REVEILLE8 and PSEUDO-REPONSE REGULATOR5 Form a Negative Feedback Loop within the Arabidopsis Circadian Clock

Abstract

Circadian rhythms provide organisms with an adaptive advantage, allowing them to regulate physiological and developmental events so that they occur at the most appropriate time of day. In plants, as in other eukaryotes, multiple transcriptional feedback loops are central to clock function. In one such feedback loop, the Myb-like transcription factors CCA1 and LHY directly repress expression of the pseudoresponse regulator TOC1 by binding to an evening element (EE) in the TOC1 promoter. Another key regulatory circuit involves CCA1 and LHY and the TOC1 homologs PRR5, PRR7, and PRR9. Purification of EE–binding proteins from plant extracts followed by mass spectrometry led to the identification of RVE8, a homolog of CCA1 and LHY. Similar to these well-known clock genes, expression of RVE8 is circadian-regulated with a dawn phase of expression, and RVE8 binds specifically to the EE. However, whereas cca1 and lhy mutants have short period phenotypes and overexpression of either gene causes arrhythmia, rve8 mutants have long-period and RVE8-OX plants have short-period phenotypes. Light input to the clock is normal in rve8, but temperature compensation (a hallmark of circadian rhythms) is perturbed. RVE8 binds to the promoters of both TOC1 and PRR5 in the subjective afternoon, but surprisingly only PRR5 expression is perturbed by overexpression of RVE8. Together, our data indicate that RVE8 promotes expression of a subset of EE–containing clock genes towards the end of the subjective day and forms a negative feedback loop with PRR5. Thus RVE8 and its homologs CCA1 and LHY function close to the circadian oscillator but act via distinct molecular mechanisms.

Author Summary

Circadian clocks help organize 24-hour rhythms in physiology and behavior so that critical organismal functions are optimally timed relative to highly predictable daily changes in the environment. Circadian clocks run at approximately the same pace across a wide range of temperatures, ensuring accurate timekeeping in all seasons. Although molecular components of the circadian clock are not conserved across higher taxa, eukaryotic circadian clocks are composed of analogous interlocked transcriptional feedback loops. In this study, we report the isolation and characterization of a new component of the plant circadian system, REVEILLE 8 (RVE8). RVE8 is a clock-regulated Myb-like transcription factor that binds with high affinity to the evening element (EE) promoter motif and helps to set the pace of the clock in a light- and temperature-dependent manner. RVE8 promotes expression of the clock component PSEUDO-RESPONSE REGULATOR 5 ( PRR5), likely via direct action at the PRR5 promoter. RVE8 expression is in turn repressed by PRR5. Thus, RVE8 is a new component of the plant circadian oscillator that takes part in a novel transcriptional feedback loop.