TIMING OF CAB EXPRESSION Research Articles

Genome-wide identification of clock-associated genes and circadian rhythms in Fragaria × ananassa seedlings

Flowering time in plants is regulated by a photoperiod-responsive mechanism. Some plant species use a circadian clock-based control mechanism to adapt to variable environments. Strawberry is a horticultural crop that responds to certain photoperiods and temperatures to induce flowering. However, clock-associated genes in octoploid cultivated strawberry (Fragaria × ananassa) have not been defined, and their regulatory mechanism for responding to photoperiods is unclear. We herein targeted 12 clock-associated genes reported in other plant species and performed a genome-wide analysis and expression comparison in F. × ananassa seedlings. Seventy-eight sequences were selected from the F. × ananassa genome. The major domains and cis-acting elements were conserved in each sequence. Transcripts were clearly expressed under continuous light conditions in F. × ananassa seedlings (‘Yotsuboshi’) acclimated to long days. Among them, 9 genes maintained their unique autonomous circadian rhythms and may function as clock genes. LHY (LATE ELONGATED HYPOCOTYL) had the Myb domain and LHY expression peaked in the dawn. PRR (PSEUDO-RESPONSE REGULATOR) family members (PRR9, PRR7, PRR5, and TOC1 (TIMING OF CAB EXPRESSION 1)) had a pseudo-receiver domain and CCT domain, and peak expression times began sequentially from the afternoon for PRR9 to the evening for TOC1. LUX (LUXARRHYTHMO) had a Myb domain, and LUX expression peaked in evening with ELF3 (EARLY FLOWERING 3). FKF1 (FLAVIN-BINDING KELCH REPEAT F BOX 1) had PAS and F-box domains, and FKF1 expression peaked in the afternoon. GI (GIGANTEA) expression also peaked in the afternoon. F. × ananassa (‘Yotsuboshi’) appears to have multiple feedback loops comprising clock-associated genes. Although the rhythmic expression of CHE (CCA1 HIKING EXPEDITION) and ZTL (ZEITLUPE) was not observed, they had conserved domains, CHE with the TCP domain and ZTL with the PAS and F-box domains. The present results provide basic information on the circadian clock for the control of F. × ananassa flowering.

Plant Circadian Clocks: Unravelling the Molecular Rhythms of Nature

The circadian clock is a fundamental biological mechanism that allows organisms to synchronize their internal processes with the external environment, thereby optimizing growth, development, and physiology. In plants, circadian rhythms govern various aspects of their life cycle, including germination, leaf movement, flowering, and responses to environmental cues such as light and temperature. Understanding the molecular mechanisms underlying plant circadian clocks is essential not only for elucidating fundamental principles of plant biology but also for applications in agriculture and biotechnology. This review delves into the intricate molecular networks that comprise plant circadian clocks, focusing on key components and their interactions. At the core of these clocks are transcription-translation feedback loops (TTFLs) involving a set of clock genes. The central oscillator, composed of genes such as CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION 1 (TOC1), and PSEUDO-RESPONSE REGULATOR (PRR) genes, drives rhythmic expression of downstream clock-controlled genes (CCGs). The interplay between positive and negative regulators within the TTFLs generates robust oscillations with a period of approximately 24 hours. Additionally, post-translational modifications, protein-protein interactions, and chromatin remodeling contribute to fine-tuning the circadian system, allowing plants to adapt to changing environmental conditions. Light perception through photoreceptors such as phytochromes and cryptochromes plays a crucial role in entraining the circadian clock to external cues, ensuring synchronization with the day-night cycle. Furthermore, recent advancements in omics technologies, including genomics, transcriptomics, proteomics, and metabolomics, have provided unprecedented insights into the complexity and plasticity of plant circadian clocks. Integration of multi-omics data has facilitated the construction of comprehensive regulatory networks and computational models, enabling predictive understanding of clock function and behavior under diverse conditions. Overall, unraveling the molecular rhythms of plant circadian clocks not only enhances our knowledge of fundamental biological processes but also holds promise for improving crop productivity, stress resilience, and sustainability in agriculture through targeted manipulation of clock components and their associated pathways. Future research endeavors will undoubtedly continue to unveil the intricacies of these fascinating timekeeping mechanisms, further enriching our understanding of the molecular rhythms of nature.

