PSEUDO-RESPONSE REGULATORS 9, 7, and 5 are transcriptional repressors in the Arabidopsis circadian clock

Abstract

An interlocking transcriptional-translational feedback loop of clock-associated genes is thought to be the central oscillator of the circadian clock in plants. TIMING OF CAB EXPRESSION1 (also called PSEUDO-RESPONSE REGULATOR1 [PRR1]) and two MYB transcription factors, CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), play pivotal roles in the loop. Genetic studies have suggested that PRR9, PRR7, and PRR5 also act within or close to the loop; however, their molecular functions remain unknown. Here, we demonstrate that PRR9, PRR7, and PRR5 act as transcriptional repressors of CCA1 and LHY. PRR9, PRR7, and PRR5 each suppress CCA1 and LHY promoter activities and confer transcriptional repressor activity to a heterologous DNA binding protein in a transient reporter assay. Using a glucocorticoid-induced PRR5-GR (glucorticoid receptor) construct, we found that PRR5 directly downregulates CCA1 and LHY expression. Furthermore, PRR9, PRR7, and PRR5 associate with the CCA1 and LHY promoters in vivo, coincident with the timing of decreased CCA1 and LHY expression. These results suggest that the repressor activities of PRR9, PRR7, and PRR5 on the CCA1 and LHY promoter regions constitute the molecular mechanism that accounts for the role of these proteins in the feedback loop of the circadian clock.

Figure 1.

Effect of PRR9, PRR7, or PRR5 on Promoter Activity of CCA1, LHY, and TOC1. Promoter activities with coexpression of CFP, PRR9-CFP, PRR7-CFP, or PRR5-CFP. Promoter activities are shown relative to values obtained with coexpression of CFP alone. Error bars indicate the sd (biological replicates, n = 6). Asterisks indicate values that are statistically different from the CFP control (Student's t test; P < 0.05). Each experiment was performed twice with similar results. (A) CCA1 promoter activity. (B) LHY promoter activity. (C) TOC1 promoter activity.

Figure 2.

CCA1, LHY, and TOC1 Expression in 35Spro:PRR5-GR-CFP. (A) Average hypocotyl lengths of 8-d-old Arabidopsis seedlings of the wild type, two independent lines of 35Spro::PRR5-GR-CFP (35Spro:5GC1 and 35Spro:5GC2), 35Spro:PRR5-CFP (35Spro:5C), and 35Spro:PRR5 (35Spro:5) grown under 10-h-light/14-h-dark conditions with or without DEX (sd; biological replicates, n = 15). (B) CCA1, LHY, and TOC1 expression in 35Spro:5GC seedlings upon DEX treatment. Relative levels of each mRNA to APX3 mRNA (internal control) under DEX-free conditions were set to 1.0. (C) CCA1, LHY, and TOC1 expression in 35Spro:5GC seedlings treated with CHX with or without DEX. Relative levels of each mRNA to APX3 mRNA in 100 μM CHX solution were set to 1.0. Error bars indicate sd (technical replicates, n = 3). Each experiment was performed twice with similar results. Asterisks indicate values that are statistically different between –DEX and +DEX (t test; P < 0.05).

Figure 3.

PRR9, PRR7, and PRR5 Act as Transcriptional Repressors. (A) GAL4 promoter activity when coexpressed with the GAL4 DNA binding domain (GAL4DB) fused to PRR9, PRR7, or PRR5. GAL4 promoter activities are shown relative to values obtained with coexpression of GAL4DB alone. (B) GAL4 promoter activity when coexpressed with the GAL4DB fused to truncated PRR5 constructs #1 to #6 (right). Schematics of truncated PRR5 constructs fused to GAL4DB (left). PR, pseudoreceiver domain; CCT, CCT motif. (C) Amino acid sequence alignment of the conserved region in PRR proteins from various plants. The 44–amino acid sequence of PRR5#6 was used for alignment. Asterisks denote amino acids conserved in all the sequences; colons denote similar amino acids. For species information, see the Accession Numbers section in Methods. (D) GAL4 promoter activity when coexpressed with the GAL4DB fused to the different motifs in the 44–amino acid region of PRR5. (E) GAL4 promoter activity when coexpressed with the GAL4DB fused to corresponding regions in PRR7 and PRR9 described in Figure 3C. (F) GAL4 promoter activity determination after coexpression of PRR5 full-length or GAL4DB-PRR5. Error bars indicate sd (biological replicates, n = 6). Asterisks indicate values that are statistically different from control (t test; P < 0.05). Each experiment was performed twice with similar results.

