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Biological material and growth conditions
Arabidopsis thaliana seeds were surface sterilized for 20 min in 20% (v/v) commercial bleach and 0.1% Triton X-100, rinsed four times with sterile, distilled water, and chilled for 2 days at 4°C. Sterile seeds were placed in square Petri plates on medium containing nutrient salts, 8 g/L of agar, and 10 g/L of Sucrose (Lincoln et al., 1990). All plants were ecotype Columbia unless stated otherwise. Petri plates were placed vertically and the seedlings were grown at 22 °C in darkness or in continuous light for 7 days. In experiments with the photoreceptor mutants, the seeds were given 16 h of white light to ensure germination before placement in the experimental light conditions. Seeds of the mutants analyzed in this study were obtained from in-house stocks or from the Arabidopsis Biological Resource Center (Ohio State University, Columbus).
Light Sources
White light at up to 150 µmol.m-2.s-1 was obtained with mixed cool-white and warm-fluorescent light bulbs. Blue light up to 30 µmol.m-2.s-1 was obtained by filtering light from a bank of cool-white fluorescent bulbs through blue plexiglass (no. 2424, Rohm and >Hass, Darmstadt, Germany). Red and far-red light up to 30 µmol.m-2.s-1 was provided by a solid-state light-emitting diode system (Qbeam 220, Quantum Devices, Barnevel, WI). Neutral- density screens were used to vary the fluence rates. Fluence rates of white, blue, and red light were measured with a quantum photometer (model LI-189, Li-Cor, Lincoln, NE) and fluence rates of far-red light were measured with a spectroradiometer (model LI-1800, Li-Cor).
Plant material and growth conditions
The Columbia (Col-0) ecotype was used as the wild-type control. The mutants used in the study described are: cry1-301, cry2-1 [S2], FRO6::XVE::YUC3 [S3], hy5-215hfr1 [S4], nph3-6 [S5], phot1-5, phot2-1, phot1-5phot2-1, phyA-211, phyB-9, phyA-211phyB-9 [S6], pif4pif5pif7 [S7], pin3-3pin4-101pin7-101 [S8], sav3-2 [S9], yuc2yuc5yuc8yuc9 [S10], 35S::PIF4 and 35S::PIF5 [S11]. The bri1-235 mutant contains a point mutation at the nucleotide position 467 (C to T) in the BRI1 protein coding sequence resulting in the substitution of S156F.
Seeds were surface sterilized and plated on half-strength Murashige and Skoog medium with 0.8 % (w/v) agar and kept at 4 °C in the dark for 3 days. Square plates were then transferred to continuous white light (50 μmol m−2 s−1) for 3 days at 22-22.5 °C in Percival AR-41L2 incubator to obtain de-etiolated seedlings. For outdoor experiments seedlings were grown in LD (16 h light / 8 h darkness) for 3 days in 50 μmol m-2 s-1 white light (fluorescent lamps) at 22 °C on vertical plates. Etiolated seedlings were obtained by inducing germination in white light (150 μmol m−2 s−1) for 6 hours after 3 days of cold and dark treatment and subsequently shifting plates to dark for 64 hours at 22 °C.
Indoor unilateral light phototropism and white light gradient experiments were performed in Percival I- 33NL and Percival SE-41L incubators, respectively. The LED light sources were from CLF Plant Climatics GmbH: blue (λmax, 462 nm), red (λmax, 664 nm) and far-red (λmax, 730 nm). Light intensities were determined with an International Light IL1400A photometer (Newburyport, MA) equipped with an SEL033 probe with appropriate light filters or with an Ocean Optics (Dunedin, FL, USA) USB2000+ spectrometer. In the field, the vertically and horizontally incident radiation (R/FR ratio, R, FR and blue light) were measured for each experiment with the SKR 1850 four-channel sensor probe of a Skye Instruments SKL 904/I SpectroSense2 meter, respectively facing upwards or towards north and south. For a more detailed characterization we produced scans with an Ocean Optics USB4000-UV-VIS spectrometer configured with a DET4-200-850 detector and QP600-2-SR optical fiber in one of the experiments (Figures S4D, S4E).