CCA1 and ELF3 Interact in the control of hypocotyl length and flowering time in Arabidopsis

Abstract

The circadian clock is an endogenous oscillator with a period of approximately 24 h that allows organisms to anticipate, and respond to, changes in the environment. In Arabidopsis (Arabidopsis thaliana), the circadian clock regulates a wide variety of physiological processes, including hypocotyl elongation and flowering time. CIRCADIAN CLOCK ASSOCIATED1 (CCA1) is a central clock component, and CCA1 overexpression causes circadian dysfunction, elongated hypocotyls, and late flowering. EARLY FLOWERING3 (ELF3) modulates light input to the clock and is also postulated to be part of the clock mechanism. elf3 mutations cause light-dependent arrhythmicity, elongated hypocotyls, and early flowering. Although both genes affect similar processes, their relationship is not clear. Here, we show that CCA1 represses ELF3 by associating with its promoter, completing a CCA1-ELF3 negative feedback loop that places ELF3 within the oscillator. We also show that ELF3 acts downstream of CCA1, mediating the repression of PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5 in the control of hypocotyl elongation. In the regulation of flowering, our findings show that ELF3 and CCA1 either cooperate or act in parallel through the CONSTANS/FLOWERING LOCUS T pathway. In addition, we show that CCA1 represses GIGANTEA and SUPPRESSOR OF CONSTANS1 by direct interaction with their promoters, revealing additional connections between the circadian clock and the flowering pathways.

Figure 1.

CCA1 and ELF3 form a negative feedback loop. A, CCA1 expression in wild-type (WT), elf3-1, and ELF3-OX plants under diurnal and continuous light (LL) conditions. B, ELF3 expression in wild-type, cca1-1, and CCA1-OX plants under diurnal and LL conditions. Ten-day-old seedlings sampled at 4-h intervals were analyzed by qRT-PCR. Day, night, and subjective night are denoted by white, black, and hatched bars, respectively. The data are presented as means of two biological replicates ± sd. All experiments were done at least twice with similar results.

Figure 2.

CCA1 binds directly to the promoter of ELF3 to repress its expression. A, Schematic drawing of genomic ELF3 and the regions examined by ChIP. Coding regions are represented by black boxes, untranslated regions are represented by gray boxes, and introns are represented by lines. The CBS (C) is represented by a white bar. B, Binding of CCA1 to the ELF3 promoter in vivo. ChIP assays were performed using CCA1 antibody (Ab) with wild-type (WT), cca1-1, and CCA1-OX (OX) seedlings collected at ZT2 and ZT14. qRT-PCR was performed on the precipitates; ELF3-P amplifies the region that contains a CBS, and ELF3-N amplifies a region in the downstream coding region. Results were normalized to the input DNA. C and D, A pulse of CCA1 expression represses the accumulation of ELF3 RNA. Control (C) and Alc::CCA1 (D) seedlings were given an ethanol (EtOH) pulse at 32 h in continuous light (circadian time [CT]-8). qRT-PCR analysis of ELF3 expression at the time of induction (0 h) and at 1, 2, and 4 h after ethanol treatment is presented. Diamonds, No ethanol treatment; squares, 1% ethanol. Means of two biological replicates ± sd are shown. All of these experiments were done at least twice with similar results.

Figure 3.

Constitutive overexpression of CCA1 protein affects hypocotyl growth in LD and SD. Wild-type (circles), elf3-1 (squares), CCA1-OX (diamonds), and elf3-1 CCA1-OX (triangles) plants were grown for 5 to 7 d under LD (16 h of light/8 h of dark) conditions (A) or under SD (8 h of light/16 h of dark) conditions (B). Average hypocotyl lengths ± sd (n = 20) are presented. All experiments were done at least twice with similar results.

Figure 4.

Transcript abundance of PIF4 and PIF5 in LD and SD. Expression levels of PIF4 (A and C) and PIF5 (B and D) transcripts in wild-type (diamonds), elf3-1 (squares), CCA1-OX (triangles), and elf3-1 CCA1-OX (circles) plants are shown. Eight-day-old seedlings grown under LD (16 h of light/8 h of dark) conditions (A and B) or 12-d-old seedlings grown under SD (8 h of light/16 h of dark) conditions (C and D) sampled at 4-h intervals were analyzed by qRT-PCR. Day and night are denoted by white and black bars, respectively. The data are presented as means of two biological replicates ± sd. All experiments were done at least twice with similar results.

Figure 5.