Arabidopsis seeds altered in the circadian clock protein TOC1 are characterized by higher level of linolenic acid

The circadian clock plays a critical role in regulating plant physiology and metabolism. However, the way in which the clock impacts the regulation of lipid biosynthesis in seeds is partially understood. In the present study, we characterized the seed fatty acid (FA) and glycerolipid (GL) compositions of pseudo-response regulator mutants. Among these mutants, toc1 (timing of cab expression 1) exhibited the most significant differences compared to control plants. These included an increase in total FA content, characterized by elevated levels of linolenic acid (18:3) along with a reduction in linoleic acid (18:2). Furthermore, our findings revealed that toc1 developing seeds showed increased expression of genes related to FA metabolism. Our results show a connection between TOC1 and lipid metabolism in Arabidopsis seeds.

Core clock genes adjust growth cessation time to day-night switches in poplar

Poplar trees use photoperiod as a precise seasonal indicator, synchronizing plant phenology with the environment. Daylength cue determines FLOWERING LOCUS T 2 (FT2) daily expression, crucial for shoot apex development and establishment of the annual growing period. However, limited evidence exists for the molecular factors controlling FT2 transcription and the conservation with the photoperiodic control of Arabidopsis flowering. We demonstrate that FT2 expression mediates growth cessation response quantitatively, and we provide a minimal data-driven model linking core clock genes to FT2 daily levels. GIGANTEA (GI) emerges as a critical inducer of the FT2 activation window, time-bound by TIMING OF CAB EXPRESSION (TOC1) and LATE ELONGATED HYPOCOTYL (LHY2) repressions. CRISPR/Cas9 loss-of-function lines validate these roles, identifying TOC1 as a long-sought FT2 repressor. Additionally, model simulations predict that FT2 downregulation upon daylength shortening results from a progressive narrowing of this activation window, driven by the phase shift observed in the preceding clock genes. This circadian-mediated mechanism enables poplar to exploit FT2 levels as an accurate daylength-meter.

The Roles of Circadian Clock Genes in Plant Temperature Stress Responses.

Plants monitor day length and memorize changes in temperature signals throughout the day, creating circadian rhythms that support the timely control of physiological and metabolic processes. The DEHYDRATION-RESPONSE ELEMENT-BINDING PROTEIN 1/C-REPEAT BINDING FACTOR (DREB1/CBF) transcription factors are known as master regulators for the acquisition of cold stress tolerance, whereas PHYTOCHROME INTERACTING FACTOR 4 (PIF4) is involved in plant adaptation to heat stress through thermomorphogenesis. Recent studies have shown that circadian clock genes control plant responses to temperature. Temperature-responsive transcriptomes show a diurnal cycle and peak expression levels at specific times of throughout the day. Circadian clock genes play essential roles in allowing plants to maintain homeostasis by accommodating temperature changes within the normal temperature range or by altering protein properties and morphogenesis at the cellular level for plant survival and growth under temperature stress conditions. Recent studies revealed that the central oscillator genes CIRCADIAN CLOCK ASSOCIATED 1/LATE ELONGATED HYPOCOTYL (CCA1/LHY) and PSEUDO-RESPONSE REGULATOR5/7/9 (PRR5/7/9), as well as the EVENING COMPLEX (EC) genes REVEILLE4/REVEILLE8 (REV4/REV8), were involved in the DREB1 pathway of the cold signaling transcription factor and regulated the thermomorphogenesis gene PIF4. Further studies showed that another central oscillator, TIMING OF CAB EXPRESSION 1 (TOC1), and the regulatory protein ZEITLUPE (ZTL) are also involved. These studies led to attempts to utilize circadian clock genes for the acquisition of temperature-stress resistance in crops. In this review, we highlight circadian rhythm regulation and the clock genes involved in plant responses to temperature changes, as well as strategies for plant survival in a rapidly changing global climate.

