(A) Effect of reduced photoperiods in the flowering time of cop1-4, DN-COP1, elf3-8, and elf3-8 cop1-4 plants. Plants were entrained in LD (L:D = 2:1) and SD (L:D = 1:2) of 24 hr (24T; LD = 16L/8D, SD = 8L/16D), 21 hr (21T; LD = 14L/7D, SD = 7L/14D), and 18 hr (18T; LD = 12L/6D, SD = 6L/12D). Asterisks indicate that WT (Col) plants grown in SD (21T and 18T) had yet not bolt when more than 55 rosette leaves were counted. Mean and standard deviation values of at least 15 plants are shown. (B) Phenotypes of cop1-4, DN-COP1, elf3-8, and elf3-8 cop1-4 mutants at bolting under SD of 18T. Plants were grown at 22°C under cool-white fluorescent light (100 μmol m⁻² s⁻¹). Bars = 2 cm.

(A–D) Rhythmic patterns of ELF3 (A), GI (B), CO (C) and FT (D) mRNA abundance in WT (Col) and cop1-4 mutant plants. Total RNA samples were collected every 2 hr from 10-d-old and 20-d-old plants entrained in LD and SD of 24 hr, respectively. mRNA abundance was quantified by semiquantitative RT-PCR and expressed relative to the abundance of TUBULIN2 (TUB2) transcripts (E). Grey areas behind the traces represent night periods.
(F) Abundance of FLC and SOC1 transcripts in WT (Col), cop1-4 and elf3-8 mutants. Equal amounts of total RNA corresponding to time points ranging from 1 to 23 hr were mixed for templates of semiquantitative RT-PCR. W, WT (Col); C, cop1-4; E, elf3-8.
(A–F) Plants were grown at 22°C under cool-white fluorescent light (100 μmol m⁻² s⁻¹). Mean and standard deviation values of three replicates are shown.

(A-C) Cycling and relative abundance of native ELF3 protein in WT and cop1-4 plants under LD (A, C) and SD (B, C). Ten μg of nuclear protein extracts from 10-d-old seedlings was loaded into each lane. Nuclear protein extracts of elf3-1 null mutant (Col) was used as negative control (A, B). Proteasome subunit RPT5 protein abundance was detected as loading control (A, B). Open and dark areas represent day and night periods, respectively.
(D) Effect of ELF3 overexpression on flowering time under cop1-4 mutation. Mean and standard deviation values were obtained from at least 20 plants. W, WT (Col); E, ELF3-OX; C, cop1-4; EC, ELF3-OX cop1-4.
(E–F) Periodic accumulation of LHY transcripts in WT, cop1-4, ELF3-OX and ELF3-OX cop1-4 plants. Ten μg of total RNA from LL-entrained plants was loaded into each lane. Radiolabeled LHY cDNA was used as a probe. LHY mRNA levels are expressed relative to the abundance of ELONGATION FACTOR 1a (EF1a) transcripts (F). White and grey areas represent subjective days and nights, respectively.

(A) COP1 and ELF3 coordinately regulate flowering time and circadian rhythms by modulating the biological activity of GI on light-input signaling to the circadian clock. At night, COP1 and ELF3 highly accumulate in the nucleus, where they interact and bind to GI to promote its degradation. In this process, ELF3 may act as a substrate adaptor to allow COP1-GI interaction, which also results in COP1-mediated degradation of ELF3, possibly to limit the extent of ELF3 function. Dark-driven GI destabilization mediated by COP1 and ELF3 plays an antagonistic role to blue-light enhanced formation of ZTL-GI and FKF1-GI complexes, whose cyclic accumulation is essential for proper clock oscillation and timely photoperiodic flowering, respectively (Kim et al., 2007; Sawa et al., 2007). By inhibiting COP1 activity, CRY may help to stabilize GI, having thus an overlapping function with blue-light sensing ZTL/FKF1/LKP2 proteins.
(B) COP1 roles in the control of flowering time involve regulation of CO function at both transcriptional and post-translational levels. Regulation of GI stability allows COP1 and ELF3 to modulate expression of floral inducer CO, a gene positively controlled by the FKF1-GI complex. Additionally, COP1 mediates degradation of CO, mainly at night, limiting thus its function in the promotion of flowering in response to seasonal changes in photoperiod.

