HOS1 Facilitates the Phytochrome B-Mediated Inhibition of PIF4 Function during Hypocotyl Growth in Arabidopsis  Ju-Heon Kim, Hyo-Jun Lee, Jae-Hoon Jung, Sangmin Lee, Chung-Mo Park  Molecular Plant  Volume 10, Issue 2, Pages 274-284 (February 2017) DOI: 10.1016/j.molp.2016.11.009 Copyright © 2017 The Author Terms and Conditions

Figure 1 HOS1 Suppresses Hypocotyl Elongation during the Light Period. Diurnal rhythms of hypocotyl growth in hos1-3 mutant were analyzed. Seedlings were grown for 4 days before measuring elongation kinetics using an infrared imaging system. Images were taken every 30 min for the measurement of hypocotyl length. Three measurements were averaged. Bars indicate standard deviation (SD) of the mean. ZT, zeitgeber time. Molecular Plant 2017 10, 274-284DOI: (10.1016/j.molp.2016.11.009) Copyright © 2017 The Author Terms and Conditions

Figure 2 HOS1 Is Functionally Linked with PIF4 in Hypocotyl Growth. (A) Yeast two-hybrid assay on HOS1-PIF4 interaction. Yeast cell growth on selective media lacking Leu, Trp, His, and Ade (-QD) represents positive interaction. The media were supplemented with 15 mM 3-amino-1,2,4-triazole. (B) BiFC assay. Partial yellow fluorescence protein (YFP) constructs were fused to HOS1 or PIF4. The fusion constructs were coexpressed transiently in Arabidopsis protoplasts. The HOS1–PIF4 interaction was visualized by fluorescence microscopy. DIC, differential interference contrast. (C) Coimmunoprecipitation assay. Two-week-old transgenic plants expressing HOS1-MYC and FLAG-PIF4 fusions driven by the endogenous HOS1 and PIF4 gene promoters, respectively, were used. The input represents 10% of the protein extracts. An anti-MYC antibody was used for immunoprecipitation (IP). An anti-FLAG antibody was used for the detection of FLAG-PIF4 proteins. (D) Hypocotyl length of hos1-3 pif4-101 double mutant. Seedlings were grown on MS agar plates for 6 days under LDs before taking photographs (upper panel). Hypocotyl lengths of 15 seedlings were averaged and statistically analyzed using Student t-test (*P < 0.01, difference from Col-0) (lower panel). Bars indicate SD. (E) Levels of YUC8 and IAA29 transcripts in hos1-3 pif4-101 double mutant. Six-day-old seedlings were used for total RNA extraction. Transcript levels were examined by RT–qPCR. Biological triplicates were averaged. Bars indicate SEM. Molecular Plant 2017 10, 274-284DOI: (10.1016/j.molp.2016.11.009) Copyright © 2017 The Author Terms and Conditions

Figure 3 HOS1 Inhibits the Transcriptional Activation Activity of PIF4. Seedlings were grown for 6 days on MS agar plates under LDs. Bars indicate SD, unless otherwise specified. (A) Diurnal patterns of PIF4 gene expression in hos1-3 mutant. Transcript levels were examined by RT–qPCR. Biological triplicates were averaged. Bars indicate SE. (B) Diurnal patterns of PIF4 accumulation in hos1-3 mutant. Transgenic seedlings expressing PIF4-MYC fusion driven by the endogenous PIF4 gene promoter in Col-0 and hos1-3 backgrounds were used. Parts of Coomassie blue-stained gels containing rubisco are displayed at the bottom (left panel). Protein levels were quantitated using the ImageJ software (right panel). Biological triplicates were averaged. (C) ChIP assay on the effects of hos1-3 mutation on DNA binding of PIF4. Transgenic seedlings described in (B) were harvested at ZT8. An anti-MYC antibody was used to immunoprecipitate PIF4-MYC fusion proteins. P1 to P4 sequence regions were assayed. White boxes indicate untranslated regions, and black boxes indicate exons. Biological triplicates were averaged. Different letters represent significant differences at P < 0.01 (one-way ANOVA with Fisher's post hoc test). (D) Transcriptional activation activity assay on PIF4. A set of reporter and effector constructs were used (left panel). The reporter and effector vectors were cotransformed into hos1-3 protoplasts. The CaMV 35S promoter-luciferase construct was included as internal control in the assays. Relative β-glucuronidase (GUS) activity was determined fluorimetrically (right panel). ARF5M was used as positive control. Six measurements were averaged and statistically analyzed. Different letters represent significant differences at P < 0.05 (one-way ANOVA with Fisher's post hoc test). (E) ChIP assay on the effects of pif4-101 mutation on DNA binding of HOS1. Transgenic seedlings expressing HOS1-MYC fusion driven by its own promoter in Col-0 and pif4-101 backgrounds were harvested at ZT8. An anti-MYC antibody was used to immunoprecipitate HOS1-MYC fusion proteins. Biological triplicates were averaged and statistically analyzed. Different letters represent significant differences at P < 0.01 (one-way ANOVA with Fisher's post hoc test). Molecular Plant 2017 10, 274-284DOI: (10.1016/j.molp.2016.11.009) Copyright © 2017 The Author Terms and Conditions

