Circadian clocks are ubiquitous molecular time-keeping mechanisms that coordinate physiology and metabolism and provide an adaptive advantage to higher plants. The central oscillator of the plant clock is composed of interlocked feedback loops that involve multiple repressive factors acting throughout the circadian cycle. PSEUDO RESPONSE REGULATORS (PRRs) comprise a five-member family that is essential to the function of the central oscillator. PRR5, PRR7, and PRR9 can bind the promoters of the core clock genes CIRCADIAN CLOCK ASSOCIATED 1 ( CCA1 ) and LATE ELONGATED HYPOCOTYL ( LHY ) to restrict their expression to near dawn, but the mechanism has been unclear. Here we report that members of the plant Groucho/Tup1 corepressor family, TOPLESS/TOPLESS-RELATED (TPL/TPR), interact with these three PRR proteins at the CCA1 and LHY promoters to repress transcription and alter circadian period. This activity is diminished in the presence of the inhibitor trichostatin A, indicating the requirement of histone deacetylase for full TPL activity. Additionally, a complex of PRR9, TPL, and histone deacetylase 6, can form in vivo, implicating this tripartite association as a central repressor of circadian gene expression. Our findings show that the TPL/TPR corepressor family are components of the central circadian oscillator mechanism and reinforces the role of this family as central to multiple signaling pathways in higher plants.

The circadian clock controls the period, phasing, and amplitude of processes that oscillate with a near 24-h rhythm. One core group of clock components in Arabidopsis that controls the pace of the central oscillator is comprised of five PRR (pseudo-response regulator) proteins whose biochemical function in the clock remains unclear. Peak expression of TOC1 (timing of cab expression 1)/PRR1, PRR3, PRR5, PRR7, and PRR9 are each phased differently over the course of the day and loss of any PRR protein alters period. Here we show that, together with TOC1, PRR5 is the only other likely proteolytic substrate of the E3 ubiquitin ligase SCF^ZTL within this PRR family. We further demonstrate a functional significance for the phosphorylated forms of PRR5, TOC1, and PRR3. Each PRR protein examined is nuclear-localized and is differentially phosphorylated over the circadian cycle. The more highly phosphorylated forms of PRR5 and TOC1 interact best with the F-box protein ZTL (ZEITLUPE), suggesting a mechanism to modulate their proteolysis. In vivo degradation of both PRR5 and ZTL is inhibited by blue light, likely the result of blue light photoperception by ZTL. TOC1 and PRR3 interact in vivo and phosphorylation of both is necessary for their optimal binding in vitro. Additionally, because PRR3 and ZTL both interact with TOC1 in vivo via the TOC1 N terminus, taken together these data suggest that the TOC1/PRR3 phosphorylation-dependent interaction may protect TOC1 from ZTL-mediated degradation, resulting in an enhanced amplitude of TOC1 cycling.

As an F-box protein, ZEITLUPE (ZTL) is involved in targeting one or more substrates for ubiquitination and degradation via the proteasome. The initial characterization of ZTL suggested a function limited largely to the regulation of the circadian clock. Here, we show a considerably broader role for ZTL in the control of circadian period and photomorphogenesis. Using a ZTL-specific antibody, we quantitated and characterized a ZTL dosage series that ranges from a null mutation to a strong ZTL overexpressor. In the dark, ztl null mutations lengthen circadian period, and overexpression causes arrhythmicity, suggesting a more comprehensive role for this protein in the clock than previously suspected. In the light, circadian period becomes increasingly shorter at higher levels of ZTL, to the point of arrhythmicity. By contrast, hypocotyl length increases and flowering time is delayed in direct proportion to the level of ZTL. We propose a novel testable mechanism by which circadian period and amplitude may act together to gate phytochrome B–mediated suppression of hypocotyl. We also demonstrate that ZTL-dependent delay of flowering is mediated through decreases in CONSTANS and FLOWERING LOCUS T message levels, thus directly linking proteasome-dependent proteolysis to flowering.

Circadian clock systems help establish the correct daily phasing of the behavioral, developmental, and molecular events needed for the proper coordination of physiology and metabolism. The circadian oscillator comprises transcription–translation feedback loops but also requires post-translational processes that regulate clock protein homeostasis. GIGANTEA is a unique plant protein involved in the maintenance and control of numerous facets of plant physiology and development. Through an unknown mechanism GIGANTEA stabilizes the F-box protein ZEITLUPE, a key regulator of the circadian clock. Here, we show that GIGANTEA has general protein chaperone activity and can act to specifically facilitate ZEITLUPE maturation into an active form in vitro and in planta. GIGANTEA forms a ternary complex with HSP90 and ZEITLUPE and its co-chaperone action synergistically enhances HSP90/HSP70 maturation of ZEITLUPE in vitro. These results identify a molecular mechanism for GIGANTEA activity that can explain its wide-ranging role in plant biology.