(A) Phenotypes of cop1 mutants (cop1-4, cop1-6, and DN-COP1), and of double and triple mutants of cop1-4 with different flowering-time mutants corresponding to the four genetic pathways of floral induction (see Table S1). Plants were grown at 22°C under cool-white fluorescent light (100 μmol m⁻² s⁻¹) in LD (16L/8D) and SD (8L/16D), and photographed at 2 to 3 d after bolting. Bars = 2 cm.
(B) Epistatic relationship between cop1-4 and cry2-1, elf3-8, gi-1, and ft-1 soc1-1 mutations in the regulation of flowering time. Flowering time was measured as the number of rosette leaves at bolting (see Table S1).
(C) Genetic model of COP1 regulation in floral induction pathways. COP1 influences flowering time by mediating light input signaling from CRY2 to GI. Based on our genetic data, epistatic relationships between COP1 and ELF3 cannot be definitively drawn. Thus, COP1 and ELF3 may act sequentially or at the same level (shown as a square enclosing both COP1 and ELF3). Genetic interactions previously described in the literature are also shown (for a review, see Komeda, 2004).

(A) COP1 interacts with ELF3 in yeast two-hybrid assays. COP1 was used as bait in pGBK vector. The following preys, cloned into pGAD vector, were used: empty pGAD, as a negative control (1), CCT1, for positive control (2), LHY (3), CCA1 (4), TOC1 (5), ELF3 (6), ZTL (7), FKF1 (8), LKP2 (9), GI (10), and CO (11). Full-length cDNAs were used except for CCT1. CCT1 (A, B) represents the C-terminal domain of CRY1 (aa 486–681)
(B) ELF3 interacts most strongly with the RING-finger domain of COP1 in yeast two-hybrid assays. Baits in pGBK vectors and preys in pGAD vectors (bait::prey) were co-transformed into yeast such as full-length COP1::empty pGAD vector for negative control (1), p53::T for positive control (Clontech) (2), COP1::ELF3 (3), RING-finger domain of COP1 (aa 1–104)::ELF3 (4), coiled-coil domain of COP1 (121–213)::ELF3 (5), seven WD-40 repeat domain of COP1 (371–675)::ELF3 (6), COP1::CCT1 (7), and COP1::CCT2 (8). CCT2 represents the C-terminal domain of CRY2 (501–612). CCT1 and CCT2 were used as positive controls.
(C) In vivo interaction between COP1 and ELF3. Immunoblots of inmunoprecipitated samples from TAP-COP1 and TAP-GFP plants (upper panels). Anti-myc (to detect TAP-tagged proteins) and anti-ELF3 antibodies were used. RbcL protein levels were visualized as input control (total soluble protein) by Coomassie Brilliant Blue staining. A comparison of ELF3 levels (only detectable in nuclear extracts; Liu et al., 2001) in three different samples of TAP-COP1 and TAP-GFP plants (20 μg nuclear protein/lane) and the effect of proteasome inhibitor MG132 (50 μM) on ELF3 stability are also shown (bottom panels). Both ELF3 and RPT5 (loading control) were detected by ECL system.
(D) COP1 ubiquitinates ELF3 in vitro. GST-ELF3 ubiquitination assays were performed using MBP-COP1 (or MBP as a negative control), rice E2 Rad6 (E2), and yeast E1 (E1; Boston Biochem). Assay conditions were as previously described (Saijo et al., 2003). Ubiquitinated GST-ELF3 was detected using anti-ELF3 (left panel), anti-GST (middle panel) or anti-Ub (right panel) antibodies. Asterisks in middle and right panels indicate the position of MBP-COP1 (non-specific reaction of anti-GST) and ubiquitinated MBP-COP1 (anti-Ub), respectively.
(E) COP1 and the proteasome control ELF3 stability in vivo. Immunoblot analysis of protein extracts corresponding to agro-infiltrated N. benthamiana leaves with indicated plasmids in the presence or absence of MG132 (25 μM). HA-ELF3 (upper panel) and HA-GFP (input control; middle lower panel) were detected using anti-HA antibody, and Flag-COP1 (middle upper panel) using anti-Flag antibody. HA-ELF3 (ELF3) and ACTIN1 (ACT1) mRNA expression levels were analyzed by competitive RT-PCR (bottom panel).

