PSEUDO-RESPONSE REGULATORS 9, 7, and 5 Are Transcriptional Repressors in the Arabidopsis Circadian Clock

Norihito Nakamichi,Hitoshi Sakakibara,Takeshi Mizuno,Takatoshi Kiba,Rossana Henriques,Nam-Hai Chua

doi:10.1105/tpc.109.072892

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.

Highlights

The circadian clock controls endogenous biological rhythms that allow a wide range of organisms to adapt to 24-h day-night cycles (Young and Kay, 2001)
PRR9, PRR7, and PRR5 associate with the promoter regions of CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) in vivo, coincident with the timing of decreased CCA1 and LHY expression. These results suggest that PRR9, PRR7, and PRR5 proteins are major transcriptional repressors of CCA1 and LHY and are essential for proper clock function
To examine whether PRR9, PRR7, and PRR5 regulate CCA1, LHY, and TIMING OF CAB EXPRESSION1 (TOC1), at the transcriptional level, we conducted transient assays using reporter plasmids harboring luciferase (LUC) under the control of the CCA1, LHY, or TOC1 promoters (CCA1pro:LUC, LHYpro:LUC, or TOC1pro:LUC) and effector plasmids harboring PRR9, PRR7, or PRR5 genes fused to a gene encoding cyan fluorescent protein (CFP) or a negative control containing CFP only, all under the control of the cauliflower mosaic virus (CaMV) 35S promoter (35Spro:PRR9-CFP, PRR7CFP, PRR5-CFP, or CFP)

Summary

Introduction

The circadian clock controls endogenous biological rhythms that allow a wide range of organisms to adapt to 24-h day-night cycles (Young and Kay, 2001). A transcriptiontranslation feedback loop connecting clock-associated genes is thought to form the central oscillator (or core) of the clock (BellPedersen et al, 2005). In Arabidopsis thaliana, reciprocal transcriptional regulation between TIMING OF CAB EXPRESSION1 (TOC1; called PSEUDO-RESPONSE REGULATOR1 [PRR1]) and CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) has been proposed as the main feedback loop. CCA1 and LHY proteins are MYB transcription factors and repress TOC1 transcription by binding directly to the TOC1 promoter around dawn (Alabadi et al, 2001; Mizoguchi et al, 2002; Perales and Mas, 2007).

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Conclusion