Tyrosine Phosphorylation Regulates the Activity of Phytochrome Photoreceptors  Kazumasa Nito, Catherine C.L. Wong, John R. Yates, Joanne Chory  Cell Reports  Volume 3, Issue 6, Pages 1970-1979 (June 2013) DOI: 10.1016/j.celrep.2013.05.006 Copyright © 2013 The Authors Terms and Conditions

Cell Reports 2013 3, 1970-1979DOI: (10.1016/j.celrep.2013.05.006) Copyright © 2013 The Authors Terms and Conditions

Figure 1 Predicted Phosphorylation Sites on phyB by Mass Spectrometry Analysis (A) Top panel: the full-length A. thaliana phyB structure. The N-terminal half is separated into four regions designated the N, PLD, GAF, and PHY domains. Two PAS domains and histidine kinase-related domain (HKRD) are located in the C-terminal half. Star: chromophore-binding site. The graphs depict the frequency of predicted phosphorylation by mass spectrometry analysis before (0 hr) and after (6 hr) white-light treatment. The gray box indicates putative light-dependent phosphorylation cluster in the N terminus. Bottom graph: enlarged 70–130 aa region of 6-hr-exposed phyB. Six groups of putative sites (S84,S86, T89–T91, S94,S95, Y104, S106, and Y113) are indicated. See also Figure S1 and Table S1. (B) Comparison of the PCSM motif in phytochromes from various species. Alignment of amino acid sequences was calculated with ClustalW software. Phosphorylation sites S84,S86, T89-T91, Y104, and S106 are indicated. Gray box indicates bacterial phytochromes. Cell Reports 2013 3, 1970-1979DOI: (10.1016/j.celrep.2013.05.006) Copyright © 2013 The Authors Terms and Conditions

Figure 2 phyBY104F Cannot Complement phyB-9 Null Mutant (A) Seedlings grown under the long-day condition with white light for 7 days. Col-0; phyB-9; WT, expressing phyBWT; Y104F, nonphospho mutant phyBY104F; Y104E, phosphomimic mutant phyBY104E. (B) Hypocotyl measurement under different red-light intensities. Two representative transgenic lines were analyzed from each line. Col-0; phyB-9; WT, expressing phyBWT; Y104F, nonphospho mutant phyBY104F; Y104E, phosphomimic mutant phyBY104E. Error bar is SE (n = 30). (C) Thirty-day-old plants of phospho mutants of Y104, grown under long-day conditions. Col-0; phyB-9; P35S::PHYB-CFP, phyB-CFP overexpressor; native promotor lines (PPHYB::PHYB-mCitrine) of WT, expressing phyBWT; Y104F, nonphospho mutant phyBY104F; Y104E, phosphomimic mutant phyBY104E. See also Figure S3. Cell Reports 2013 3, 1970-1979DOI: (10.1016/j.celrep.2013.05.006) Copyright © 2013 The Authors Terms and Conditions

Figure 3 Y104 on phyB Is Phosphorylated in a Light-Dependent Manner (A) phyB-YFP was purified from etiolated seedlings (D) of phyBWT (WT) and phyBY104F (Y104F) overexpressor lines and exposed to 3 hr of white light (L), and then detected by anti-phosphotyrosine antibody (α-phospho-Y) and anti-GFP antibody (α-GFP). phyB-9 was used as a negative control. (B) Graph indicating relative values of the band intensities of phosphotyrosine. The data were derived from three independently performed experiments. For comparison of each blot, the values were normalized by the value from etiolated seedlings of phyBWT. Error bar is SD; p values were calculated. Cell Reports 2013 3, 1970-1979DOI: (10.1016/j.celrep.2013.05.006) Copyright © 2013 The Authors Terms and Conditions

Figure 4 phyBY104E Mutants Cannot Interact with PIF3 In vitro pull-down assay of Y104 mutants with PIF3. Input panel indicates the protein levels of GST-PIF3 and phyB-FLAG (phyBWT, phyBY104F, and phyBY104E) in the in vitro translation system. The pull-down fraction indicates the interaction of each phyB with PIF3 under far-red (F) and red (R) light. The protein complex was precipitated by GST tag. See also Figure S4. Cell Reports 2013 3, 1970-1979DOI: (10.1016/j.celrep.2013.05.006) Copyright © 2013 The Authors Terms and Conditions