(A) ELF3 interacts with GI in yeast two-hybrid assays. As preys, full-length ELF3 (F; aa 1–695) or ELF3 N-terminal (N; 1–261), middle (M; 261–440), C-terminal (C; 440–695), NM (1–440), and MC (261–695) regions (Liu et al., 2001). For baits, GI was divided by three parts, such as N-terminal (N; 1–507), middle (M; 401–907), and C-terminal (C; 801–1173). p53::T indicates a positive control (Clontech). Empty pGBK (bait) and pGAD (prey) plasmids were used as negative controls.
(B) RING-finger and coiled-coil domains of COP1 interact with N-terminal region of GI in yeast two-hybrid assays. Full-length and three domains of COP1 were used as baits (see Figure 4B), and full-length (1–1173) and three parts of GI in (A) as preys. R, RING-finger; CC, coiled-coil; W, WD40 repeat domains of COP1.
(C) BiFC visualization of COP1-ELF3, ELF3-GI and COP1-GI interactions in the nucleus of onion epidermal cells. Empty BiFC plasmids were used as a negative control. For COP1-ELF3 (positive control) and ELF3-GI interactions, two BiFC constructs encoding the indicated partial-YFP fusions were co-bombarded into cell layers. For COP1-GI interaction, HA-ELF3-expressing plasmids were co-bombarded with the cYFP-COP1 and nYFP-GI constructs. In all cases, YFP signals were only detectable in the presence of MG132 (50 μM) and upon dark incubation. Transient expression of nYFP-COP1 and cYFP-ELF3, cYFP-ELF3 and GI-nYFP, and nYFP-COP1, cYFP-GI with or without HA-ELF3 showed the same results (data not shown). These experiments were repeated at least three times with similar results. Numbers in bars =μm. DIC, differential interference contrast.
(D) GI accumulation is controlled by COP1 and ELF3 in a proteasome-dependent manner. Immunoblots of protein extracts corresponding to agro-infiltrated N. benthamiana leaves with indicated plasmids in the presence or absence of MG132 (25 μM). GI-GFP (two upper panels, corresponding to short and long immunoblot exposures), Flag-COP1, and HA-ELF3 and HA-GFP (input control) were detected using anti-GFP, anti-Flag and anti-HA antibodies, respectively. GI-GFP (GI) and ACTIN1 (ACT1) mRNA expression levels in agro-infiltrated leaves were analyzed by competitive RT-PCR (bottom panel).
(E) Degradation of ³⁵S-labelled GI (TNT GI) after incubation for the indicated times (min) with cellular extracts from WT (Col), MG132-treated WT (WT+MG132), cop1-4, elf3-8 or elf3-8 cop1-4 plants grown under LD and harvested at ZT22. Mean and standard deviation values of three replicates are shown.
(F) Cycling and relative abundance of GI-GFP protein in WT, cop1-4 and elf3-8 plants grown under SD. Total protein extracts (100 μg) from 20-d-old seedlings were loaded into each lane. Anti-GFP antibody was used to detect GI-GFP. Proteasome subunit RPT5 protein abundance was detected as loading control. Open and dark areas represent day and night periods, respectively.