Light-Dependent Degradation of PIF3 by SCFEBF1/2 Promotes a Photomorphogenic Response in Arabidopsis  Jie Dong, Weimin Ni, Renbo Yu, Xing Wang Deng, Haodong Chen, Ning Wei  Current Biology  Volume 27, Issue 16, Pages 2420-2430.e6 (August 2017) DOI: 10.1016/j.cub.2017.06.062 Copyright © 2017 The Author(s) Terms and Conditions

Current Biology 2017 27, 2420-2430.e6DOI: (10.1016/j.cub.2017.06.062) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 EBF1/2 Inhibit Hypocotyl Elongation by Reducing PIF3 Activity in Red Light (A) Overexpression of EBF1 or EBF2 suppressed PIF3-Myc-induced hypocotyl elongation in red light. Seedlings grown under 10 μmol/m2/s red light for around 4 days were measured for hypocotyl length. Representative seedlings are shown on the left, and the mean hypocotyl lengths (mean ± SEM) are shown as bar graphs on the right. Statistical significance was calculated by Student’s t test. ∗p < 0.05; ∗∗∗p < 0.001. (B) The ebf1 ebf2 double mutant exhibited longer hypocotyls in red light compared to Col, and the phenotype could be suppressed by the pif3 mutation. Seedling growth and data analyses were performed as in (A). The scale bars represent 2 mm. See also Figures S1 and S2. Current Biology 2017 27, 2420-2430.e6DOI: (10.1016/j.cub.2017.06.062) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 PIF3 Interacts with EBF1 and EBF2 (A) PIF3 interacts with both full-length and LRR domains of EBF1 and EBF2 in yeast. Full-length or partial EBF1 and EBF2 were fused with the binding domain (BD) as baits. Full-length PIF3 was fused with the activation domain (AD) as prey. Empty vectors were used as negative controls. (B) PIF3 interacts with EBF1 and EBF2 in vitro. The proteins of EBF1-flag, EBF2-flag, and PIF3-Myc were synthesized using an in vitro translation system. The flag antibody was used for in vitro pull down, and pellets were analyzed by western blots using antibodies to flag and Myc. (C) EBF1 and EBF2 co-immunoprecipitate PIF3 in Arabidopsis. Total proteins were extracted from 3-day-old dark-grown PIF3-Myc or EBF-GFP/PIF3-Myc seedlings. GFP antibody was used for immunoprecipitation, and pellets were analyzed by western blots using antibodies to GFP and Myc. (D) PIF3 interacts with EBF1 and EBF2 in LCI assays. PIF3 was fused to nLUC, and EBF1/2 without an F box domain (EBF1/2 ΔF) were fused to cLUC, which were co-expressed in tobacco. Empty vectors were used as negative controls. See also Figure S3. Current Biology 2017 27, 2420-2430.e6DOI: (10.1016/j.cub.2017.06.062) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 EBF1/2 Promote PIF3 Degradation after Light Exposure (A and B) Mutation of EBF1 and EBF2 slowed down the light-induced degradation rate of endogenous PIF3 protein (A) and ectopic PIF3-GFP protein (B). Dark-grown seedlings (“D”) were treated with a red-light pulse (Rp) and kept in the dark for the indicated times before protein extraction and western blotting with antibodies to PIF3 and RPN6. (A) Top: representative western blot result. Bottom: quantification results of PIF3 degradation kinetics from three independent biological repeats. PIF3 protein level was normalized to RPN6 loading control, and the dark PIF3 level was set as 100%. Data are shown as mean ± SEM. (B) S.exp, short exposure time; L.exp, long exposure time. (C and D) Overexpression of EBF1-TAP and EBF2-TAP accelerated light-induced degradation of endogenous PIF3 (C) or ectopic PIF3-Myc protein (D). Dark-grown seedlings (“D”) were treated with either pulse (Rp) or continuous (Rc) red light. Western blots were performed with antibodies to PIF3, RPN6, Myc, or RPT5. See also Figure S4. Current Biology 2017 27, 2420-2430.e6DOI: (10.1016/j.cub.2017.06.062) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 EBF1/2 Promote