Genome-wide identification, characterization, and expression analysis of CCT transcription factors in poplar

The CCT [CONSTANS (CO), CO-like, and TIMING OF CAB EXPRESSION1 (TOC1)] gene family is involved in photoperiodic flowering and adaptation to different environments. In this study, 39 CCT family genes from the poplar genome were identified and characterized, including 18 COL, 7 PRR, and 14 CMF TFs. Phylogenetics analysis showed that the PtrCCT gene family could be classified into five classes (Classes I–V) that have close relationships with Arabidopsis thaliana. Eight pairs of PtrCCTs had collinear relationships through interchromosomal synteny analysis in poplar, suggesting segmental duplication played a vital role in the expansion of the poplar CCT gene family. Besides, synteny analyses of the CCT members among poplar and different species provided more clues for PtrCCT gene family evolution. Cis-acting elements in the promoters of PtrCCTs predicted their involvement in light responses, hormone responses, biotic/abiotic stress responses, and plant growth and development. Eight members of the PpnCCT gene family were differentially expressed in the apical buds and leaves of triploid poplar compared to diploids. We then focused on PpnCCT39 upregulated in triploid poplars and showed that PpnCCT39 was localized in the nucleus, chloroplast, and cytoplasm and could interact with CLPP1 in the chloroplast. Overexpression of PpnCCT39 in poplar increased chlorophyll contents and enhanced photosynthetic rate. This study provided comprehensive information for the CCT gene family and set up a basis for its function identification in poplar.

Circadian Clock Controls Root Hair Elongation through Long-Distance Communication.

Plants adapt to periodic environmental changes, such as day and night, by using circadian clocks. Cell division and elongation are primary steps to adjust plant development according to their environments. In Arabidopsis, hypocotyl elongation has been studied as a representative model to understand how the circadian clock regulates cell elongation. However, it remains unknown whether similar phenomena exist in other organs, such as roots, where circadian clocks regulate physiological responses. Here, we show that root hair elongation is controlled by both light and the circadian clock. By developing machine-learning models to automatically analyze the images of root hairs, we found that genes encoding major components of the central oscillator, such as TIMING OF CAB EXPRESSION1 (TOC1) or CIRCADIAN CLOCK ASSOCIATED1 (CCA1), regulate the rhythmicity of root hair length. The partial illumination of light to either shoots or roots suggested that light received in shoots is mainly responsible for the generation of root hair rhythmicity. Furthermore, grafting experiments between wild-type (WT) and toc1 plants demonstrated that TOC1 in shoots is responsible for the generation of root hair rhythmicity. Our results illustrate the combinational effects of long-distance signaling and the circadian clock on the regulation of root hair length.

OsTOC1 plays dual roles in the regulation of plant circadian clock by functioning as a direct transcription activator or repressor

Plant clock function relies on precise timing of gene expression through complex regulatory networks consisting of activators and repressors at the core of oscillators. Although TIMING OF CAB EXPRESSION 1 (TOC1) has been recognized as a repressor involved in shaping oscillations and regulating clock-driven processes, its potential to directly activate gene expression remains unclear. In this study, we find that OsTOC1 primarily acts as a transcriptional repressor for core clock components, including OsLHY and OsGI. Here, we show that OsTOC1 possesses the ability to directly activate the expression of circadian target genes. Through binding to the promoters of OsTGAL3a/b, transient activation of OsTOC1 induces the expression of OsTGAL3a/b, indicating its role as an activator contributing to pathogen resistance. Moreover, TOC1 participates in regulating multiple yield-related traits in rice. These findings suggest that TOC1's function as a transcriptional repressor is not inherent, providing flexibility to circadian regulations, particularly in outputs.

