Explore the vast range of titles available.

This Review examines the molecular mechanisms underlying the plant circadian clock, highlighting the functions of transcriptional circuits and post-translational regulation in timing and describing how clock components integrate and respond to environmental signals. Circadian clocks are endogenous timekeeping networks that allow organisms to align their physiology with their changing environment and to perform biological processes at the most relevant times of the day and year. Initial feedback-loop models of the oscillator have been enriched by emerging evidence highlighting the increasing variety of factors and mechanisms that contribute to the generation of rhythms. In this Review, we consider the two major input pathways that connect the circadian clock of the model plant Arabidopsis thaliana to its environment and discuss recent advances in understanding of how transcriptional, post-translational and post-transcriptional mechanisms contribute to clock function.

Plants perceive environmental signals such as day length and temperature to determine optimal timing for the transition from vegetative to floral stages. Arabidopsis flowers under long-day conditions through the CONSTANS (CO)-FLOWERING LOCUS T (FT) regulatory module. It is thought that the environmental cues for photoperiodic control of flowering are initially perceived in the leaves. We have previously shown that GIGANTEA (GI) regulates the timing of CO expression, together with FLAVIN-BINDING, KELCH REPEAT, F BOX protein 1. Normally, CO and FT are expressed exclusively in vascular bundles, whereas GI is expressed in various tissues. To better elucidate the role of tissue-specific expression of GI in the flowering pathway, we established transgenic lines in which GI is expressed exclusively in mesophyll, vascular bundles, epidermis, shoot apical meristem, or root. We found that GI expressed in either mesophyll or vascular bundles rescues the late-flowering phenotype of the gi-2 loss-of-function mutant under both short-day and long-day conditions. Interestingly, GI expressed in mesophyll or vascular tissues increases FT expression without up-regulating CO expression under short-day conditions. Furthermore, we examined the interaction between GI and FT repressors in mesophyll. We found that GI can bind to three FT repressors: SHORT VEGETATIVE PHASE (SVP), TEMPRANILLO (TEM)1, and TEM2. Finally, our chromatin immunoprecipitation experiments showed that GI binds to FT promoter regions that are near the SVP binding sites. Taken together, our data further elucidate the multiple roles of GI in the regulation of flowering time.

Transcription modulated by the circadian clock is diverse across cell types, underlying circadian control of peripheral metabolism and its observed perturbation in human diseases. We report that knockout of the lineage-specifying Hnf4a gene in mouse liver causes associated reductions in the genome-wide distribution of core clock component BMAL1 and accessible chromatin marks (H3K4me1 and H3K27ac). Ectopically expressing HNF4A remodels chromatin landscape and nucleates distinct tissue-specific BMAL1 chromatin binding events, predominantly in enhancer regions. Circadian rhythms are disturbed in Hnf4a knockout liver and HNF4A-MODY diabetic model cells. Additionally, the epigenetic state and accessibility of the liver genome dynamically change throughout the day, synchronized with chromatin occupancy of HNF4A and clustered expression of circadian outputs. Lastly, Bmal1 knockout attenuates HNF4A genome-wide binding in the liver, likely due to downregulated Hnf4a transcription. Our results may provide a general mechanism for establishing circadian rhythm heterogeneity during development and disease progression, governed by chromatin structure. Genome-wide occupancy of the master circadian clock transcription factor BMAL1::CLOCK varies across tissues and is reprogrammed in cancers, but how specificity is governed is not known. Here the authors show BMAL1::CLOCK in liver tissue is guided by chromatin accessibility remodeled by HNF4A, shedding new lights onto mechanisms of dysregulated circadian rhythms in hepatocarcinoma.

Either expression level or transcriptional activity of various nuclear receptors (NRs) have been demonstrated to be under circadian control. With a few exceptions, little is known about the roles of NRs as direct regulators of the circadian circuitry. Here we show that the nuclear receptor HNF4A strongly transrepresses the transcriptional activity of the CLOCK:BMAL1 heterodimer. We define a central role for HNF4A in maintaining cell-autonomous circadian oscillations in a tissue-specific manner in liver and colon cells. Not only transcript level but also genome-wide chromosome binding of HNF4A is rhythmically regulated in the mouse liver. ChIP-seq analyses revealed cooccupancy of HNF4A and CLOCK: BMAL1 at a wide array of metabolic genes involved in lipid, glucose, and amino acid homeostasis. Taken together, we establish that HNF4A defines a feedback loop in tissue-specific mammalian oscillators and demonstrate its recruitment in the circadian regulation of metabolic pathways.

