Regulation of Arabidopsis photoreceptor CRY2 by two distinct E3 ubiquitin ligases

Abstract

Cryptochromes (CRYs) are photoreceptors or components of the molecular clock in various evolutionary lineages, and they are commonly regulated by polyubiquitination and proteolysis. Multiple E3 ubiquitin ligases regulate CRYs in animal models, and previous genetics study also suggest existence of multiple E3 ubiquitin ligases for plant CRYs. However, only one E3 ligase, Cul4^COP1/SPAs, has been reported for plant CRYs so far. Here we show that Cul3^LRBs is the second E3 ligase of CRY2 in Arabidopsis. We demonstrate the blue light-specific and CRY-dependent activity of LRBs (Light-Response Bric-a-Brack/Tramtrack/Broad 1, 2 & 3) in blue-light regulation of hypocotyl elongation. LRBs physically interact with photoexcited and phosphorylated CRY2, at the CCE domain of CRY2, to facilitate polyubiquitination and degradation of CRY2 in response to blue light. We propose that Cul4^COP1/SPAs and Cul3^LRBs E3 ligases interact with CRY2 via different structure elements to regulate the abundance of CRY2 photoreceptor under different light conditions, facilitating optimal photoresponses of plants grown in nature.

Fig. 1. LRBs are required for blue light responses.

a 6-day-old seedlings grown in darkness (D), red light (R, 20 μmol m⁻² s⁻¹), far-red light (FR, 6 μmol m⁻² s⁻¹), long days (LD, 16 h light / 8 h dark), or short days (SD, 8 h light / 16 h dark). b Measurements of hypocotyl length of seedlings shown in (a), (mean ± SD). c 6-day-old seedlings grown in darkness or blue light (10 μmol m⁻² s⁻¹). d Measurements of hypocotyl length of indicated genotypes grown under blue light of different intensities (0, 5, 10, 20, and 40 μmol m⁻² s⁻¹) for 6 days, (mean ± SD, n = 25). e, g 6-day old seedlings grown in darkness or blue light (20 μmol m⁻² s⁻¹). f, h Measurements of hypocotyl length of seedlings shown in (e) and (g), (mean ± SD). The above experiments were repeated at least three times with similar results.

Fig. 2. LRBs and COP1 regulate the rapid or prolonged proteolysis of CRY2, respectively.

a Representative immunoblots showing the abundance of endogenous CRY2 in the 7-day-old etiolated wild type (WT) and lrb123 mutants irradiated with 30 μmol m⁻² s⁻¹ of blue light for the indicated time. b Quantitative analysis of CRY2 abundance from immunoblots shown in (a). Data are presented as mean ± SD (n = 3 individual immunoblots). c Representative immunoblots showing the abundance of endogenous CRY2 in seedlings of indicated genotypes grown in darkness (D) or continuous blue light (cB, 30 μmol m⁻² s⁻¹). d Quantitative analysis of CRY2 abundance from immunoblots shown in (c). Data are presented as mean ± SD (n = 3 individual immunoblots). e Representative immunoblots showing degradation of the endogenous CRY2 in 7-day-old etiolated seedlings irradiated with 30 μmol m⁻² s⁻¹ of blue light for indicated time. f Quantitative analysis of CRY2 degradation from immunoblots shown in (e). Data are presented as mean ± SD (n = 3 individual immunoblots). CRY2 (B/D) = [CRY2/HSP90]^blue / [CRY2/HSP90]^dark. The best fitted curves with one phase decay of nonlinear regression were shown. CRY2 and HSP90 were detected with anti-CRY2 antibody and anti-HSP90 antibody, respectively. HSP90 is used as a loading control. Arrows indicate phosphorylated CRY2. The above experiments were repeated at least three times with similar results.

Fig. 3. LRBs interact with phosphorylated CRY2 in the HEK293T cells.

