Photoactivation and inactivation of Arabidopsis cryptochrome 2

Abstract

Cryptochromes are blue-light receptors that regulate development and the circadian clock in plants and animals. We found that Arabidopsis cryptochrome 2 (CRY2) undergoes blue light-dependent homodimerization to become physiologically active. We identified BIC1 (blue-light inhibitor of cryptochromes 1) as an inhibitor of plant cryptochromes that binds to CRY2 to suppress the blue light-dependent dimerization, photobody formation, phosphorylation, degradation, and physiological activities of CRY2. We hypothesize that regulated dimerization governs homeostasis of the active cryptochromes in plants and other evolutionary lineages.

Fig. 1.. BIC1 and BIC2 suppress the functions of Arabidopsis cryptochromes.

(A) Five-day-old seedlings overexpressing BIC1 (bic1D-1) or BIC2-GFP (BIC2-GFP) and cry1cry2 or bic1bic2 mutant seedlings grown in continuous blue light (4 μmol m⁻² s⁻¹) or dark; WT, wild type. (B) Hypocotyl lengths of 5-day-old seedlings grown under continuous blue light with fluence rates of 0.1 to 100 μmol m⁻² s⁻¹. (C) Hypocotyl lengths of indicated genotypes grown in the dark (D), under blue light (B; 10 μmol m⁻² s⁻¹), under red light (R; 10 μmol m⁻² s⁻¹), or under far-red light (FR; 5 μmol m⁻² s⁻¹) for 5 days. (D) Plants of indicated genotypes grown in LD photoperiods (16 hours light, 8 hours dark) for 31 days. (E and F) Days to flowering (E) and number of rosette leaves at the time of flowering (F) of the indicated genotypes grown in LD. (G to J) Immunoblots of samples prepared from seedlings grown in continuous blue light (30 μ;mol m⁻² s⁻¹) (G), LD photoperiods (H), darkness (I), or continuous red light (30 μmol m⁻² s⁻¹) (J) for 6 or 9 days were probed with antibodies to CRY2, GFP, or HSP90 (loading control). Data in (B), (C), (E), and (F) are means ± SD (n > 20); **P < 0.01.

Fig. 2.. CRY and BIC regulate similar transcriptome changes in response to blue light.

(A) Venn diagram depicting overlaps among blue light–regulated, CRY-regulated, and BIC-regulated genes determined by RNA sequencing. Five-day-old etiolated seedlings were exposed to blue light (20 μmol m⁻² s⁻¹) or kept in the dark for 2 hours.The blue light–regulated genes are defined as those that showed a factor of >2 change of mRNA [fold change (FC) > 2, P < 0.01, false discovery rate <*** 0.01; see table S2 for details] between light- and dark-treated wild-type plants. The blue light–regulated genes that showed 200% reduction of FC or changed significance to P>0.01 in cry1cry2 or bic1D-1 are defined as CRY-regulated and BIC-regulated genes, respectively; those that showed similar change in both cry1cry2 and bic1D-1 are defined as CRY- and BIC-regulated (“CB-reg”) genes. (B) Scatter-plot showing plots of log2 (FC) of wild-type (x axis) versus log2 (FC) of cry1cry2 or bic1D-1 (y axis). The dashed lines indicate log2 (FC) = 1 and −1. The colored dots (see key at top) indicate up-regulated or down-regulated genes not co-regulated by CRY and BIC1 (“Non CB-reg”) and genes that are co-regulated by CRY and BIC1 (“CB-reg”). (C and D) Hierarchical clustering (GENE-E) of the change of expression of blue light–induced genes (C) and blue light-repressed genes (D) detected in the indicated genotypes. Scale bars at top indicate relative expression levels of mRNA.

Fig. 3.. BIC1 and BIC2 inhibit early photoreactions of cryptochromes.

(A and B) BIC1 inhibits blue light–dependent phosphorylation of CRY1 and CRY2 and blue light–dependent degradation of CRY2. Immunoblots of samples prepared from 7-day-old etiolated seedlings (WT, wild type; +BIC1, BIC1-overexpressing) exposed to blue light (30 μmol m⁻² s⁻¹) for the indicated times were probed with antibodies to CRY1, CRY2, or HSP90 (loading control). Arrowheads indicate upshift bands of the phosphorylated CRYs. (C to E) Quantification of band intensities, showing CRY phosphorylation [(C), CRY1Pi/CRY1; (D), CRY2Pi/CRY2] or CRY2 degradation [(E), CRY2^B/CRY2^D]. (F) BIC inhibition of CRY2 photobodies. Protoplasts isolated from 4-week-old plants of indicated genotypes were transfected to express CRY2-YFP, exposed to blue light (20 μmol m⁻²s⁻¹) for 0 to 60 min, fixed in 1% formaldehyde, and examined by a fluorescence microscope. Scale bar, 5 μm. (G) BiFC assay showing the blue light-independent (dispersed fluorescence) and blue light–dependent (photobodies) interactions between nYFP-CRY2 and cCFP-CRY2. Protoplasts transfected to express nYFP-CRY2 and cCFP-CRY2 were exposed to blue light and analyzed as in (F). (H) The CRY2-YFP photobody of the experiment shown in (F) was digitized and quantified; 30 nuclei per sample were counted, and the percentage of protoplasts containing photobodies was calculated (±SD; n = 3). (I) Same as (H) but for the nYFP-CRY2/cCFP-CRY2 photobody shown in (G).

Fig. 4.. BIC1 interacts with CRY2 to inhibit blue light–dependent CRY2 dimerization and CRY2-CIB1 interaction.

(A) Seven-day-old etiolated seedlings expressing 35S::BIC1-GFP or 35S::GFP (control) were exposed to blue light (30 μmol m⁻² s⁻¹) for 0 to 120 min and immunoprecipitated by GFP-trap beads. The IP (BIC1) and co-IP signals (CRY2) were detected by immunoblots probed with antibodies to GFP and CRY2, respectively. (B) HEK293T cells were cotransfected to express the indicated proteins, exposed to blue light (180 μmol m⁻² s⁻¹) for 0 to 120 min, and immunoprecipitated by antibody to Flag (Flag-IP).The IP signal (Flag-CRY2) or the co-IP signals (Myc-CRY2 and BIC1) were detected by immunoblots probed with antibodies to Flag or to Myc and GFP, respectively. (C) Co-IP assays showing light-independent dimerization of human CRY2 (hCRY2) in HEK293T cells. (D) Seven-day-old etiolated transgenic seedlings coexpressing the indicated proteins were exposed to blue light (30 μmol m⁻² s⁻¹) for 0 to 10 min and immunoprecipitated by the GFP-trap beads. The IP signal (GFP-CRY2) and the co-IP signal (Myc-CRY2) were detected by immunoblots probed with antibodies to GFP or Myc, respectively. (E) HEK293T cells transfected to express the indicated fusion proteins were kept in the dark (–Blue) or exposed to blue light (180 μmol m⁻² s⁻¹) for 2 hours (+Blue) and analyzed by co-IP assay as in (B). (F) HEK293T cells coexpressing the indicated proteins were exposed to blue light (180 μmol m⁻² s⁻¹) for 0 to 120 min. The IP signal (CRY2) or the co-IP signals (CIB1 and BIC1) were detected by immunoblots probed with antibodies to Flag or to GFP and Myc, respectively.