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

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.


Supplementary Figure 5. PCH1 and PCHL do not alter phyB protein levels. a-c
Three day-old etiolated wild type (Col-0), pch1, pchl, pch1 pchl, PCH1ox (p35S:HA-YFP-PCH1), PCHLox (p35S:HA-YFP-PCHL), and phyB-9 seedlings were exposed to red light (R, 3 µmol m -2 s -1 ) for 24 h or kept in darkness (D). Total protein was extracted and analysed by immunoblotting. α-phyB and α-actin antibodies were used to detect endogenous phyB, and actin as loading control. The molecular size marker (kDa) is shown on the left. a-c show three independent experiments with independent protein extracts. Bands have been quantified in each experiment; numbers indicate relative signal strength compared to Col-0 (shown in bold). In b and c the Col-0 protein extract was loaded twice to assess the reliability of quantification. Figure 6. Genotyping of pch1 and pchl T-DNA insertion lines. a T-DNA insertion in pch1 and pchl. Genomic DNA was isolated from wild type (Col-0), pch1 (SALK_024229), pchl (SALK_206946), and pch1 pchl seedlings. Primers specifically detecting wild type PCH1 or PCHL or the T-DNA insertion in the respective genes were used for genotyping by PCR. The ethidium bromide stained agarose gel is shown. b PCH1 and PCHL transcript levels are strongly reduced in pch1 pchl. Wild type (Col-0), pch1, pchl, and pch1 pchl seedlings were grown for 4 days in the dark on filter paper soaked with water. 24 h prior to harvesting they were transferred to far-red light (740 nm, 25 µmol m -2 s -1 ) to induce expression of PCH1 and PCHL. Total RNA was extracted and qRT-PCR was performed using probes specific for either PCH1 or PCHL. ACT1 was used as internal control and expression of PCH1 and PCHL is shown relative to expression of ACT1. Data are means of three technical replicates; error bars indicate ±s.d. c Gene models for PCH1 (At2g16365) and PCHL (At4g34550). At2g16365.1 and At2g16365.4 encode a predicted F-box domain, indicated in grey, which is not present in the isoform At2g16365.2 used in this and a previous study 3 . At4g34550 does not encode a predicted F-box domain. The sites for the T-DNA insertions in pch1 and pchl are indicated. Regions targeted for qRT-PCR analysis are indicated by a red line.

Supplementary Figure 8. Western blot quantification of phyB-GFP and endogenous phyB in lines used for in vivo measurement of Pfr.
Four day-old etiolated seedlings expressing phyB-GFP in PCH1 PCHL wild type (phyA phyB phyB-GFP) or pch1 pchl mutant background (phyA pch1 pchl phyB-GFP) were exposed to red light (R, 3 µmol m -2 s -1 ) for 24 h or kept in darkness (D). Total protein was extracted and analysed by immunoblotting. α-phyB and α-actin antibodies were used to detect endogenous phyB, and actin as loading control. Replicate 1 and 2 are experiments with independent protein extracts. Bands have been quantified for each replicate; numbers indicate relative signal strength compared to dark-grown phyA phyB phyB-GFP of the respective replicate (shown in bold).

