Led chamber

The Arabidopsis mutants phyB-9^{14 (link)}, phyA-211^{68 (link)}, phyA-211/phyB-9^{68 (link)}, phyABCDE^{11 (link)}, YHB^{36 (link)}, and pifq^{24 (link)} in the Columbia (Col-0) background were used for the characterization of plastidial gene expression and the assembly of the PEP complex. The PBG and YHB lines in the Landsberg erecta (Ler) background have been previously reported^{17 (link),36 (link)}. Additional Arabidopsis transgenic lines and other mutants generated in this study are described below. Seeds were surface-sterilized and plated on half-strength Murashige and Skoog (MS) media with Gamborg’s vitamins (MSP0506, Caisson Laboratories, North Logan, UT), 0.5 mM MES pH 5.7, and 0.8% agar (w/v) (A038, Caisson Laboratories, North Logan, UT)^{62 (link)}. Seeds were stratified in the dark at 4 °C for 4 days before being placed in an LED chamber (Percival Scientific, Perry, IA) in the indicated light conditions. The fluence rate of light was determined using an Apogee PS200 spectroradiometer (Apogee Instruments Inc., Logan, UT) and SpectraWiz Software (StellarNet, Tampa, FL). Representative seedlings in the indicated light conditions were imaged using a Leica MZ FLIII stereo microscope (Leica Microsystems Inc., Buffalo Grove, IL) and the images were processed using Adobe Photoshop CC (Adobe Systems, Mountain View, CA).

Spores of the strains used for photoinduction assay were plated in PDA and incubated at 28°C during 48 h. For gene expression analysis plugs of mycelia (0.5 cm diameter) were obtained from the colony edges and placed on the center of PDA plates with cellophane overlay and incubated during 36 h in the dark before light exposure. Colonies were subjected to a 5 min pulse of blue light (450 nm; 1,200 µmol m⁻²) in a light emitting diode (LED) chamber (Percival) at 28°C and plates taken back to darkness. Samples of mycelium were collected at 15, 30, 60, and 120 min, frozen immediately in liquid nitrogen, and stored until used for RNA extraction. Gene expression was also analyzed upon continuous exposure of the colonies for 5, 15, and 30 min to blue light (3.6 µmol m⁻² s⁻¹), and samples collected after exposure to light, frozen in liquid nitrogen, and stored for later analysis. All manipulations of the mycelium were carried out in a dark room using red light of security.

The PBG^{36 (link)}, PBC^{55 (link)}, YHB^{53 (link)} and BCY^{55 (link)} lines of Arabidopsis were previously described. Seeds were surface sterilized^{65 (link)} and plated on half-strength Murashige and Skoog media with Gamborg’s Vitamins (MSP0506, Caisson Laboratories, North Logan, UT), 0.5 mM MES (pH 5.7), and 0.8% (w/v) agar (A038, Caisson Laboratories, North Logan, UT). Seeds were stratified in the dark at 4 °C for 5 days before treatment of specific light and temperature in an LED chamber (Percival Scientific, Perry, IA). For temperature experiments, seedlings were grown first in 21 °C for 2 days (48 h), and then either kept at 21 °C or transferred to 12, 16, or 27 °C for 2 additional days (48 h) before characterization. The light condition was maintained constant at 10 or 50 μmol m⁻² s⁻¹ R light for the entire 4 days. For the shade condition, seedlings were grown in a mixture of 10 μmol m⁻² s⁻¹ R and 10 μmol m⁻² s⁻¹ FR light at 21 °C for 4 days. For dark treatment, BCY and YHB seeds were exposed to 10 μmol m⁻² s⁻¹ FR light for 3 h immediately after stratification to deactivate the remaining phys and induce germination^{65 (link)}, and then kept in the dark at 21 °C for 45 h before a treatment of an indicated temperature for 2 additional days. Fluence rates of light were measured using an Apogee PS200 spectroradiometer (Apogee Instruments Inc., Logan, UT).