ZTL regulates thermomorphogenesis through TOC1 and PRR5.

Plants adapt to high temperature stresses through thermomorphogenesis, a process that includes stem elongation and hyponastic leaf growth. Thermomorphogenesis is gated by the circadian clock through two evening-expressed clock components, TIMING OF CAB EXPRESSION1(TOC1) and PSEUDO-RESPONSE REGULATORS5(PRR5). These proteins directly interact with and inhibit PHYTOCHROME INTERACTING FACTOR4(PIF4), a basic helix-loop-helix transcription factor that promotes thermoresponsive growth. PIF4-mediated thermoresponsive growth is positively regulated by ZEITLUPE(ZTL), a central clock component, but the molecular mechanisms underlying this are poorly understood. Here, we show that ZTL regulates thermoresponsive growth through TOC1 and PRR5. Genetic analyses reveal that ZTL regulates PIF4 activity as well as PIF4 expression. In Arabidopsis thaliana, ztl mutants exhibit highly accumulated TOC1 and PRR5 and unresponsive expression of PIF4 target genes under exposure to high temperatures. Mutations in TOC1 and PRR5 restore thermoactivation of PIF4 target genes and thermoresponsive growth in ztl mutants. We also show that the molecular chaperone heat-shock protein90 promotes thermoresponsive growth through the ZTL-TOC1/PRR5 signaling module. Further, we show that ZTL protein stability is increased at high temperatures. Taken together, our results demonstrate that ZTL-mediated degradation of TOC1 and PRR5 enhances the sensitivity of hypocotyl growth to high temperatures.

Universal Stress Protein regulates the circadian rhythm of central oscillator genes in Arabidopsis.

Environmental stresses restrict plant growth and development and decrease crop yield. The circadian clock is associated with the ability of a plant to adapt to daily environmental fluctuations and the production and consumption of energy. Here, we investigated the role of Arabidopsis Universal Stress Protein (USP; At3g53990) in the circadian regulation of nuclear clock genes. The Arabidopsis usp knockout mutant line exhibited critically diminished circadian amplitude of the central oscillator CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) but enhanced the amplitude of TIMING OF CAB EXPRESSION 1 (TOC1). However, the expression of USP under the control of its own promoter restored the circadian timing of both genes, suggesting that USP regulates the circadian rhythm of Arabidopsis central clock genes, CCA1 and TOC1.

Arabidopsis Circadian Clock Repress Phytochrome a Signaling.

The plants’ internal circadian clock can strongly influence phytochrome signaling in response to the changes in the external light environment. Phytochrome A (phyA) is the photoreceptor that mediates various far-red (FR) light responses. phyA signaling is modulated by FHY3 and FAR1, which directly activate the transcription of FHY1 and FHL, whose products are essential for light-induced phyA nuclear accumulation and subsequent light responses. However, the mechanisms by which the clock regulates phyA signaling are poorly understood. Here, we discovered that FHY1 expression is diurnally regulated, peaking in the middle of the day. Two Arabidopsis core clock components, CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and TIMING OF CAB EXPRESSION1 (TOC1), repress FHY3/FAR1-mediated FHY1/FHL activation. Consistently, the specific expression pattern of FHY1 under diurnal conditions is altered in cca1-1, toc1-101, CCA1, and TOC1 overexpression plants. Furthermore, far-red induced gene expression and particularly nuclear accumulation of phyA are compromised in TOC1 and CCA1 overexpression seedlings. Our results therefore revealed a previously unidentified FHY1 expression pattern in diurnal cycles, which is negatively regulated by CCA1 and TOC1.