Figure 4.

PRR9, PRR7, and PRR5 Associate with CCA1 and LHY Promoters in Vivo. (A) Schematics of CCA1, LHY, TOC1, and APX3 loci and the locations of the target DNA fragments (amplicons) used in the ChIP assays. Positions of the 10 amplicons are shown as short horizontal black bars. Arrows indicate full-length coding sequences, with the ATG (translation initiation codon) being located at the tail of the arrow. The open triangle, closed triangle, black line, and diamond are G-box, TBS, 5A, and evening element (EE), respectively. (B) to (D) ChIP assays, with the percentage of DNA fragments coimmunoprecipitated with anti-GFP antibody relative to input DNA presented. (B) ChIP assays for PRR9pro:FLAG-GUS-GFP (9pro:FGG) and PRR9pro:FLAG-PRR9-GFP (9pro:F9G) seedlings (C) ChIP assays for PRR7pro:FLAG-GUS-GFP (7pro:FGG) and PRR7pro:FLAG-PRR7-GFP (7pro:F7G) seedlings (D) ChIP assays for PRR5pro:FLAG-GUS-GFP (5pro:FGG) and PRR5pro:FLAG-PRR5-GFP (5pro:F5G) seedlings (E) ChIP assays for prr5-11 and 35Spro:PRR5 etiolated seedlings. Five-day-old etiolated seedlings exposed to white light for 10 h were used. Immunoprecipitation was performed with anti-PRR5 antibody. Error bars indicate sd (technical replicates, n = 3). Each experiment was performed twice with similar results.

Figure 5.

Association Patterns of PRR9, PRR7, and PRR5 with the Promoter Regions of CCA1 and LHY in 12-h-Light/12-h-Dark Conditions. (A) F9G, F7G, and F5G protein levels in 12-h-light/12-h-dark conditions. 9pro:F9G, 7pro:F7G, and 5pro:F5G plants were grown in 12-h-light/12-h-dark conditions for 2 weeks and cross-linked at 2-h intervals starting at ZT0 (light on). Total protein was immunoblotted by anti-FLAG antibody (top panel). F9G, F7G, and F5G protein amounts normalized with the total protein (bottom). Peak levels were set to 1.0. White and gray areas represent white light and dark conditions, respectively. (B) Percentages of the amplicon 3 of CCA1 region (top) and amplicon 7 LHY region (bottom) coimmunoprecipitated with anti-GFP antibody relative to input DNA in 9pro:F9G, 7pro:F7G, and 5pro:F5G plants were plotted. (C) CCA1 and LHY mRNA expression in 5pro:F5G plants. Expression levels were determined relative to APX3 mRNA. Peak levels were set to 1.0. Error bars indicate sd (technical replicates, n = 3). Each experiment was performed twice with similar results.

Figure 6.

Expression Patterns of PRR9 and PRR5 Proteins, and CCA1 and LHY Expression in prr7 prr5 and prr9 prr7 Mutants. (A) The wild-type waveforms of PRR9, PRR7, and PRR5 protein levels and CCA1 and LHY mRNA expression. These data were also presented in Figures 5A and 5C. (B) PRR9 protein levels and CCA1 and LHY mRNA expression in the prr7 prr5 double mutant. The amount of PRR9 protein was normalized with total protein, and the amounts of CCA1 and LHY mRNA were normalized with APX3 mRNA (sd; technical replicates, n = 3). Each experiment was performed twice with similar results. (C) PRR5 protein levels and CCA1 and LHY mRNA expression in the prr9 prr7 mutant. The amount of PRR5 protein was normalized with total protein, and the amounts of CCA1 and LHY mRNA were normalized with APX3 mRNA (sd; technical replicates, n = 3). Each experiment was performed twice with similar results. In all panels, peak levels were set to 1.0, and white and gray areas represent white light and dark conditions, respectively.

Figure 7.

Arabidopsis Clock Model Incorporating the Transcriptional Repressors PRR9, PRR7, and PRR5. CCA1 and LHY repress TOC1 transcription (blue bar) by binding to the TOC1 promoter. In turn, TOC1 activates CCA1 expression (blue arrow) by antagonizing CHE1, the repressor of CCA1. As a new addition to this circuit (red), PRR9, PRR7, and PRR5 proteins repress CCA1 and LHY transcription directly from morning until midnight (ZT2 to ZT16). CCA1 and LHY proteins activate PRR9 and PRR7 transcription.