The circadian clock, an endogenous time-keeping mechanism common to most species, allows organisms to coordinate biological processes with specific times of day. In plants, the role of the clock extends to almost every aspect of growth and development, including responses to biotic and abiotic stresses. The core molecular components and circuits of the clock have been well studied in the model organism Arabidopsis thaliana ; however, how this mechanism connects to clock-controlled outputs remains poorly understood. Here, we performed a genome-wide characterization of the direct targets of a key clock component in Arabidopsis . Our results emphasize the broad role of the plant clock in regulating multiple biological functions and provide direct links between the oscillator and clock-regulated outputs. The circadian clock in Arabidopsis exerts a critical role in timing multiple biological processes and stress responses through the regulation of up to 80% of the transcriptome. As a key component of the clock, the Myb -like transcription factor CIRCADIAN CLOCK ASSOCIATED1 ( CCA1 ) is able to initiate and set the phase of clock-controlled rhythms and has been shown to regulate gene expression by binding directly to the evening element (EE) motif found in target gene promoters. However, the precise molecular mechanisms underlying clock regulation of the rhythmic transcriptome, specifically how clock components connect to clock output pathways, is poorly understood. In this study, using ChIP followed by deep sequencing of CCA1 in constant light (LL) and diel (LD) conditions, more than 1,000 genomic regions occupied by CCA1 were identified. CCA1 targets are enriched for a myriad of biological processes and stress responses, providing direct links to clock-controlled pathways and suggesting that CCA1 plays an important role in regulating a large subset of the rhythmic transcriptome. Although many of these target genes are evening expressed and contain the EE motif, a significant subset is morning phased and enriched for previously unrecognized motifs associated with CCA1 function. Furthermore, this work revealed several CCA1 targets that do not cycle in either LL or LD conditions. Together, our results emphasize an expanded role for the clock in regulating a diverse category of genes and key pathways in Arabidopsis and provide a comprehensive resource for future functional studies.

The plant hormone salicylic acid (SA) is essential for local defense and systemic acquired resistance (SAR). When plants, such as Arabidopsis , are challenged by different pathogens, an increase in SA biosynthesis generally occurs through transcriptional induction of the key synthetic enzyme isochorismate synthase 1 (ICS1). However, the regulatory mechanism for this induction is poorly understood. Using a yeast one-hybrid screen, we identified two transcription factors (TFs), NTM1-LIKE 9 (NTL9) and CCA1 HIKING EXPEDITION (CHE), as activators of ICS1 during specific immune responses. NTL9 is essential for inducing ICS1 and two other SA synthesis-related genes, PHYTOALEXIN-DEFICIENT 4 ( PAD4 ) and ENHANCED DISEASE SUSCEPTIBILITY 1 ( EDS1 ), in guard cells that form stomata. Stomata can quickly close upon challenge to block pathogen entry. This stomatal immunity requires ICS1 and the SA signaling pathway. In the ntl9 mutant, this response is defective and can be rescued by exogenous application of SA, indicating that NTL9-mediated SA synthesis is essential for stomatal immunity. CHE, the second identified TF, is a central circadian clock oscillator and is required not only for the daily oscillation in SA levels but also for the pathogen-induced SA synthesis in systemic tissues during SAR. CHE may also regulate ICS1 through the known transcription activators CALMODULIN BINDING PROTEIN 60g (CBP60g) and SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) because induction of these TF genes is compromised in the che-2 mutant. Our study shows that SA biosynthesis is regulated by multiple TFs in a spatial and temporal manner and therefore fills a gap in the signal transduction pathway between pathogen recognition and SA production. Biosynthesis of the plant immune signal salicylic acid (SA) is normally induced upon pathogen challenge through transcriptional activation of the key SA synthetic enzyme gene, ICS1 . However, how different pathogenic signals trigger SA synthesis in both local and systemic tissues and during different immune responses is poorly understood. Our study filled this knowledge gap by the identification of two transcription factors (TFs): one is required for SA biosynthesis in stomata to prevent pathogen entry through these epidermal openings, and the other is essential for both the circadian oscillation in SA levels and the accumulation of SA in distal tissue during systemic acquired resistance. Our study shows that SA biosynthesis is regulated by multiple TFs in a spatial and temporal manner.

Photoperiod is one of the most reliable environmental cues for plants to regulate flowering timing. In Arabidopsis thaliana, CONSTANS (CO) transcription factor plays a central role in regulating photoperiodic flowering. In contrast to posttranslational regulation of CO protein, still little was known about CO transcriptional regulation. Here we show that the CINCINNATA (CIN) clade of class II TEOSINTE BRANCHED 1/ CYCLOIDEA/ PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR (TCP) proteins act as CO activators. Our yeast one-hybrid analysis revealed that class II CIN-TCPs, including TCP4, bind to the CO promoter. TCP4 induces CO expression around dusk by directly associating with the CO promoter in vivo. In addition, TCP4 binds to another flowering regulator, GIGANTEA (GI), in the nucleus, and induces CO expression in a GI-dependent manner. The physical association of TCP4 with the CO promoter was reduced in the gi mutant, suggesting that GI may enhance the DNA-binding ability of TCP4. Our tandem affinity purification coupled with mass spectrometry (TAP-MS) analysis identified all class II CIN-TCPs as the components of the in vivo TCP4 complex, and the gi mutant did not alter the composition of the TCP4 complex. Taken together, our results demonstrate a novel function of CIN-TCPs as photoperiodic flowering regulators, which may contribute to coordinating plant development with flowering regulation.