Supplementary Figure 9. Upregulation of PCH1 and PCHL expression by monochromatic lights. a-h
Four day-old dark-grown wild type seedlings were either kept in darkness (D) or exposed to red (R, 7.5 µmol m -2 s -1 ), blue (B, 5.5 µmol m -2 s -1 ), or far-red light (FR, 6.7 µmol m -2 s -1 ) for the time indicated. Total RNA was extracted and qRT-PCR was performed using probes specific for either PCH1 or PCHL. ACT1 was used as internal control and expression of PCH1 and PCHL is shown relative to expression of ACT1. Three biological replicates are shown for PCH1 (a-c) and PCHL (e-g). Data of each biological replicate are means (±s.d.) of three technical replicates. d, h Bars show means (±s.d.) of the biological replicates. Figure 10. Spatio-temporal analysis of PCH1 and PCHL promoter activity. a Three day-old etiolated Col-0 pPCH1:GUS or Col-0 pPCHL:GUS seedlings were exposed to red (R, 25 µmol m -2 s -1 ), far-red (FR, 25 µmol m -2 s -1 ), blue (B, 25 µmol m -2 s -1 ), or white light (W, 100 µmol m -2 s -1 ) for 24 h or kept in the dark and then stained for GUS activity. Apical hook and cotyledons in dark-grown seedlings were closed when the seedlings were harvested and opened due to the staining/destaining procedure. b Col-0, Col-0 pPCH1:GUS, and Col-0 pPCHL:GUS plants were grown for 14 days under either long day (16 h W, 100 µmol m -2 s -1 /8 h D) or short day conditions (8 h W, 100 µmol m -2 s -1 /16 h D), and then stained for GUS activity. a, b Black scale bars = 5 mm; grey scale bars = 1 mm. Figure 11. Regulation of PCH1 and PCHL expression by red and far-red light requires phyA. a-h Four day-old dark-grown wild type (Col-0), phyA-211, phyB-9, and phyA-211 phyB-9 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 or PCHL. ACT1 was used as internal control and expression of PCH1 and PCHL is shown relative to expression of ACT1.  FR and B. a, b Dark-grown wild type (Col-0), pch1, pchl, pch1 pchl, phyA-211, and phyB-9 seedlings were pre-treated with either FR (25 µmol m -2 s -1 ) (a) or B (25 µmol m -2 s -1 ) (b) and given a R pulse (5 min, 50 µmol m -2 s -1 ), either followed by a FR pulse (5 min, 50 µmol m -2 s -1 ) or not; the light pulses were repeated after one day. See Fig. 4e and 4f for quantification of hypocotyl growth. A schematic drawing illustrating the light treatments is shown on the right.
For expression of PCH1 from its native promoter, a promoterless vector pPPOv3 (pDS75) was first generated. pCHF5v1 4 was cut with HindIII/XmaI, treated with Klenow fragment, and re-ligated to remove additional sequence between the terminator and Basta resistance marker. Oligos DS342/DS341/DS331/DS343 were used to amplify and remove an NheI site present within the replication origin by overlap-extension PCR, cut and ligated into flanking NdeI sites. Subsequently the CaMV 35S promoter was replaced with annealed oligonucleotides DS356/DS357 ligated into the NcoI/SalI sites, generating a new multiple cloning site. Finally DS332/DS340/DS333/DS330 were used to amplify the Butafenacil resistance marker from pPPO70v1HA 4 , removing a NcoI site, by overlap extension PCR, cut with SalI/XhoI, and cloned into the new SalI/XhoI sites to generate pPPOv3. The upstream region of the PCH1 promoter (1455 nt upstream of ATG start codon) was amplified using primers BE006/BE007, cut with NcoI/XmaI, and cloned into the NcoI/XmaI sites of pPPOv3.

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For expression of mCerulean (mCer)-tagged phyB from its native promoter we first generated a T-DNA vector containing a p35S:BamHI-XbaI-mCer:terRbcS cassette. To this end, we amplified the CDS of mCer from pCHF80 5 using the primers ah042/ah043, cut the PCR product with BamHI/NheI and ligated it into the BamHI/XbaI sites of pCHF5 9 , resulting in pCHF43. Then we cut pWCO35 10 with PvuII/PstI and ligated the fragment containing the CDS of modified PPO into pCHF43 cut with SbfI/PmlI to obtain pPPO43. pCHF43 contains a selection marker conferring resistance to Basta, while pPPO43 contains modified PPO that confers resistance to Butafenacil/Inspire 5 . To generate pPPO43-PHYB we cut phyB from pCHF40-PHYB 4 using XbaI and ligated it into pPPO43 cut with XbaI. Finally, we amplified a fragment containing the PHYB promoter (1825 nt upstream of the ATG start codon) and part of the PHYB CDS from Arabidopsis thaliana (Ler-0 ecotype) genomic DNA using the primers ah944/ah945, cut the fragment with PmeI/BamHI and ligated it into pPPO43-PHYB digested with PmeI/BamHI, resulting in pPPO43B-PHYB (pPHYB:PHYB-mCer:terRbcS).
To assess the spatio-temporal activity of the PCH1 and PCHL promoters in planta, pPCH1:GUS and pPCHL:GUS plant vectors were generated. First, we amplified the GUS CDS using primers ah1159/ah1160, cut the PCR fragment with BamHI/XbaI and ligated it into the BamHI/SpeI sites of pPPOv3. Second, we amplified promoter fragments of PCH1 (1455 nt upstream of the start ATG codon) and PCHL (1268 nt upstream of the start ATG 26/29