Volume 1, Issue 1, Pages 4-14 (January 2008) Chemically Induced and Light-Independent Cryptochrome Photoreceptor Activation  Rosenfeldt Gesa , Viana Rafael Muñoz , Mootz Henning D. , von Arnim Albrecht G. , Batschauer Alfred   Molecular Plant  Volume 1, Issue 1, Pages 4-14 (January 2008) DOI: 10.1093/mp/ssm002 Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 1 Coimmunoprecipitation of cry2 Probed with Anti-cry2 Antibody. (A) Western-blot analysis of protein extract from the Arabidopsis wild type (Ler) and transgenic line (cry2-GFP) expressing cry2–GFP. The arrow marks cry2; the arrow with an asterisk marks cry2–GFP. (B) Control pull-down experiment with anti-GFP antibody and protein extract from wild type. Total protein extract (Extract), supernatant (SN), and pellet fractions were probed with anti-cry2 antibody. Cry2 remains in the supernatant fraction as expected. (C) Pull-down experiment with anti-GFP antibody and protein extract from the cry2–GFP line. The extracts were incubated either with protein G–agarose in the absence of anti GFP-antibody (No antibody) as control, or with anti-GFP antibody and protein G–agarose (+ anti-GFP). Supernatants (SN), pellet fractions (Pellet), and the protein extract that was used for the pull-down experiments (Extract) were analyzed for the presence of cry2 and cry2–GFP with anti-cry2 antibody. Bands of mobility lower than cry2 most probably represent cry2 degradation products since they are not detectable in extracts from the cry2 mutant fha-1 (data not shown). Molecular Plant 2008 1, 4-14DOI: (10.1093/mp/ssm002) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 2 Induced Heterodimerization of Luciferase and YFP Fusion Proteins in Onion Epidermal Cells. Epidermal cells were cobombarded with equal amounts of gene constructs expressing the following proteins under control of the CaMV 35S promoter (from left to right), Renilla luciferase and yellow fluorescent protein (RLUC + YFP); FKBP fused to the N-terminus of RLUC and FRB fused to the N-terminus of YFP (FKBP-RLUC + FRB-YFP); FRB fused to the N-terminus of RLUC and FKBP fused to the N-terminus of YFP (FRB-RLUC + FKBP-YFP); and full-length COP1 fused to RLUC and to YFP, respectively (RLUC-COP1 + YFP-COP1). The FKBP and FRB proteins were separated from their fusion partners by nine alanine residues. After bombardment, cells were incubated for 40 h to allow expression of the transfected genes. Bioluminescence resonance energy transfer (BRET) signals were measured before (white columns) and 60 min (gray columns) or 120 min (black columns) after incubation with 2 μM inducer AP21967. The RLUC + YFP combination served as negative, and the RLUC-COP1 + YFP-COP1 combination as positive control. Data are averages from four independent experiments ± SD. Molecular Plant 2008 1, 4-14DOI: (10.1093/mp/ssm002) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 3 Induced Heterodimerization of Luciferase and YFP Fusion Proteins in Arabidopsis Protoplasts. The combination of FRB-RLUC and FKBP-YFP plasmids was transfected into Arabidopsis protoplasts, and BRET signals monitored before and after addition of AP21967 inducer in concentrations of 10 nM (squares), 100 nM (triangles), and 1000 nM (crosses) for up to 240 min. Protoplasts treated with a mock solution (diamonds) served as controls. Data are averages from three experiments ± SD. Molecular Plant 2008 1, 4-14DOI: (10.1093/mp/ssm002) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 4 Immunoblot Analysis of cry2 C-Terminal Domain (CCT2) Fusions with Either FKBP or FRB Expressed in Transfected Arabidopsis Protoplasts. (A) Protoplasts were transformed either with the plasmids FKBP-CCT2 and FRB-CCT2 together (lanes 1–3) or with only one plasmid (lanes 4 and 5). Untransformed cells (lane 6) served as a control. Protoplasts were kept in darkness (D) or blue light (B, λmax = 436 nm, 80 μmol m−2 s−1) in the absence (−) or presence (+) of 1 μM AP21967. Non-transfected and singly transfected protoplasts were kept in darkness without inducer. Proteins were isolated 16 h after transfection and equal amounts (10 μg) per lane were separated by SDS–PAGE, western-blotted, and probed with cry2-specific antibodies. The upper band with a molecular weight of approximately 70 kDa corresponds to endogenous cry2 and is strongly reduced in cells treated with blue light for 16 h (lane 3), as expected. Compared with the untransformed control cells (lane 6), single transformed protoplasts showed additional signals of the FKBP–CCT2 protein (lane 4) and the FRB–CCT2 protein (lane 5) that also appeared in the double transformants (lanes 1–3). As estimated from signal strengths, FKBP–CCT2 and FRB–CCT2 proteins were expressed to similar levels (lane 1–3) and the protein levels were not affected by incubation of the protoplasts with 1 μM AP21967 after transfection (compare lane 1 with lane 2). (B) The blot was stripped and reprobed with antibody against constitutively expressed mitochondrial POM 34 protein (Heins et al., 1994) to check for equal loading and transfer. Molecular Plant 2008 1, 4-14DOI: (10.1093/mp/ssm002) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 5 Light-Independent Activation of a Blue Light-Responsive Reporter Gene Construct by CCT2 Dimerization. Arabidopsis protoplasts were cotransfected with the LRU4:GUS reporter gene as well as with 35S:RLUC as a reference gene to normalize for transformation efficiency. In addition, the combinations of either FKBP–CCT2 with FRB–CCT2 (columns 1–4) or FKBP–YFP with FRB–YFP (columns 5 and 6) were cotransfected. After transfection, protoplasts were kept in darkness (D, columns 1, 2, and 5) or under blue light (B, 436 nm, 80 μmol m−2 s−1columns 3, 4, and 6) for 16 h in the absence (−, columns 1, 3, and 6) or presence of 1 μM AP21967 (+, columns 2, 4, and 5). Normalized GUS activity refers to the ratio of GUS units to RLUC units. Data are averages from three independent experiments ± SD. Molecular Plant 2008 1, 4-14DOI: (10.1093/mp/ssm002) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 6 Induced Activation of Endogenous CHS and CHI by CCT2 Dimerization. Transcript levels were quantified by real-time PCR. RNA was isolated from protoplasts transfected with FKBP–CCT2 and FRB–CCT2 constructs (black columns), or from non-transfected protoplasts (gray columns) that were kept in darkness (D, part A, C) or blue light (B, part B, D) for 16 h in either the presence (+) or absence (−) of AP21967 inducer (1 μM). CHS (part A B) and CHI (part C D) transcript levels were normalized to the UBQ signals. Data are averages from three repeats ± SD. Molecular Plant 2008 1, 4-14DOI: (10.1093/mp/ssm002) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions