Volume 5, Issue 3, Pages 726-733 (May 2012) A Study of the Blue-Light-Dependent Phosphorylation, Degradation, and Photobody Formation of Arabidopsis CRY2  Ze-Cheng Zuo, Ying-Ying Meng, Xu-Hong Yu, Zeng-Lin Zhang, De-Shun Feng, Shih-Fan Sun, Bin Liu, Chen-Tao Lin  Molecular Plant  Volume 5, Issue 3, Pages 726-733 (May 2012) DOI: 10.1093/mp/sss007 Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 1 The Lysine Residues of the Putative NLS of CRY2 Are Important for the Nuclear Importation of CRY2. (A) The putative NLS of CRY2. The diagram at the bottom shows the structure of CRY2 protein containing the N-terminal PHR domain and C-terminal CCE domain. The putative NLS of CRY2 (residues from K541 to R557) is aligned with the NLSs of Nucleoplasmin and Nipah virus matrix (NiV-M). Two clusters of basic residues are boxed by blue rectangles. The sequences of the K541R and K554/5R mutants are shown in red. (B) Immunoblot showing the level of protein expression of the CRY2–GFP and mutant proteins of the indicated transgenic lines. Seven-day-old seedlings were grown in the ½ MS medium in continuous red light; total protein extracts were fractioned by a 10% SDS–PAGE gel, blotted, and probed with the anti-CRY2 (CRY2). The Ponceau S staining of the Rubisco band is included as the loading control. (C) REU (Relative Expression Unit) is calculated by the formula [CRY2mt/Rubiscomt]/[CRY2wt/Rubiscowt], in which ‘CRY2’ and ‘Rubisco’ designate the digitized band intensity of CRY2 or Rubisco of the respective CRY2 mutants (mt) or the wild-type CRY2–GFP control (wt). (D) Representative fluorescence images showing the subcellular localization of CRY2–GFP, K541R, and K554/5R in epidermal cells at the top half of the hypocotyls of seedlings grown in dark. (E) The nuclear/cytoplasmic distribution of the indicated proteins is represented by RIU (Relative Intensity Unit), which is calculated by the formula [nuclear GFP fluorescence intensity]/[cytoplasmic GFP fluorescence intensity], n ≥ 20, bar = 5 μm. Molecular Plant 2012 5, 726-733DOI: (10.1093/mp/sss007) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 2 The Hypocotyl Inhibition Response of Transgenic Seedlings Expressing the CRY2 and Mutant Fusion Proteins. (A–E) Images (left) and hypocotyl lengths (right, n ≥ 20) of 7-day-old seedlings of indicated genotypes grown in compound soil under continuous white light (90 μmol m−2 s−1) (A), blue light (15 μmol m−2 s−1) (B), red light (10 μmol m−2 s−1) (C), far-red light (3 μmol m−2 s−1) (D), or in darkness (E). (F) A fluence-rate response of hypocotyl growth of the indicated genotypes grown under continuous blue light for 5 d in ½ MS medium. Hypocotyl lengths and standard deviations (n ≥ 20) are shown. All transgenic lines are in the cry1cry2 background. Molecular Plant 2012 5, 726-733DOI: (10.1093/mp/sss007) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 3 Blue-Light-Dependent Phosphorylation and Degradation of the CRY2 Mutant Proteins. (A–D) Immunoblots showing the level of the CRY2–GFP (A, C), K541R (A), and K554/5R (C) proteins and semi-quantification of degradation (B, D). Seedlings were grown in dark for 7 d, transferred to blue light (∼22 μmol m−2 s−1) for the indicated time (h), and the total protein extracts were analyzed in a 10% SDS–PAGE for immunoblot analyses, which were probed with anti-CRY2 (CRY2); the vPPase detected by vPPase antibody is included as the loading control. Arrows and arrowheads indicate the unphosphorylated or hyperphosphorylated CRY2 fusion proteins (B, E). The relative expression level of CRY2 represented by ‘REU’ (Relative Expression Unit) is calculated by the formula [CRY2t/vPPase t]/[CRY20/vPPase 0], in which ‘CRY2’ and ‘vPPase’ denote digitized band intensities of CRY2 or vPPase of the respective samples collected at time zero (0) or the indicated time after blue-light exposure (t). (E, F) The immunoblot showing the level of CRY2–GFP and CRY2 mutant proteins (E) and a semi-quantification of phosphorylation (F). Seven-day-old etiolated seedlings were transferred to blue light (∼22 μmol m−2 s−1) for the indicated time (h), and analyzed as in (A) and (C). The relative level of CRY2 phosphorylation is calculated by the formula [CRY2Pi]/[CRY2+CRY2Pi], in which CRY2Pi and CRY2 denote digitized band intensities of phosphorylated and unphosphorylated CRY2 signals, respectively. Molecular Plant 2012 5, 726-733DOI: (10.1093/mp/sss007) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 4 Blue-Light-Dependent Formation of Photobodies and Cytoplasmic Photobody-Like Protein Bodies of the K541R and K554/5R Mutant Proteins. Upper half of hypocotyls of 5-day-old etiolated seedlings exposed to blue light (36 μmol m−2 s−1) for the time (0, 1, 5, 30, and 60 min) indicated. Samples were fixed in 4% paraformaldehyde and examined by a fluorescence microscope. The white arrowheads indicate representative cytoplasmic photobody-like protein bodies. Molecular Plant 2012 5, 726-733DOI: (10.1093/mp/sss007) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 5 Formation of the Cytoplasmic CRY2K541R or CRY2K554/5R Photobody-Like Structures. (A) The percentage of nucleus-containing photobodies decreases over time. PCN (Photobody Containing Nucleus) was measured as described in Figure 4, with the standard deviations (n ≥ 200) shown. (B) The number of CPLPB (Cytoplasmic Photobody-Like Protein Bodies) decreases over time. CPLPB per cell is calculated by the formula [total number of CPLPBs in a field of view]/[the number of cells in the same field view]. The standard deviation is derived from three measurements. (C) The size of CPLPB increases over time. Molecular Plant 2012 5, 726-733DOI: (10.1093/mp/sss007) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions