PCH1 and PCHL promote photomorphogenesis in plants by controlling phytochrome B dark reversion

Abstract

Phytochrome B (phyB) is the primary red light photoreceptor in plants, and regulates both growth and development. The relative levels of phyB in the active state are determined by the light conditions, such as direct sunlight or shade, but are also affected by light-independent dark reversion. Dark reversion is a temperature-dependent thermal relaxation process, by which phyB reverts from the active to the inactive state. Here, we show that the homologous phyB-binding proteins PCH1 and PCHL suppress phyB dark reversion, resulting in plants with dramatically enhanced light sensitivity. Moreover, far-red and blue light upregulate the expression of PCH1 and PCHL in a phyB independent manner, thereby increasing the response to red light perceived by phyB. PCH1 and PCHL therefore provide a node for the molecular integration of different light qualities by regulation of phyB dark reversion, allowing plants to adapt growth and development to the ambient environment.

Fig. 1

Light-activated phyB interacts with PCH1 and PCHL. a, b Y2H detection of interaction of phyB with PCH1 and PCHL. The phyB-GAL4-activation domain (phyB-AD) fusion was co-expressed with GAL4-DNA-binding domain (BD-) fusions of PCH1 or PCHL. a X-Gal filter-lift assay. Yeast cells were lifted from chromophore-supplemented plates pre-incubated for 48 h in either constant darkness (D), red (R), or far-red light (FR). b ONPG assay. Yeast cultures supplemented with chromophore were exposed to red light, to convert phyB to Pfr, or to far-red light for phyB Pr. n = 3; error bars indicate ± s.e.m. c, d PCH1 and PCHL co-purify with phyB from native plant extracts. Four-day-old dark-grown seedlings over-expressing HA-YFP-PCH1 (PCH1ox) c or HA-YFP-PCHL (PCHLox) d in the Col-0 wild type background were either treated with red light (R, 7 μmol m⁻² s⁻¹) for 10 min or kept in darkness (D). Native total protein extracts were prepared and used for co-immunoprecipitation (Co-IP) assays. IP was performed using α-GFP antibody. α-phyB and α-GFP antibodies were used to detect endogenous phyB and YFP-tagged PCH1 and PCHL, respectively

Fig. 2

PCH1 and PCHL stabilise phyB in the active state in planta. a, b PCH1ox and PCHLox seedlings respond to red light pulse treatments (Rp). Wild type (Col-0) and mutant seedlings expressing either HA-YFP-PCH1 (PCH1ox) (a) or HA-YFP-PCHL (PCHLox) (b) were grown for 4 days in darkness on filter paper soaked with water. The seedlings were either treated with a single red light pulse (Rp, 5 min, 50 μmol m⁻² s⁻¹) per day, or a Rp followed by a long-wavelength FR pulse (FRp, 776 nm, 5 min, 50 μmol m⁻² s⁻¹) (Rp → FRp). Control seedlings were kept in darkness (D). See Supplementary Fig. 4 for quantification of hypocotyl lengths and experiments with seedlings grown on 0.5× MS medium. c High levels of active phyB are maintained during the dark phase in PCH1ox and PCHLox seedlings. Wild type (Col-0), PCH1ox, PCHLox, and phyB-9 seedlings were grown for 4 days in 8 h red (R, 50 μmol m⁻² s⁻¹)/16 h dark (D) cycles and given a long-wavelength far-red light pulse (FRp, 776 nm, 5 min, 50 μmol m⁻² s⁻¹) at time points after lights-off. Control seedlings were kept in darkness (D). d The end-of-day far-red (EOD-FR) response requires PCH1 and PCHL. Wild type (Col-0) and mutant seedlings were grown as in c, except either constant red light (Rc), an immediate far-red light pulse (8 h R → FRp → 16 h D), or no far-red light pulse (8 h R → 16 h D) were used. c, d Mean hypocotyl length relative to dark-grown seedlings is shown. Error bars indicate ± s.e.m.; n ≥ 20. e, f Subnuclear localisation of PCH1 and phyB was analysed by fluorescence microscopy. Scale bar = 5 µm. e PCH1 stabilises phyB photobodies. Four-day-old etiolated seedlings expressing phyB-mCer in phyB-9 or HA-YFP-PCH1 (PCH1ox) backgrounds were exposed to red light (R, 10 μmol m⁻² s⁻¹) for 8 h, followed either by 0 or 14 h incubation in darkness (D). Data for phyB-mCer single transgenic seedlings are duplicated in Supplementary Fig. 7a. f PhyB photobodies are highly unstable in the pch1 pchl mutant. Four-day-old etiolated seedlings expressing phyB-GFP in wild type or pch1 pchl backgrounds were exposed to red light (R, 50 μmol m⁻² s⁻¹) for 12 h followed by incubation in darkness (D)

Fig. 3

PCH1 and PCHL inhibit phyB dark reversion. Dual-wavelength ratiospectrophotometer quantification of the relative abundance of phyB in the active state (Pfr/Ptot) (a, c) and total phyB (Ptot) (b, d) in planta. a, b PhyB dark reversion is accelerated in the absence of PCH1 and PCHL. Four-day-old etiolated seedlings expressing phyB-GFP in PCH1/PCHL wild type (WT; phyA-211 phyB-9 phyB-GFP) or the mutant background (pch1 pchl; phyA-211 pch1 pchl phyB-GFP) were exposed to red light (R, 10 μmol m⁻² s⁻¹) for 3 h followed by incubation in darkness (D) for indicated times. c, d PhyB dark reversion is reduced in PCH1ox and PCHLox seedlings. Four-day-old etiolated seedlings expressing c-Myc-mCherry-PCH1 (PCH1ox) or c-Myc-mCherry-PCHL (PCHLox) in the phyB-GFP background were pre-irradiated with red light (R, 20 μmol m⁻² s⁻¹) for either 20 min (black lines and symbols) or 3 h (grey lines and symbols) followed by incubation in darkness (D). a–d Data are means of ≥ 4 biological replicates; error bars indicate ±s.e.m.

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

Fig. 5

Model for PCH1-dependent and PCHL-dependent regulation of phyB responses. PCH1 and PCHL reduce phyB dark reversion. Blue and far-red light perceived by phyA induce expression of PCH1 and PCHL, and thereby allow seedlings to respond to red light pulses (phyB responsiveness amplification by far-red and blue light). We speculate that also other signalling pathways could control the activity of phyB through regulation of PCH1 and PCHL, which might play an important role in integration of phyB-dependent light signalling with other signalling pathways