controlled culture room at 22°C with relative humidity of 55% under long day conditions (16-h light/8-h dark) with white light illumination provided by fluorescent FLR40D/A tubes (120 μmol photons/m2s, Osram).
5
Cultivation of Arabidopsis thaliana Ecotypes
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Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used, unless otherwise specified. The upf1-5 and upf3-1 mutants, which have been previously described [26 (link),34 (link)], were kindly provided by Dr. Jeong Sheop Shin (Korea University, Seoul, Korea) and Dr. Hee-Jeong Jeong (Kyung Hee University, Yongin, Korea). Plants were grown on ½ X Murashige & Skoog media containing 0.6% (w/v) agar (hereafter referred to as MS-agar plates) in a growth chamber set at 23°C with relative humidity of 60% under either long day conditions (LDs, 16-h light and 8-h dark) or short day conditions (SDs, 8-h light and 16-h dark) with white light illumination (120 μM photons m-2 s-1) provided by fluorescent FLR40D/A tubes (Osram, Seoul, Korea).
6
Arabidopsis Thermomorphogenesis Assay Protocol
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Sterilized Arabidopsis seeds were cold-stratified for 3 days and allowed to germinate on 1/2 × Murashige and Skoog agar (MS agar) plates under long days (LDs, 16-h light and 8-h dark) with white light illumination (120 µmol m -2 s -1 ) provided by fluorescent FLR40D/A tubes (Osram, Seoul, Korea) in a controlled growth chamber set at 23°C. For hypocotyl growth assays, 3-day-old seedlings grown on MS-agar plates at 23°C were further grown at either 23°C or 28°C under LDs for 4 additional days. To prepare total RNA and protein samples, 5-dayold seedlings grown on MS-agar plates at 23°C were transferred to either 23°C or 28°C for varying durations before harvesting whole seedlings.
To examine potential effects of light quality on SMAX1-mediated hypocotyl thermomorphogenesis, seeds were pre-exposed to white light (120 µmol m -2 s -1 ) at 23°C for 6 h to trigger seed germination on MS-agar plates. Seedlings were incubated under different light wavelengths, such as continuous illumination of red (80 µmol m -2 s -1 ), blue (50 µmol m -2 s -1 ), and far-red (20 µmol m -2 s -1 ) lights, or under darkness for 7 days either at 23°C or 28°C.
To examine potential effects of light quality on SMAX1-mediated hypocotyl thermomorphogenesis, seeds were pre-exposed to white light (120 µmol m -2 s -1 ) at 23°C for 6 h to trigger seed germination on MS-agar plates. Seedlings were incubated under different light wavelengths, such as continuous illumination of red (80 µmol m -2 s -1 ), blue (50 µmol m -2 s -1 ), and far-red (20 µmol m -2 s -1 ) lights, or under darkness for 7 days either at 23°C or 28°C.
7
Arabidopsis Growth and Transgenic Lines
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All Arabidopsis thaliana lines used were in a Columbia (Col-0) background. Arabidopsis plants were grown either in the soil or on 1/2 3 Murashige and Skoog agar (MS agar) plates in a controlled culture room at 23 C with relative humidity of 55% under LDs or 28 T cycles. Plants were illuminated with white light (120 mmol photons m À2 s À1 ) provided by fluorescent FLR40D/A tubes (Osram).
The hos1-3, phyb-9, and pif4-101 mutants and pHOS1:HOS1-MYC transgenic plants have been described previously (Jung et al., 2013) .
To generate pPIF4:PIF4-MYC transgenic plants, a PIF4 genomic fragment containing a 4.1-kbp promoter sequence upstream of the translational start site was subcloned into the modified myc-pBA vector from which the CaMV 35S promoter was deleted. To produce pphyB:phyB-MYC transgenic plants, a phyB-coding sequence was expressed driven by its endogenous promoter containing a 4.0-kbp sequence upstream of the translational start site in Col-0 plants.
The expression constructs were transformed into appropriate Arabidopsis genotypes by a modified floral dip method using Agrobacterium tumefaciens (Zhang et al., 2006) .
The hos1-3, phyb-9, and pif4-101 mutants and pHOS1:HOS1-MYC transgenic plants have been described previously (Jung et al., 2013) .
To generate pPIF4:PIF4-MYC transgenic plants, a PIF4 genomic fragment containing a 4.1-kbp promoter sequence upstream of the translational start site was subcloned into the modified myc-pBA vector from which the CaMV 35S promoter was deleted. To produce pphyB:phyB-MYC transgenic plants, a phyB-coding sequence was expressed driven by its endogenous promoter containing a 4.0-kbp sequence upstream of the translational start site in Col-0 plants.
The expression constructs were transformed into appropriate Arabidopsis genotypes by a modified floral dip method using Agrobacterium tumefaciens (Zhang et al., 2006) .
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