Figure 5 Subcellular Localization of phyBY104E (A–C) Localization of phyB-mCitrine was observed in cotyledons of 7-day-old seedlings grown under (A) the long-day condition (16 hr light/8 hr dark), (B) continuous white light (24 hr light), and (C) continuous white light, and then transferred to the dark for 10 min (24 hr light + 10 min dark). Enlarged images of the nuclei are indicated. Scale bar = 50 μm. See also Figure S5. Cell Reports 2013 3, 1970-1979DOI: (10.1016/j.celrep.2013.05.006) Copyright © 2013 The Authors Terms and Conditions

Figure 6 phyBY104E Possesses Normal Photochemical Properties (A) phyBWT and phyBY104E were expressed and purified as a native protein. Purified phyBs were stained by Coomassie brilliant blue. Arrow indicates phyB protein. (B) Spectra of phyBWT and phyBY104E. Purified phyBs were exposed to each light condition for 5 min followed by dark treatment, and their spectra were measured (500–800 nm). Top panel: phyBWT. Bottom panel: phyBY104E. F, far-red light; R, red light; R to D10, red + dark for 10 min; R to D30, red + dark for 30 min; R to D60, red + dark for 60 min; R to D180, red + dark for 180 min; R to F, red following far-red light exposure. The peaks of the Pr form (650 nm) and Pfr form (710 nm) are indicated. (C) Graph showing the remaining Pfr form of phyB after a 30-min-dark treatment. Spectra of both phyBWT and phyBY104E were repeatedly measured five times in the dark for 30 min after red-light exposure. The integrated values from 701 nm to 720 nm are normalized to red-light-exposed samples. WT, phyBWT; Y104E, phyBY104. Error bar is SD. (D) EOD-FR treatments of WT and phospho mutants of phyB. Seedlings were grown under the 10 hr light/14 hr dark cycling condition for 4 days. For EOD-FR treatment, seedlings were exposed to far-red light for 15 min at the end of light period (gray) and compared with nontreated seedlings (white). The ratio of effect (+FR/−FR) is indicated on the right (diamond). Error bar is SE (n = 30). See also Figure S6. Cell Reports 2013 3, 1970-1979DOI: (10.1016/j.celrep.2013.05.006) Copyright © 2013 The Authors Terms and Conditions

Figure S1 Tandem Mass Spectrum and Expressing Phospho Mutant phyBs in the Transgenic Arabidopsis, Related to Figure 1 (A) A tandem mass spectrum of the peptide ApYLpSRIQRGGYIQPFGCMIAVDESSFR is shown. pY and pS represent the sites of phosphorylation in the sequence identified in the spectrum. Fragment ions in spectrum are labeled with C-terminally related fragment ions labeled in red as yn with the subscript indicating the position in the sequence where the fragment ion is located. N-terminally related fragment ions are labeled in blue as bn ions with the subscript indicating where in the sequence the fragment ion is located. (B and C) Protein levels of phospho mutant phyBs in transgenic Arabidopsis seedlings. phyB levels were determined by immunoblot with anti-phyB (mBA2) antibody in 7-day-old seedlings grown under long day conditions. Actin was used as a loading control. Cell Reports 2013 3, 1970-1979DOI: (10.1016/j.celrep.2013.05.006) Copyright © 2013 The Authors Terms and Conditions

Figure S2 Hypocotyl Measurement under Red Light, Related to Table 1 Seedlings were grown under dark condition or under continuous 1 μE/m2/sec of red light for 4 days, and were measured by Image J software. Error bar is standard error (n = 30). Cell Reports 2013 3, 1970-1979DOI: (10.1016/j.celrep.2013.05.006) Copyright © 2013 The Authors Terms and Conditions