Noise reduction by upstream open reading frames

Gene expression is prone to burst production, making it a highly noisy process that requires additional controls. Upstream open reading frames (uORFs) are widely present in the 5′ leader sequences of 30–50% of eukaryotic messenger RNAs1–3. The translation of uORFs can repress the translation efficiency of the downstream main coding sequences. Whether the low translation efficiency leads to a different variation, or noise, in gene expression has not been investigated, nor has the direct biological impact of uORF-repressed translation. Here we show that uORFs achieve low but precise protein production in plant cells, possibly by reducing the protein production rate. We also demonstrate that, by buffering a stable TIMING OF CAB EXPRESSION 1 (TOC1) protein production level, uORFs contribute to the robust operation of the plant circadian clock. Our results provide both an action model and the biological impact of uORFs in translational control to mitigate transcriptional noise for precise protein production.

A molecular link between autophagy and circadian rhythm in plants.

Extremely high or low autophagy levels disrupt plant survival under nutrient starvation. Recently, autophagy has been reported to display rhythms in animals. However, the mechanism of circadian regulation of autophagy is still unclear. Here, we observed that autophagy has a robust rhythm and that various autophagy-related genes (ATGs) are rhythmically expressed in Arabidopsis. Chromatin immunoprecipitation (ChIP) and dual-luciferase (LUC) analyses showed that the core oscillator gene TIMING OF CAB EXPRESSION 1 (TOC1) directly binds to the promoters of ATG (ATG1a, ATG2, and ATG8d) and negatively regulates autophagy activities under nutritional stress. Furthermore, autophagy defects might affect endogenous rhythms by reducing the rhythm amplitude of TOC1 and shortening the rhythm period of CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1). Autophagy is essential for the circadian clock pattern in seedling development and plant sensitivity to nutritional deficiencies. Taken together, our studies reveal a plant strategy in which the TOC1-ATG axis involved in autophagy-rhythm crosstalk to fine-tune the intensity of autophagy.

GUN4 Affects the Circadian Clock and Seedlings Adaptation to Changing Light Conditions.

The chloroplast is a key organelle for photosynthesis and perceiving environmental information. GENOME UNCOUPLED 4 (GUN4) has been shown to be required for the regulation of both chlorophyll synthesis, reactive oxygen species (ROS) homeostasis and plastid retrograde signaling. In this study, we found that growth of the gun4 mutant was significantly improved under medium strong light (200 μmol photons m−2s−1) compared to normal light (100 μmol photons m−2s−1), in marked contrast to wild-type (WT). Further analysis revealed that GUN4 interacts with SIGNAL RECOGNITION PARTICLE 54 KDA SUBUNIT (SRP43) and SRP54. RNA-seq analysis indicated that the expression of genes for light signaling and the circadian clock is altered in gun4 compared with (WT). qPCR analysis confirmed that the expression of the clock genes CLOCK-RELATED 1 (CCA1), LATE ELONGATION HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION 1 (TOC1) and PSEUDO RESPONSE REGULATOR 7 (PRR7) is significantly changed in the gun4 and srp54 mutants under normal and medium strong light conditions. These results suggest that GUN4 may coordinate the adaptation of plants to changing light conditions by regulating the biological clock, although it is not clear whether the effect is direct or indirect.

TOC1 clock protein phosphorylation controls complex formation with NF‐YB/C to repress hypocotyl growth

Plant photoperiodic growth is coordinated by interactions between circadian clock and light signaling networks. How post‐translational modifications of clock proteins affect these interactions to mediate rhythmic growth remains unclear. Here, we identify five phosphorylation sites in the Arabidopsis core clock protein TIMING OF CAB EXPRESSION 1 (TOC1) which when mutated to alanine eliminate detectable phosphorylation. The TOC1 phospho‐mutant fails to fully rescue the clock, growth, and flowering phenotypes of the toc1 mutant. Further, the TOC1 phospho‐mutant shows advanced phase, a faster degradation rate, reduced interactions with PHYTOCHROME‐INTERACTING FACTOR 3 (PIF3) and HISTONE DEACETYLASE 15 (HDA15), and poor binding at pre‐dawn hypocotyl growth‐related genes (PHGs), leading to a net de‐repression of hypocotyl growth. NUCLEAR FACTOR Y subunits B and C (NF‐YB/C) stabilize TOC1 at target promoters, and this novel trimeric complex (NF‐TOC1) acts as a transcriptional co‐repressor with HDA15 to inhibit PIF‐mediated hypocotyl elongation. Collectively, we identify a molecular mechanism suggesting how phosphorylation of TOC1 alters its phase, stability, and physical interactions with co‐regulators to precisely phase PHG expression to control photoperiodic hypocotyl growth.

