Volume 5, Issue 3, Pages 629-641 (May 2012) A Short Amino-Terminal Part of Arabidopsis Phytochrome A Induces Constitutive Photomorphogenic Response  András Viczián, Éva Ádám, Iris Wolf, János Bindics, Stefan Kircher, Marc Heijde, Roman Ulm, Eberhard Schäfer, Ferenc Nagy  Molecular Plant  Volume 5, Issue 3, Pages 629-641 (May 2012) DOI: 10.1093/mp/sss035 Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 1 Expression of PHYA406 Protein Results in Constitutive Photomorphogenic Phenotype. Images of seedlings grown under different monochromatic light fields for 4 d. R: 50 μmol m−2 s−1 red light; FR: 10 μmol m−2 s−1 far-red light; B: 5 μmol m−2 s−1 blue light; D: darkness. Every chimeric protein is expressed under the control of the Pro35S promoter in phyA-201 background. Black bar represents 10 mm. Molecular Plant 2012 5, 629-641DOI: (10.1093/mp/sss035) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 2 Fluence Rate-Dependent Hypocotyl Elongation Inhibition in Seedlings Expressing PHYA406. Hypocotyl length of 4-day-old seedlings grown under far-red (A), red (B), or blue (C) light was measured and plotted against the applied fluence rate. The indicated chimeric proteins are expressed under the control of the Pro35S promoter in phyA-201 background. Dark values: Ler: 12.29 ± 0.14 mm; phyA-201: 10.48 ± 0.27 mm; PHYA406–YFP–DD: 6.61 ± 0.23 mm; PHYA406–YFP–DD–NLS: 4.81 ± 0.1 mm; PHYA406–YFP–DD–NES: 9.35 ± 0.27 mm. Error bars indicate standard error. Molecular Plant 2012 5, 629-641DOI: (10.1093/mp/sss035) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 3 Intracellular Localization of PHYA406 Protein under Different Light Conditions. (A, B) Four-day-old phyA-201 seedlings harboring Pro35S:PHYA406–YFP–DD (A) or Pro35S:PHYA406–YFP–DD–NLS (B) were grown under different monochromatic light fields. Epifluorescence microscopy was applied to quantify the signal of examined nuclei located in the upper third of the hypocotyl. Nuclear fluorescence was corrected to the background and normalized to the corresponding dark control. D: dark control; FR: 10 μmol m−2 s−1 far-red light; R: 50 μmol m−2 s−1 red light; B: 10 μmol m−2 s−1 blue light. (C) Epifluorescence images of hypocotyl cells of 4-day-old seedlings, grown under different monochromatic light fields. Every chimeric protein is expressed under the control of the Pro35S promoter in phyA-201 background. D: dark control; R: 20 μmol m−2 s−1 red light; FR: 10 μmol m−2 s−1 far-red light; B: 10 μmol m−2 s−1 blue light. White bar represents 10 μm. Molecular Plant 2012 5, 629-641DOI: (10.1093/mp/sss035) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 4 The PHYA406 Protein Acts in the Nucleus. (A) Epifluorescence microscopy of PHYA406–YFP–DD expressed under the control of the Pro35S promoter in phyA-201 (a, b) and fhy1-3 fhl1 (c, d) mutant backgrounds. The examined seedlings were grown for 4 d in darkness (a, c) or under 10 μmol m−2 s−1 far-red light (b, d). Dotted contour lines encircle nuclei; white bars indicate 10 μm. (B, C) Fluence rate response curves of hypocotyl elongation inhibition of 4-day-old seedlings grown in far-red (B) or red (C) light. The obtained values were normalized to the hypocotyl length of the corresponding dark-grown seedlings. Error bars indicate standard error. Hypocotyl length values of etiolated seedlings: Col-0: 10.69 ± 0.19 mm; fhy1-3 fhl1: 11.09 ± 0.14 mm; Pro35S:PHYA406–YFP–DD in fhy1-3 fhl1: 10.89 ± 0.21 mm. (D) Representative 35-day-old plants grown under short-day conditions. Plant 1: Col-0; Plant 2: Pro35S:PHYA406–YFP–DD in fhy1-3 fhl1; Plant 3: cop1-4. White bar represents 10 mm. Molecular Plant 2012 5, 629-641DOI: (10.1093/mp/sss035) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 5 Adult Phenotype of Plants Expressing PHYA406. (A) Number of leaves of plants grown under short-day (8-h light/16-h dark) conditions was counted at the time of flowering. Error bars indicate standard error. (B) Images