A study of the blue-light-dependent phosphorylation, degradation, and photobody formation of Arabidopsis CRY2

Abstract

Arabidopsis cryptochrome 2 (CRY2) is a blue-light receptor mediating blue-light inhibition of hypocotyl elongation and photoperiodic promotion of floral initiation. CRY2 is a constitutive nuclear protein that undergoes blue-light-dependent phosphorylation, ubiquitination, photobody formation, and degradation in the nucleus, but the relationship between these blue-light-dependent events remains unclear. It has been proposed that CRY2 phosphorylation triggers a conformational change responsible for the subsequent ubiquitination and photobody formation, leading to CRY2 function and/or degradation. We tested this hypothesis by a structure-function study, using mutant CRY2-GFP fusion proteins expressed in transgenic Arabidopsis. We show that changes of lysine residues of the NLS (Nuclear Localization Signal) sequence of CRY2 to arginine residues partially impair the nuclear importation of the CRY2K541R and CRY2K554/5R mutant proteins, resulting in reduced phosphorylation, physiological activities, and degradation in response to blue light. In contrast to the wild-type CRY2 protein that forms photobodies exclusively in the nucleus, the CRY2K541R and CRY2K554/5R mutant proteins form protein bodies in both the nucleus and cytosol in response to blue light. These results suggest that photoexcited CRY2 molecules can aggregate to form photobody-like structure without the nucleus-dependent protein modifications or the association with the nuclear CRY2-interacting proteins. Taken together, the observation that CRY2 forms photobodies markedly faster than CRY2 phosphorylation in response to blue light, we hypothesize that the photoexcited cryptochromes form oligomers, preceding other biochemical changes of CRY2, to facilitate photobody formation, signal amplification, and propagation, as well as desensitization by degradation.

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 [CRY2^mt/Rubisco^mt]/[CRY2^wt/Rubisco^wt], 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.

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.

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 [CRY2^t/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.

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.

Figure 5.

Formation of the Cytoplasmic CRY2^K541R or CRY2^K554/5R 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.