Phytochrome A elevates plant circadian-clock components to suppress shade avoidance in deep-canopy shade

Shade-avoiding plants can detect the presence of neighboring vegetation and evoke escape responses before canopy cover limits photosynthesis. Rapid stem elongation facilitates light foraging and enables plants to overtop competitors. A major regulator of this response is the phytochrome B photoreceptor, which becomes inactivated in light environments with a low ratio of red to far-red light (low R:FR), characteristic of vegetational shade. Although shade avoidance can provide plants with a competitive advantage in fast-growing stands, excessive stem elongation can be detrimental to plant survival. As such, plants have evolved multiple feedback mechanisms to attenuate shade-avoidance signaling. The very low R:FR and reduced levels of photosynthetically active radiation (PAR) present in deep canopy shade can, together, trigger phytochrome A (phyA) signaling, inhibiting shade avoidance and promoting plant survival when resources are severely limited. The molecular mechanisms underlying this response have not been fully elucidated. Here, we show that Arabidopsis thaliana phyA elevates early-evening expression of the central circadian-clock components TIMING OF CAB EXPRESSION 1 (TOC1), PSEUDO RESPONSE REGULATOR 7 (PRR7), EARLY FLOWERING 3 (ELF3), and ELF4 in photocycles of low R:FR and low PAR. These collectively suppress stem elongation, antagonizing shade avoidance in deep canopy shade.

Circadian Control of Metabolism by the Clock Component TOC1.

Photosynthesis in chloroplasts during the day and mitochondrial respiration during the night execute nearly opposing reactions that are coordinated with the internal cellular status and the external conditions. Here, we describe a mechanism by which the Arabidopsis clock component TIMING OF CAB EXPRESSION1 (TOC1) contributes to the diurnal regulation of metabolism. Proper expression of TOC1 is important for sustaining cellular energy and for the diel and circadian oscillations of sugars, amino acids and tricarboxylic acid (TCA) cycle intermediates. TOC1 binds to the promoter of the TCA-related gene FUMARASE 2 to repress its expression at night, which results in decreased fumarate accumulation in TOC1 over-expressing plants and increased in toc1-2 mutant. Genetic interaction studies confirmed that over-expression of FUMARASE 2 in TOC1 over-expressing plants alleviates the molecular and physiological energy-deprivation phenotypes of TOC1 over-expressing plants. Thus, we propose that the tandem TOC1-FUMARASE 2 is one of the mechanisms that contribute to the regulation of plant metabolism during the day and night.

Identification and expression profiling of genes involved in circadian clock regulation in red dragon fruit (Hylocereus polyrhizus) by full-length transcriptome sequencing