The first described feedback loop of the Arabidopsis circadian clock is based on reciprocal regulation between TIMING OF CAB EXPRESSION 1 (TOC1) and CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY). CCA1 and LHY are Myb transcription factors that bind directly to the TOC1 promoter to negatively regulate its expression. Conversely, the activity of TOC1 has remained less well characterized. Genetic data support that TOC1 is necessary for the reactivation of CCA1/LHY, but there is little description of its biochemical function. Here we show that TOC1 occupies specific genomic regions in the CCA1 and LHY promoters. Purified TOC1 binds directly to DNA through its CCT domain, which is similar to known DNA-binding domains. Chemical induction and transient overexpression of TOC1 in Arabidopsis seedlings cause repression of CCA1/LHY expression, demonstrating that TOC1 can repress direct targets, and mutation or deletion of the CCT domain prevents this repression showing that DNA-binding is necessary for TOC1 action. Furthermore, we use the Gal4/UAS system in Arabidopsis to show that TOC1 acts as a general transcriptional repressor, and that repression activity is in the pseudoreceiver domain of the protein. To identify the genes regulated by TOC1 on a genomic scale, we couple TOC1 chemical induction with microarray analysis and identify previously unexplored potential TOC1 targets and output pathways. Taken together, these results define a biochemical action for the core clock protein TOC1 and refine our perspective on how plant clocks function.

The evening routine In plants, the circadian clock functions as an endogenous pacemaker that anticipates and responds to a changing environment in order to optimize the timing of physiological and developmental events. Nusinow et al . elucidate the mechanism by which the circadian clock controls growth of the model plant Arabidopsis thaliana . A novel trimeric complex called the evening complex is regulated by the clock and has a peak of expression at dusk. The complex represses the expression of two transcription factors, PIF4 and PIF5, which are part of a light-signalling cascade that controls the timing of plant growth in response to light conditions. The circadian clock is required for adaptive responses to daily and seasonal changes in environmental conditions 1 , 2 , 3 . Light and the circadian clock interact to consolidate the phase of hypocotyl cell elongation to peak at dawn under diurnal cycles in Arabidopsis thaliana 4 , 5 , 6 , 7 . Here we identify a protein complex (called the evening complex)—composed of the proteins encoded by EARLY FLOWERING 3 ( ELF3 ), ELF4 and the transcription-factor-encoding gene LUX ARRHYTHMO ( LUX ; also known as PHYTOCLOCK 1 )—that directly regulates plant growth 8 , 9 , 10 , 11 , 12 . ELF3 is both necessary and sufficient to form a complex between ELF4 and LUX, and the complex is diurnally regulated, peaking at dusk. ELF3 , ELF4 and LUX are required for the proper expression of the growth-promoting transcription factors encoded by PHYTOCHROME INTERACTING FACTOR 4 ( PIF4 ) and PIF5 (also known as PHYTOCHROME INTERACTING FACTOR 3-LIKE 6 ) under diurnal conditions 4 , 6 , 13 . LUX targets the complex to the promoters of PIF4 and PIF5 in vivo . Mutations in PIF4 and/or PIF5 are epistatic to the loss of the ELF4–ELF3–LUX complex, suggesting that regulation of PIF4 and PIF5 is a crucial function of the complex. Therefore, the evening complex underlies the molecular basis for circadian gating of hypocotyl growth in the early evening.

Organisms have evolved endogenous biological clocks as internal timekeepers to coordinate metabolic processes with the external environment. Here, we seek to understand the mechanism of synchrony between the oscillator and products of metabolism known as Reactive Oxygen Species (ROS) in Arabidopsis thaliana . ROS-responsive genes exhibit a time-of-day–specific phase of expression under diurnal and circadian conditions, implying a role of the circadian clock in transcriptional regulation of these genes. Hydrogen peroxide production and scavenging also display time-of-day phases. Mutations in the core-clock regulator, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), affect the transcriptional regulation of ROS-responsive genes, ROS homeostasis, and tolerance to oxidative stress. Mis-expression of EARLY FLOWERING 3 , LUX ARRHYTHMO , and TIMING OF CAB EXPRESSION 1 affect ROS production and transcription, indicating a global effect of the clock on the ROS network. We propose CCA1 as a master regulator of ROS homeostasis through association with the Evening Element in promoters of ROS genes in vivo to coordinate time-dependent responses to oxidative stress. We also find that ROS functions as an input signal that affects the transcriptional output of the clock, revealing an important link between ROS signaling and circadian output. Temporal coordination of ROS signaling by CCA1 and the reciprocal control of circadian output by ROS reveal a mechanistic link that allows plants to master oxidative stress responses.