Constitutive overexpression of CCA1 protein delays flowering in LD and SD. A, Photograph of plants of various genotypes grown for 25 d under LD (16 h of light/8 h of dark) conditions. B and C, Flowering time of seedlings of various genotypes under LD (16 h of light/8 h of dark) conditions. D, Photograph of plants of various genotypes grown for 50 d under SD (8 h of light/16 h of dark) conditions. E and F, Flowering time of seedlings of various genotypes under SD (8 h of light/16 h of dark) conditions. Flowering time is expressed as either days to bolting or rosette leaf number. Data are means ± sd (n = 18–25). WT, Wild type.

Figure 6.

Transcript abundance of flowering time genes under LD (16 h of light/8 h of dark) conditions. Expression levels of FT (A), SOC1 (B), FLC (C), CO (D), and GI (E) transcripts in wild-type (WT; diamonds), elf3-1 (squares), CCA1-OX (triangles), and elf3-1 CCA1-OX (circles) plants are shown. Eight-day-old seedlings grown under LD (16 h of light/8 h of dark) conditions sampled at 4-h intervals were analyzed by qRT-PCR. Day and night are denoted by white and black bars, respectively. The data are presented as means of two biological replicates ± sd. All experiments were done at least twice with similar results.

Figure 7.

Transcript abundance of flowering time genes under SD (8 h of light/16 h of dark) conditions. Expression levels of FT (A), SOC1 (B), FLC (C), CO (D), and GI (E) transcripts in wild-type (WT; diamonds), elf3-1 (squares), CCA1-OX (triangles), and elf3-1 CCA1-OX (circles) plants are shown. Twelve-day-old seedlings grown under SD (8 h of light/16 h of dark) conditions sampled at 4-h intervals were analyzed by qRT-PCR. Day and night are denoted by white and black bars, respectively. The data are presented as means of two biological replicates ± sd. All experiments were done at least twice with similar results.

Figure 8.

CCA1 binds directly to the promoter of GI to repress its expression. A, Schematic drawing of genomic GI and the regions examined by ChIP. Coding regions are represented by black boxes, untranslated regions are represented by gray boxes, and introns are represented by lines. The CBS (C) is represented by a white bar. B, Binding of CCA1 to the GI promoter in vivo. ChIP assays were performed with wild-type (WT), cca1-1, and CCA1-OX (OX) seedlings collected at ZT2 and ZT14. qRT-PCR was performed on the precipitates; GI-P amplifies the region that contains a CBS, and GI-N amplifies a region in the downstream coding region. Results were normalized to the input DNA. C and D, A pulse of CCA1 expression represses the accumulation of GI RNA. Control (C) and Alc::CCA1 (D) seedlings were given an ethanol (EtOH) pulse at 32 h in continuous light (CT-8). qRT-PCR analysis of GI expression at the time of induction (0 h) and at 1, 2, and 4 h after ethanol treatment is presented. Diamonds, No ethanol treatment; squares, 1% ethanol. Means of two biological replicates ± sd are shown. All of these experiments were done at least twice with similar results.

Figure 9.

CCA1 binds directly to the promoter of SOC1 to repress its expression. A, Schematic drawing of genomic SOC1 and the regions examined by ChIP. Coding regions are represented by black boxes, untranslated regions are represented by gray boxes, and introns are represented by lines. The CBS (C) is represented by a white bar. B, Binding of CCA1 to the SOC1 promoter in vivo. ChIP assays were performed with wild-type (WT), cca1-1, and CCA1-OX (OX) seedlings collected at ZT2 and ZT14. qRT-PCR was performed on the precipitates; SOC1-P amplifies the region that contains a CBS, and SOC1-N amplifies a region in the downstream coding region. Results were normalized to the input DNA. C and D, A pulse of CCA1 expression represses the accumulation of SOC1 RNA. Control (C) and Alc::CCA1 (D) seedlings were given an ethanol (EtOH) pulse at 32 h in continuous light (CT-8). qRT-PCR analysis of SOC1 expression at the time of induction (0 h) and at 1, 2, and 4 h after ethanol treatment is presented. Diamonds, No ethanol treatment; squares, 1% ethanol. Means of two biological replicates ± sd are shown. All of these experiments were done at least twice with similar results.

Figure 10.

A proposed model for how the clock-associated factors CCA1, ELF3, and PRR9 affect the expression of members of the photoperiodic flowering pathway. Solid lines indicate direct regulation. Connections that were confirmed in this study are represented by thick lines. Dotted lines indicate indirect regulation (i.e. additional steps and/or components are involved but not shown here).