ABSTRACT Crassulacean acid metabolism (CAM) plants fix CO2 at night, exhibiting a reversed regulatory pattern of metabolomic pathways compared with most model plants, which have C3 and C4 pathways. In this study, we used a valuable tropic fruit, red dragon fruit (Hylocereus polyrhizus), as model plant to identify and analyze the circadian regulation genes. Due to the absence of red dragon fruit’s whole-genome dataset, we established a full-length transcriptome dataset using single-molecule real-time (SMRT) sequencing method. A 7.66-Gb dataset with 4,552,474 subreads was generated, with an average length of 1,683 bp and an N50 of 2,446 bp. Using this dataset, we identified center oscillator genes: CCA1 (CIRCADIAN CLOCK ASSOCIATED1), ELF3 (EARLY FLOWERING 3), GI (GIGANTEA), LHY (LATE ELONGATED HYPOCOTYL), LNK1 (NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED 1), and TOC1 (TIMING OF CAB EXPRESSION 1); a gene for the input pathway: CRY1 (CRYPTOCHROME); a gene for the output pathway: CO (CONSTANS); and genes related to the CAM pathway: MDH (MALATE DEHYDROGENASE), ME (MALIC ENZYMES), and PPDK (PYRUVATE PHOSPHATE DIKINASE). We further established the 24-h rhythmic expression pattern of these genes and classified these into three groups: HpCCA1, HpELF3, HpLHY, HpLNK1, and HpGI have expression peaks during the day; HpTOC1, HpCO, and HpCRY1 have highest expression levels at night; The genes involved in the CAM pathways, namely, HpMDH, HpME1, and HpPPDK, have double expression peaks in the day and night. Comparison of these expression patterns between red dragon fruit and model plants could provide clues in understanding the circadian clock regulation and the activity of the CAM pathways in cactus plants.

Circadian Clock in Arabidopsis thaliana Determines Flower Opening Time Early in the Morning and Dominantly Closes Early in the Afternoon.

Many plant species exhibit diurnal flower opening and closing, which is an adaptation influenced by the lifestyle of pollinators and herbivores. However, it remains unclear how these temporal floral movements are modulated. To clarify the role of the circadian clock in flower movement, we examined temporal floral movements in Arabidopsis thaliana. Wild-type (accessions; Col-0, Ler-0 and Ws-4) flowers opened between 0.7 and 1.4 h in a 16-h light period and closed between 7.5 and 8.3 h in a diurnal light period. In the arrhythmic mutants pcl1-1 and prr975, the former flowers closed slowly and imperfectly and the latter ones never closed. Under continuous light conditions, new flowers emerged and opened within a 23-26 h window in the wild-type, but the flowers in pcl1-1 and prr975 developed straight petals, whose curvatures were extremely small. Anti-phasic circadian gene expression of CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYLE (LHY) and TIMING OF CAB EXPRESSION 1 (TOC1) occurred in wild-type flowers, but non-rhythmic expression was observed in pcl1-1 and prr975 mutants. Focusing on excised petals, bioluminescence monitoring revealed rhythmic promoter activities of genes expressed (CCA1, LHY and PHYTOCLOCK 1/LUX ARRHYTHMO, PCL1/LUX) in the morning and evening. These results suggest that the clock induces flower opening redundantly with unknown light-sensing pathways. By contrast, flower closing is completely dependent on clock control. These findings will lead to further exploration of the molecular mechanisms and evolutionary diversity of timing in flower opening and closing.

SDC mediates DNA methylation-controlled clock pace by interacting with ZTL in Arabidopsis.

Molecular bases of eukaryotic circadian clocks mainly rely on transcriptional-translational feedback loops (TTFLs), while epigenetic codes also play critical roles in fine-tuning circadian rhythms. However, unlike histone modification codes that play extensive and well-known roles in the regulation of circadian clocks, whether DNA methylation (5mC) can affect the circadian clock, and the associated underlying molecular mechanisms, remains largely unexplored in many organisms. Here we demonstrate that global genome DNA hypomethylation can significantly lengthen the circadian period of Arabidopsis. Transcriptomic and genetic evidence demonstrate that SUPPRESSOR OF drm1 drm2 cmt3 (SDC), encoding an F-box containing protein, is required for the DNA hypomethylation-tuned circadian clock. Moreover, SDC can physically interact with another F-box containing protein ZEITLUPE (ZTL) to diminish its accumulation. Genetic analysis further revealed that ZTL and its substrate TIMING OF CAB EXPRESSION 1 (TOC1) likely act downstream of DNA methyltransferases to control circadian rhythm. Together, our findings support the notion that DNA methylation is important to maintain proper circadian pace in Arabidopsis, and further established that SDC links DNA hypomethylation with a proteolytic cascade to assist in tuning the circadian clock.