a Co-immunoprecipitation (co-IP) assays showing the blue light and phosphorylation-dependent interaction of LRB1 and CRY2 in heterologous HEK293T cells. Immunoprecipitations (IP) were performed with Flag-conjugated beads. The IP (LRB1) and co-IP (CRY2) products were detected with anti-Flag and anti-CRY2 antibodies, respectively. PPK1 was detected with anti-HA antibody. b Co-IP assays showing the blue light and phosphorylation-dependent interaction of LRB2 and CRY2 in HEK293T cells. The experiments were performed as in (a). c Co-IP assays showing that LRB2 failed to interact with the photo-insensitive CRY2^D387A mutant. The experiments were performed as in (a), except that the IP (CRY2 and CRY2^D387A) and co-IP (LRB2) products were detected with anti-CRY2 and anti-Myc antibodies, respectively. d Co-IP assays showing the phosphorylation-dependent interaction of LRB2 and CRY2 interaction in HEK293T cells. The experiments were performed as in (a). PPK1^D267N, catalytically inactive PPK1. e–f Co-IP assays showing the interaction between CRY2 CCE domain and LRB2. IP was performed with Flag-conjugated beads. The IP (PHR2 and CCE2) and co-IP (LRB2) products were detected with anti-Flag and anti-Myc antibodies, respectively. PHR2, photolyase homologous region of CRY2; CCE2, CRY2 C-terminal extension. The cells were treated with blue light (+ Blue; 100 μmol m⁻² s⁻¹) for 2 h or kept in the dark (-Blue). Arrows show phosphorylated CRY2. The above experiments were repeated three times with similar results.

Fig. 4. LRBs interact with CRY2 in vivo.

a Confocal microscopic images showing BiFC signals of indicated protein pairs transiently expressed in Arabidopsis leaves. The Hoechst 33342 (which is used to stain the nuclei) and GFP (BiFC) signals are shown. The relative intensity of CRY2/LRB interaction was presented as BiFC ratio, calculated as BiFC ratio = [GFP intensity]^nuclei / [Hoechst 33342 intensity]^nuclei. BiFC ratio (mean ± SD), total number of quantified images and nuclei are shown blow the images. CRY2^D387A, photo-insensitive CRY2, is used as the negative control. Scale bars, 10 μm. b Split-luciferase complementation assays showing the interactions of LRB1 and CRY2 or LRB2 and CRY2 in tobacco. Indicated split-LUC protein pairs were transiently co-expressed in N. benthamiana with FGFP-PPK1. LRB1ΔBTB, LRB1 with deletion of BTB domain (143-212 aa); LRB2ΔBTB, LRB2 with deletion of BTB domain (145-250 aa). c Quantification of split-luciferase complementation assays shown in (b). Data was shown as mean ± SD (n = 3 individual experiments). d–e Co-immunoprecipitation assays showing the interactions of LRB1 and CRY2 (d) or LRB2 and CRY2 (e) in Arabidopsis. 7-day-old seedlings expressing Myc-LRB or co-expressing FGFP-CRY2 and Myc-LRB were grown in the dark and then treated with blue light (100 μmol m⁻² s⁻¹) for the indicated time, or grown in long days (LD, 16 h light/ 8 h dark) and continuous white light (cWL). Plant extracts were immunoprecipitated by GFP-trap beads. The IP and co-IP products were detected with anti-Flag and anti-Myc antibodies, respectively. Co-IP experiments of 7-day-old seedlings co-expressing FGFP-CRY2 and Myc-CRY2 were performed in parallel as positive controls. The relative interaction intensity of CRY2/LRB or CRY2/CRY2 is calculated by [Co-IP intensity] / [IP intensity] and shown blow the immunoblots. The above experiments were repeated twice with similar results.

Fig. 5. LRBs and COP1 are both required for CRY2 ubiquitination.

Immunoblots showing the ubiquitination of FGFP-CRY2 in indicated genotypes. 7-day-old etiolated seedlings constitutively expressing FGFP-CRY2 in indicated genotypes were pretreated with MG132 and kept in the dark or exposed to 30 μmol m⁻² s⁻¹ of blue light for 5, 10, and 15 min. The blue light treated samples (5, 10, and 15 min) were pooled together while performing immunoprecipitation for (a–e). a–c Total ubiquitinated proteins were purified by TUBE2-conjugated beads. Immunoprecipitated proteins were analyzed by immunoblots probed with anti-ubiquitin antibody (α-Ubq) or anti-CRY2 antibody (α-CRY2). The extent of CRY2 ubiquitination was shown below the immunoblots, calculated as [CRY2-Ubq intensity]^IP/[Ubq intensity]^IP. d–e FGFP-CRY2 proteins were purified with GFP-trap beads. Immunoprecipitated proteins were analyzed by immunoblots probed with anti-ubiquitin antibody (α-Ubq), anti-Flag antibody (α-Flag) or anti-Myc antibody (α-Myc) for detecting epitope-tagged proteins. The extent of CRY2 ubiquitination is calculated as [CRY2-Ubq intensity]^IP/[Flag intensity]^IP with the short exposure immunoblots and shown below the immunoblots. S. exp or L. exp: short or long chemiluminescence exposures of immunoblots. D, dark treatment; B, blue light treatment. The above experiments were repeated at least twice with similar results.