Light-Induced PIF3 Ubiquitination Total polyubiquitinated proteins were precipitated using tandem ubiquitin-binding entities 2 (TUBE2) from 3-day dark-grown seedlings without/with R light pulse (D/Rp) treatment, and then analyzed by western blots using antibodies to ubiquitin (A), Myc (B and C), or PIF3 (D). Both Ub and Ubi are the abbreviations for ubiquitin. See also Figure S5. Current Biology 2017 27, 2420-2430.e6DOI: (10.1016/j.cub.2017.06.062) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 EBF1- and EBF2-Mediated PIF3 Degradation Is Dependent on Phytochrome-Induced PIF3 Phosphorylation (A and B) EBF1-promoted (A) or EBF2-promoted (B) PIF3 degradation upon light exposure is dependent on phyA and phyB. Dark-grown seedlings (“D”) were treated with a Rp and kept in the dark for the indicated times before total proteins were extracted and analyzed by western blots using antibodies to PIF3 or RPN6. (C and D) The interaction between PIF3 and phytochromes is necessary for EBF1-promoted (C) or EBF2-promoted (D) PIF3 degradation. The GFP-fused PIF3 transgenic lines PIF3 WT (wild-type PIF3) and PIF3 mAPAmAPB (PIF3 with mutations in the APA and APB domains) in wild-type (Col) or EBF1/2-TAP backgrounds were used. Dark-grown seedlings (“D”) were treated with continuous red light (Rc) for the indicated times before western blotting using antibodies to GFP or RPN6. (E and F) EBF1-promoted (E) or EBF2-promoted (F) PIF3 degradation is dependent on light-induced PIF3 phosphorylation. The Myc-fused PIF3 transgenic lines PIF3 WT (wild-type PIF3) and PIF3 A20 (PIF3 protein with 20 mutations of light-induced phosphorylation sites) in wild-type (Col) or EBF1/2-TAP backgrounds were used. Protein preparation was as described in (C), and western blots were performed with antibodies to Myc or RPN6. Current Biology 2017 27, 2420-2430.e6DOI: (10.1016/j.cub.2017.06.062) Copyright © 2017 The Author(s) Terms and Conditions

Figure 6 PIF3 Phosphorylation Promotes the Assembly of PIF3-EBF1/2 with CUL1 (A) EBF interacts with PIF3 under both dark and light conditions in Arabidopsis. Dark-grown (“D”) seedlings (3-day-old) were treated with a Rp and then kept in darkness for 10 min, and total proteins were extracted. IgG Sepharose was used for immunoprecipitation, and pellets were analyzed by western blot using a Myc antibody. (B) EBF1/2 interact with wild-type and phospho-site mutants of PIF3 in yeast two-hybrid assays. Full-length EBF1, EBF2, and LRB2 were fused to DNA-binding domains (BD) as baits. Phospho-dead (A6 and A20), wild-type, and phospho-mimic (D6 and D19) PIF3s were fused to activation domains (AD) as prey. Empty vectors were used as negative controls. (C) Light induces the association of CUL1 and PIF3 in Arabidopsis. Experimental conditions were the same as in (A). GFP antibody was used for immunoprecipitation (IP), and the pellets were analyzed by western blots using antibodies to GFP or CUL1. CUL1 was co-precipitated with YFP-PIF3 after light (Rp), but not in the dark (“D”). (D) EBF1 and EBF2 are essential for light-induced association of CUL1 and PIF3 in Arabidopsis. Experimental conditions were the same as in (A). GFP antibody was used for IP, and the pellets were analyzed by western blots using antibodies to CUL1 and PIF3. (E) PIF3 phosphorylation can enhance its association with CUL1 without light activation. Dark-grown seedlings (3-day-old) were used for IP with Myc antibody. The pellets were analyzed by western blots using antibodies to Myc or CUL1. Endogenous CUL1 was pulled down by proteins of PIF3 phospho-mimic (PIF3-D6), but not PIF3 phospho-dead (PIF3-A20) mutant. (F) Phospho-mimic PIF3 promotes EBF1 and CUL1 association. Experimental procedures were the same as in (E). IgG Sepharose was used for IP of