representative 35-day-old plants grown under short-day conditions. White bar represents 10 mm. (C) Epifluorescence microscopy images were taken of plant leaves on the 35th day of their growth under short-day conditions. Arrows point to selected nuclei. White bars indicate 10 μm. Legend: 1: Ler; 2: phyA-201; 3: Pro35S:PHYA406–YFP–DD in phyA-201; 4: Pro35S:PHYA406–YFP–DD–NLS in phyA-201; 5: Pro35S:PHYA406–YFP–DD–NES in phyA-201; 6: Col-0; 7: cop1-4. Molecular Plant 2012 5, 629-641DOI: (10.1093/mp/sss035) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 6 The PHYA406 Protein Is Stable in Light. (A) Western blot analysis was performed to examine the levels of different PHYA406–YFP proteins compared with the WT PHYA–YFP. Four-day-old etiolated seedlings were treated with 0, 1, 3, or 6 h of strong R light (20 μmol m−2 s−1) irradiation and transgenic fusion protein levels were determined using anti-YFP antibody (upper panels). Anti-ACTIN antibody was used to show the uniformity of sample loading (lower panels). (B) Comparison of PHYA and PHYA406 protein levels between etiolated and adult plants. Transgenic plants expressing PHYA–YFP or different PHYA406–YFP proteins under the control of Pro35S promoter in phyA-201 background were grown for 4 d in the dark (lanes 1, 3, 5, and 7) or for 35 d under 8-h light/16-h dark photocycles (lanes 2, 4, 6, and 8). Total protein extracts isolated from these plants were subjected to Western blot analysis. Transgenic proteins were visualized using anti-YFP antibody (upper panel), whereas anti-ACTIN antibody was used for loading control (lower panel). Molecular Plant 2012 5, 629-641DOI: (10.1093/mp/sss035) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 7 The PHYA406 Binds to COP1. (A) The examined proteins were fused to either GAL4 activation or GAL4 DNA-binding domain (AD or BD, respectively) and expressed in yeast cells (strain AH109). Equal amounts (5 μl) of overnight liquid cultures of co-transformed yeast cells grown in L-W- medium were dropped on either non-selective (L-W-) or selective (H-L-W-, containing 1 mM 3-aminotiazole, 3-AT) synthetic dropout plates. H-L-W- plates also contained 10 μM PCB, unless indicated (–PCB). Plates were incubated at 28°C for 2 d under 1 μmol m−2 s−1 red light (R) or 10 μmol m−2 s−1 far-red light (FR) or in darkness (D). (B) The indicated proteins fused to AD and BD domains were co-expressed in yeast strain Y187. Liquid cultures were propagated in non-selective medium (L-W-) overnight. β-galactosidase activity was measured using ortho-Nitrophenyl-β-galactoside substrate. Triplicates were assayed and mean values are plotted. Error bars indicate standard error. (C) Co-immunoprecipitation of PHYA406 and COP1 in vivo. Total protein extract from wild-type Ler (lane 1) and from plants expressing PHYA406–YFP–DD–NES (lane 2) or PHYA406–YFP–DD–NLS (lane 3) was immunoprecipitated with anti-GFP antibody and the samples were subsequently probed with anti-GFP (top panel) or anti-COP1 (middle panel) antibodies, respectively. The source of IgG signal is the anti-GFP antibody used in the immunoprecipitations. Bottom panel (Input) shows the non-immunoprecipitated raw extract probed with anti-GFP antibody. Molecular Plant 2012 5, 629-641DOI: (10.1093/mp/sss035) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 8 COP1 Co-Localizes with PHYA406 But Not with PHYA In Planta. Epifluorescence microscope images were taken of 4-day-old etiolated phyA-201 cop1-4 double mutant seedlings harboring Pro35S:CFP–COP1 together with either Pro35S:PHYA406–YFP–DD–NLS or 35S:PHYA–YFP. Hypocotyl cells were examined directly without any light treatment (D) or after a 5-min R light pulse (5' R) using YFP-specific (YFP) and subsequently CFP-specific (CFP) microscope filter sets. Arrows point to nuclear bodies; scale bar represents 10 μm. Molecular Plant 2012 5, 629-641DOI: (10.1093/mp/sss035) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions