Fig. 4.

PCH1 and PCHL contribute to molecular signal integration. a, b PCH1 protein accumulates in far-red and blue light. a Four-day-old etiolated pch1 seedlings expressing HA-YFP-PCH1 from its native promoter were either kept in darkness (D), or exposed to far-red (FR, 25 μmol m⁻² s⁻¹), blue light (B, 25 μmol m⁻² s⁻¹), or red light (R, 25 μmol m⁻² s⁻¹) for 8 h and harvested. Etiolated HA-YFP-PCH1 over-expressing seedlings (PCH1ox) were used for comparison. Total protein was extracted and used for immunoblotting; α-HA and α-actin antibodies were used for detection of HA-YFP-PCH1 and actin (loading control). b pch1 seedlings expressing HA-YFP-PCH1 from its native promoter were grown as described in a and used for fluorescence microscopy. Scale bar = 5 µm. c, d Regulation of PCH1 and PCHL expression by red and far-red light requires phyA. Four-day-old dark-grown wild type (Col-0) and mutant seedlings were either kept in darkness (D) or exposed to red (R, 7.5 μmol m⁻² s⁻¹) or far-red light (FR, 6.7 μmol m⁻² s⁻¹) for 1 h. Total RNA was extracted and qRT-PCR was performed using probes specific for either PCH1 (c) or PCHL (d). ACT1 was used as internal control and expression of PCH1 and PCHL is shown relative to expression of ACT1. Data are means of three biological replicates; error bars indicate ± s.d. Biological replicates are shown in Supplementary Fig. 11; data in c and d correspond to Supplementary Fig. 11d and h. e, f PCH1 and PCHL are required for phyB responsiveness amplification by far-red and blue light. Dark-grown wild type (Col-0) and mutant seedlings were pre-treated with either far-red (FR, 25 μmol m⁻² s⁻¹) (e) or blue light (B, 25 μmol m⁻² s⁻¹) (f) and given a red light pulse (Rp, 5 min, 50 μmol m⁻² s⁻¹), either followed by a long-wavelength far-red light pulse (FRp, 776 nm, 5 min, 50 μmol m⁻² s⁻¹) or darkness; these light pulses were repeated after 24 h. Mean hypocotyl length relative to dark-grown seedlings is shown. Error bars indicate ± s.e.m.; n ≥ 20. See Supplementary Fig. 13 for seedling photographs and detailed explanation of light treatments