Fig. 6. LRBs and COP1 interact with different structural elements of CRY2.

a A schematic diagram depicting the CRY2^P532L mutation. PHR, photolyase homologous region; CCE, CRY C-terminal extension; DQQVPSAV, VP motif in CRY2; numbers, amino acid positions. b Hypocotyl length of 6-day-old seedlings of indicated genotypes grown under blue light of different light intensities. Data were shown as mean ± SD. c Immunoblots showing degradation of FGFP-CRY2 or FGFP-CRY2^P532L in 7-day-old etiolated seedlings irradiated with blue light (100 μmol m⁻² s⁻¹) for the indicated time. Immunoblots were probed with anti-CRY2 and anti-HSP90 antibodies. d Quantitative analysis of CRY2 degradation from immunoblots shown in (c), n = 3 individual immunoblots. The best fitted curves with one phase decay of nonlinear regression were shown. CRY2 (B/D) = [CRY2/HSP90]^blue / [CRY2/HSP90]^dark. T_1/2 indicates the time required for 50% degradation. e–f Co-IP assays showing the lack of CRY2^P532L/COP1 interaction (e) and the blue light- and phosphorylation-dependent CRY2^P532L/LRB2 interaction (f) in HEK293T cells. Transfected cells were either kept in the dark (− Blue) or treated with 100 μmol m⁻² s⁻¹ blue light for 2 h (+ Blue). CRY2^D387A is used as a negative control. Anti-Flag, anti-Myc or anti-HA antibodies were used for detecting indicated tagged proteins. Arrows indicate phosphorylated CRY2. g Co-IP results showing the photooligomerization of CRY2^P532L in HEK293T cells. Transfected cells were irradiated with blue light (30 μmol m⁻² s⁻¹) for indicated time. CRY2^P532L tagged with Flag or Myc were detected with anti-Flag and anti-Myc antibodies, respectively. h Comparison of photooligomerization kinetics of CRY2 and CRY2^P532L in HEK293T cells. Co-IP (α-Myc) signals was normalized to the corresponding IP (α-Flag) signals, and the dark oligomerization level was set as 1. O₅₀ indicates the time required to reach 50% saturation of CRY2 photooligomerization. The best fitted curves with one phase association of nonlinear regression were shown. i Co-IP results showing the dark reversion of CRY2^P532L photooligomers in HEK293T cells. The cells were treated with 30 μmol m⁻²s⁻¹ blue light for 5 min then moved to the darkness for indicated time. j Comparison of the dark reversion dynamics of CRY2 and CRY2^P532L photooligomers in HEK293T cells. Similar analyses were performed as in (h), except that the oligomerization level in blue was set as 1. T_1/2 indicates the time required to reverse 50% of CRY2 photooligomers into monomers. The best fitted curves analyzed with one phase decay of nonlinear regression were shown. CRY2 photooligomerization and dark reversion data shown in (h) and (j) were extracted from the published paper with permission. The above experiments were repeated at least twice with similar results.

Fig. 7. Blue light positively regulates LRBs protein abundance in plants.

a A hypothetic model depicting the degradation of CRY2 by LRBs and COP1 E3 ubiquitin ligases. CRY2 exists as inactive monomers in darkness (grey). Upon blue light exposure, CRY2 undergoes photoexcitation, oligomerization, and phosphorylation to become photoactive. Photoactivated CRY2 then were targeted by two distinct E3 ubiquitin ligases, LRBs, and COP1, for degradation. Minus sign in a circle, indicates negative charge; dashed line, indicates a hypothetical mechanism. b–e Representative immunoblots (top) and a quantitative analysis (bottom) showing the expression of recombinant LRB proteins in transgenic plants driven by native promoters (b, d) or the ACT2 constitutive promoter (c, e). Seedlings were grown in darkness or continuous blue light (50 μmol m⁻² s⁻¹) for 5, 6, or 7 days. Proteins were extracted and subjected to western blot analysis. LRBs and HSP90 were detected with anti-Flag and anti-HSP90 antibodies, respectively. HSP90 is used as a loading control. Data were shown as mean ± SD (n = 3 individual immunoblots). The above experiments were repeated at least three times with similar results.