In many organisms, the circadian clock is composed of functionally coupled morning and evening oscillators. In Arabidopsis, oscillator coupling relies on a core loop in which the evening oscillator component TIMING OF CAB EXPRESSION 1 (TOC1) was proposed to activate a subset of morning-expressed oscillator genes. Here, we show that TOC1 does not function as an activator but rather as a general repressor of oscillator gene expression. Repression occurs through TOC1 rhythmic association to the promoters of the oscillator genes. Hormone-dependent induction of TOC1 and analysis of RNA interference plants show that TOC1 prevents the activation of morning-expressed genes at night. Our study overturns the prevailing model of the Arabidopsis circadian clock, showing that the morning and evening oscillator loops are connected through the repressing activity of TOC1.

In Arabidopsis, the root clock regulates the spacing of lateral organs along the primary root through oscillating gene expression. The core molecular mechanism that drives the root clock periodicity and how it is modified by exogenous cues such as auxin and gravity remain unknown. We identified the key elements of the oscillator (AUXIN RESPONSE FACTOR 7, its auxin-sensitive inhibitor IAA18/POTENT, and auxin) that form a negative regulatory loop circuit in the oscillation zone. Through multilevel computer modeling fitted to experimental data, we explain how gene expression oscillations coordinate with cell division and growth to create the periodic pattern of organ spacing. Furthermore, gravistimulation experiments based on the model predictions show that external auxin stimuli can lead to entrainment of the root clock. Our work demonstrates the mechanism underlying a robust biological clock and how it can respond to external stimuli.

Circadian clocks sustain 24-h rhythms in physiology and metabolism that are synchronized with the day/night cycle. In plants, the regulatory network responsible for the generation of rhythms has been broadly investigated over the past years. However, little is known about the intersecting pathways that link the environmental signals with rhythms in cellular metabolism. Here, we examine the role of the circadian components REVEILLE8/LHY-CCA1-LIKE5 (RVE8/LCL5) and NIGHT LIGHT-INDUCIBLE AND CLOCK-REGU-LATED genes (LNK) shaping the diurnal oscillation of the anthocyanin metabolic pathway. Around dawn, RVE8 up-regulates anthocyanin gene expression by directly associating to the promoters of a subset of anthocyanin biosynthetic genes. The upregulation is overcome at midday by the repressing activity of LNK proteins, as inferred by the increased anthocyanin gene expression in lnk1/lnk2 double mutant plants. Chromatin immunoprecipitation assays using LNK and RVE8 misexpressing plants show that RVE8 binding to target promoters is precluded in LNK overexpressing plants and conversely, binding is enhanced in the absence of functional LNKs, which provides a mechanism by which LNKs antagonize RVE8 function in the regulation of anthocyanin accumulation. Based on their previously described transcriptional coactivating function, our study defines a switch in the regulatory activity of RVE8-LNK interaction, from a synergic coactivating role of eveningexpressed clock genes to a repressive antagonistic function modulating anthocyanin biosynthesis around midday.circadian clock | anthocyanin accumulation | transcriptional regulation | protein-protein interaction | Arabidopsis thaliana C ircadian clocks are broadly present in nature and allow organisms to anticipate and prepare for the predictable changes that occur during the day/night cycles (1). Synchronization by the environmental signals ensures proper coordination of metabolism and physiology in many organisms, including plants (2). In Arabidopsis, the molecular architecture depends on a complex regulatory network, in which the morning-expressed single Myb-like transcription factors, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) (3) and LATE ELONGATED HYPOCOTYL (LHY) (4) repress the expression (5) of the evening-phased pseudoresponse regulator, TIMING OF CAB EXPRESSION 1 (TOC1/ PRR1) (6, 7). TOC1 in turn represses CCA1 and LHY (8, 9) as well as the other members of the PRR family (PRR9, 7, and 5) (10) that in turn act as repressors of CCA1 and LHY expression (11). TOC1 also represses LUX ARRHYTHMO (LUX) and EARLY FLOWERING 4 (ELF4) (9), whose protein products interact with EARLY FLOWERING 3 (ELF3) to form the socalled Evening Complex (EC) (12).Chromatin changes at the promoters of the core oscillator genes also play an important role modulating clock gene expression and function (13,14). The single Myb-like transcription factor REVEILLE8/LHY-CCA1-LIKE5 (RVE8/LCL5) (15) antagonizes CCA1 repressing function in the regulation of Histone3 acetylation at TOC1 promoter (16). RVE8 overexpressio...