Figure S3 Phenotypic Analysis of Phospho Mutants of Y104, Related to Figure 2 (A) Flowering time in phospho mutants of Y104. Plants were grown under long day conditions. Time of bolting and concurrent leaf number are shown. Error bar is standard error (n = 24). (B) Chlorophyll levels in phospho mutants of Y104. Chlorophyll levels were measured in 4 expanded leaves of 30-day-old plants. Error bar is standard deviation. Col-0, Columbia-0; phyB-9; WT, expressing phyBWT; Y104F, non-phosphorylated mutant phyBY104F; Y104E, phospho-mimic mutant phyBY104E; phyB-CFPox and phyB-YFPox are expressed by 35S promotor. (C–F) Phenotype of phyBY104E overexpressor line. (C) phyB levels were determined by immunoblot with anti-phyB antibody in 7-day-old seedlings grown under long day conditions. Actin was used as a loading control. (D) Seedlings grown in long day conditions for 7 days. Col-0, Columbia-0; phyB-9; WT, expressing phyBWT; Y104F, non-phosphorylated mutant phyBY104F; Y104E, phospho-mimic mutant phyBY104E. All transgenes are expressed by a 35S promotor. (E) Hypocotyl measurements of the overexpressor lines under 1 μE/m2/sec of red light. Col-0; phyB-9; WT, expressing phyBWT; Y104F, non-phosphorylated mutant phyBY104F; Y104E, phospho-mimic mutant phyBY104E. Error bar is standard error (n = 30). (F) Thirty-day-old plants of the overexpressor lines. Arabidopsis plants were grown under long day conditions. Cell Reports 2013 3, 1970-1979DOI: (10.1016/j.celrep.2013.05.006) Copyright © 2013 The Authors Terms and Conditions

Figure S4 The Phosphomimic Mutant phyAY70E Does Not Have Activity, Related to Figure 4 (A) In vitro pull down assay of phyA Y70 mutants to PIF3. Input indicates the protein levels of GST-PIF3 and phyA-FLAG (phyAWT, phyAY70F and phyAY70E) in the in vitro translation system. Pull down fraction indicates interaction of each phyA to PIF3 under far-red (F) and red (R) light. Protein complex was precipitated by GST tag. (B) Seedlings grown under continuous far-red light condition for 4 days. Col-0, Columbia-0; phyA-211; WT, expressing phyAWT; Y70F, non-phospho mutant phyAY70F; Y104E, phospho-mimic mutant phyAY70E. Cell Reports 2013 3, 1970-1979DOI: (10.1016/j.celrep.2013.05.006) Copyright © 2013 The Authors Terms and Conditions

Figure S5 Protein Stability of phyBY104E in the Dark Condition, Related to Figure 5 The seedlings were grown under continuous white light for 7 days, and were transferred to the dark for 0, 10, 30 or 60 min. To eliminate de novo synthesis, seedlings were treated with cycloheximide prior to shifting to the dark. phyB-mCitrine was detected by anti-phyB antibody. Actin was used as a loading control. WT, expressing phyBWT; Y104F, non-phosphorylated mutant phyBY104F; Y104E, phospho-mimic mutant phyBY104E. Cell Reports 2013 3, 1970-1979DOI: (10.1016/j.celrep.2013.05.006) Copyright © 2013 The Authors Terms and Conditions

Figure S6 Schematic Model for the Role of Tyrosine Phosphorylation at Amino Acid 104 in phyB Signaling, Related to Figure 6 phyB is synthesized in its Pr form and remains in the cytoplasm in dark-grown seedlings. After red light exposure, Pr is rapidly converted to Pfr and translocates into the nucleus to promote degradation of PIFs for photomorphogenesis. The site of this interaction is likely to be in nuclear bodies. Depending on light conditions, Excess Pfr form of phyB is phosphorylated at Y104 to inactivate phyB and phyB signaling is relieved regardless of its photochemical states. phyB with the phosphotyrosine cannot interact with PIFs. The phosphorylation on Y104 seems not to affect nuclear import of phyB, however it is still unclear at where Y104 is phosphorylated in the cell. To reactivate the phosphorylated phyB, protein tyrosine phosphateses (PTPs) may need to dephosphorylate Y104 on phyB. Cell Reports 2013 3, 1970-1979DOI: (10.1016/j.celrep.2013.05.006) Copyright © 2013 The Authors Terms and Conditions