Figure 4 HOS1 Is Required for the phyB-Mediated Inhibition of PIF4 Function. Seedlings were grown for 6 days on MS agar plates under LDs. (A) Diurnal patterns of PIF4 protein accumulation in phyb-9 mutant. A PIF4-MYC gene fusion was expressed driven by the endogenous PIF4 gene promoter in Col-0 and phyb-9 backgrounds. Parts of Coomassie blue-stained gels containing rubisco were displayed at the bottom. Protein levels were quantitated using ImageJ software. Biological triplicates were averaged. Bars indicate SD. (B) Hypocotyl growth of hos1-3 phyb-9 double mutant. Hypocotyl lengths of 15 seedlings were averaged and statistically analyzed using Student t-test (*P < 0.01). Numbers indicate fold changes. Bars indicate SD. (C and D) Diurnal expression patterns of PIF4 target genes in hos1-3 phyb-9 double mutant. Transcript levels were measured as described in Figure 2E (C). Bars indicate SE. Since the rhythms of YUC8 and IAA29 expressions were different in phyb-9 and hos1-3 phyb-9 backgrounds, the rhythms were synchronized to more systematically compare the transcript levels (D). The data in (D) were replotted from those in (C). Molecular Plant 2017 10, 274-284DOI: (10.1016/j.molp.2016.11.009) Copyright © 2017 The Author Terms and Conditions

Figure 5 phyB Activates HOS1 to Inhibit PIF4 Function. (A) Yeast two-hybrid assay on HOS1–phyB interaction. Yeast cell growth on selective media lacking Leu, Trp, His, and Ade (-QD) represents positive interaction. (B) ChIP assay on the effects of phyb-9 mutation on DNA binding of HOS1. Transgenic seedlings expressing a HOS1-MYC gene fusion driven by the endogenous HOS1 gene promoter in Col-0 and phyb-9 backgrounds were assayed. ChIP assays were performed as described in Figure 3C. Biological triplicates were averaged and statistically analyzed using Student t-test (*P < 0.01, difference from mock). Bars indicate SD. (C and D) ChIP assay on phyB binding to the promoter regions of PIF4 target genes. Production of phyB-MYC protein in pphyB:phyB-MYC seedlings was verified by immunoblot assays before assays (C). ChIP assays were performed as described in Figure 3C (D). Immunoprecipitation using an anti-H3 antibody was performed in parallel as positive control. *P < 0.01. (E) The phyB-HOS1-PIF4 module plays a role in photomorphogenesis. Photoactivated phyB mediates the HOS1–PIF4 interactions, which subsequently inhibits the transcriptional activation activity of PIF4, thus inducing a photomorphogenic response of hypocotyl growth in Arabidopsis. Molecular Plant 2017 10, 274-284DOI: (10.1016/j.molp.2016.11.009) Copyright © 2017 The Author Terms and Conditions