EBF1-TAP, and the pellets were analyzed by western blots using antibodies to Myc or CUL1. CUL1 was strongly pulled down with EBF1-TAP in the presence of PIF3 phospho-mimic transgenic products, compared to those in PIF3 wild-type, PIF3 phospho-dead, or no PIF3 transgene. The relative amounts of CUL1 protein to EBF1-TAP protein in pellets are shown as mean ± SD from three independent experiments, in which the ratio of EBF1-TAP transgenic plants was set as 1.0. (G) ASK1 is necessary for the association of EBF1 and CUL1 promoted by phospho-mimic PIF3 in vitro. EBF1-flag, CUL1-His, PIF3 A20-Myc, and PIF3 D6-Myc proteins were expressed using an in vitro translation system. GST and GST-ASK1 proteins were expressed and purified from E. coli. Anti-flag antibody was used to immunoprecipitate EBF1-flag, and then the pellets were analyzed by western blots using antibodies against flag, His, Myc, and GST. See also Figure S6. Current Biology 2017 27, 2420-2430.e6DOI: (10.1016/j.cub.2017.06.062) Copyright © 2017 The Author(s) Terms and Conditions

Figure 7 EBF and LRB E3 Ligases Have Distinct Functions (A) LRBs regulate light-induced degradation of phyB whereas EBFs do not. 3-day-old dark-grown (“D”) seedlings were transferred to 60 μmol/m2/s red light for 72 hr (Rc) and then total proteins were analyzed by western blots using antibodies to phyB or RPN6. Light-induced decline in phyB was prohibited only in lrb mutants, not in the other lines tested. (B) lrb mutants show light hypersensitivity whereas ebf mutants show hyposensitivity with regard to the hypocotyl response. Hypocotyl lengths of 3-day-old seedlings growing under dark (Dc) or 5 μmol/m2/s red light (Rc) were measured (mean ± SEM). Statistical significance was calculated by Student’s t test. n.s, p > 0.05; ∗∗∗p < 0.001. The lrb mutants displayed shorter hypocotyls whereas ebf mutants displayed longer hypocotyls under Rc. The scale bars represent 2 mm. (C) ebf mutants showed defects in PIF3 degradation under both strong and weak light conditions, whereas lrb mutants showed defects in PIF3 degradation predominantly under strong light condition. 3-day-old dark-grown seedlings were treated with either 3,000 μmol/m2 or 100 μmol/m2 Rp and then kept in darkness for 15 min before total proteins were extracted and analyzed by western blots using antibodies to PIF3 and RPN6. (D) EBFs promote PIF3 degradation under both strong and weak light conditions, whereas LRB’s effect on PIF3 is detected predominantly in strong light. 3-day-old dark-grown seedlings were treated with either 3,000 μmol/m2 (upper) or 100 μmol/m2 (lower) Rp and kept in the dark for the indicated times before western blotting using antibodies to PIF3 or RPN6. (E) A model illustrating the distinct functions of EBF1/2 and LRBs in the plant light signaling pathway through their control of PIF3. In the dark, PIF3 accumulates to promote skotomorphogenesis. PIF3 binds EBF1/2 but the complex is not recruited to the SCF core, and PIF3 does not bind LRBs. When plants sense weak light at the beginning of light exposure, PIF3 undergoes multisite phosphorylation (represented by the circles with “-”). The phospho-PIF3-EBF complex and core SCF scaffold assemble to form SCFEBF1/2-PIF3 complexes, resulting in the ubiquitination and degradation of PIF3. This PIF3 degradation pathway promotes photomorphogenesis. When plants perceive strong light, another PIF3 degradation pathway is triggered in addition to SCFEBF1/2-PIF3, in which the phyB-PIF3 complex is targeted by CUL3LRBs E3 ligases. The LRB pathway results in the degradation of both phyB and PIF3 to attenuate plant light responses. See also Figure S7. Current Biology 2017 27, 2420-2430.e6DOI: (10.1016/j.cub.2017.06.062) Copyright © 2017 The Author(s) Terms and Conditions