The intrinsically disordered N-terminal region of mouse DNA polymerase alpha mediates its interaction with POT1a/b at telomeres.

Mouse telomerase and the DNA polymerase alpha-primase complex elongate the leading and lagging strands of telomeres, respectively. To elucidate the molecular mechanism of lagging strand synthesis, we investigated the interaction between DNA polymerase alpha and two paralogs of the mouse POT1 telomere-binding protein (POT1a and POT1b). Yeast two-hybrid analysis and a glutathione S-transferase pull-down assay indicated that the C-terminal region of POT1a/b binds to the intrinsically disordered N-terminal region of p180, the catalytic subunit of mouse DNA polymerase alpha. Subcellular distribution analyses showed that although POT1a, POT1b, and TPP1 were localized to the cytoplasm, POT1a-TPP1 and POT1b-TPP1 coexpressed with TIN2 localized to the nucleus in a TIN2 dose-dependent manner. Coimmunoprecipitation and cell cycle synchronization experiments indicated that POT1b-TPP1-TIN2 was more strongly associated with p180 than POT1a-TPP1-TIN2, and this complex accumulated during the S phase. Fluorescence in situ hybridization and proximity ligation assays showed that POT1a and POT1b interacted with p180 and TIN2 on telomeric chromatin. Based on the present study and a previous study, we propose a model in which POT1a/b-TPP1-TIN2 translocates into the nucleus in a TIN2 dose-dependent manner to target the telomere, where POT1a/b interacts with DNA polymerase alpha for recruitment at the telomere for lagging strand synthesis.

Modeling and Validation of Vertical Direction Force Estimation with a Three-Dimensional Force Measurement Instrument Based on a Zero-Compliance Mechanism.

The principle of a zero-compliance mechanism was used to develop a three-dimensional force measurement instrument. In each axis, the point of force is suspended by a zero-compliance mechanism. A vertical axis force estimation operation imitates the structure of a double series magnetic suspension system. An electromagnet directly controls the movement of the first suspended object (floator), which is denoted as a detection point, and indirectly controls the motion of the second floator, which is denoted as a point of force. Indirect control of the point of force is executed by the attractive force of a permanent magnet that is fixed to the bottom part of the detection point. To achieve zero-compliance, a Proportional-Integral-Derivative (PID) control is applied to the point of force, and to make the system stable, a Proportional-Derivative (PD) control is also applied to the detection point. In such suspension conditions, when force is exerted on the point of force, the displacement of the second floator is regulated to maintain its primary position while the detection point displaces in proportion to the applied force. Thus, a zero-compliance condition is maintained at the point of force, and the external force is measured from the linear displacement of the detection point. To restrict the motions of the detection point and the point of force in translation only, they are supported with leaf springs. This paper presents the modeling of the vertical direction force measurement operation of the developed three-axis force estimation instrument, and the theoretical analyses were validated by experiments of force measurement in both the millinewton and micronewton ranges.

The Mcm2-7-interacting domain of human mini-chromosome maintenance 10 (Mcm10) protein is important for stable chromatin association and origin firing.

The protein mini-chromosome maintenance 10 (Mcm10) was originally identified as an essential yeast protein in the maintenance of mini-chromosome plasmids. Subsequently, Mcm10 has been shown to be required for both initiation and elongation during chromosomal DNA replication. However, it is not fully understood how the multiple functions of Mcm10 are coordinated or how Mcm10 interacts with other factors at replication forks. Here, we identified and characterized the Mcm2-7-interacting domain in human Mcm10. The interaction with Mcm2-7 required the Mcm10 domain that contained amino acids 530-655, which overlapped with the domain required for the stable retention of Mcm10 on chromatin. Expression of truncated Mcm10 in HeLa cells depleted of endogenous Mcm10 via siRNA revealed that the Mcm10 conserved domain (amino acids 200-482) is essential for DNA replication, whereas both the conserved and the Mcm2-7-binding domains were required for its full activity. Mcm10 depletion reduced the initiation frequency of DNA replication and interfered with chromatin loading of replication protein A, DNA polymerase (Pol) α, and proliferating cell nuclear antigen, whereas the chromatin loading of Cdc45 and Pol ϵ was unaffected. These results suggest that human Mcm10 is bound to chromatin through the interaction with Mcm2-7 and is primarily involved in the initiation of DNA replication after loading of Cdc45 and Pol ϵ.

Insight into a Physiological Role for the EC Night-Time Repressor in the Arabidopsis Circadian Clock.

Life cycle adaptation to seasonal variation in photoperiod and temperature is a major determinant of ecological success of widespread domestication of Arabidopsis thaliana. The circadian clock plays a role in the underlying mechanism for adaptation. Nevertheless, the mechanism by which the circadian clock tracks seasonal changes in photoperiod and temperature is a longstanding subject of research in the field. We previously showed that a set of the target genes (i.e. GI, LNK1. PRR9 and PRR7) of the Evening Complex (EC) consisting of LUX-ELF3-ELF4 is synergistically induced in response to both warm-night and night-light signals. Here, we further show that the responses occur within a wide range of growth-compatible temperatures (16-28°C) in response to a small change in temperature (Δ4°C). A dim light pulse (<1 µmol m(-2) s(-1)) causes the enhanced effect on the transcription of EC targets. The night-light pulse antagonizes against a positive effect of the cool-night signal on the EC activity. The mechanism of double-checking external temperature and light signals through the EC nighttime repressor might enable plants to ignore (or tolerate) daily fluctuation of ambient temperature within a short time interval in their natural habitats. Taken together, the EC night-time repressor might play a physiological role in tracking seasonal variation in photoperiod and temperature by conservatively double-checking both the light and temperature conditions. Another EC target output gene PIF4 regulating plant morphologies is also regulated by both the temperature and light stimuli during the night. Hence, the EC night-time repressor is also implicated in a physiological output of the PIF4-mediated regulation of morphologies in response to seasonal variation in photoperiod and ambient temperature.

Direct single-molecule observations of local denaturation of a DNA double helix under a negative supercoil state.

Effects of a negative supercoil on the local denaturation of the DNA double helix were studied at the single-molecule level. The local denaturation in λDNA and λDNA containing the SV40 origin of DNA replication (SV40ori-λDNA) was directly observed by staining single-stranded DNA regions with a fusion protein comprising the ssDNA binding domain of a 70-kDa subunit of replication protein A and an enhanced yellow fluorescent protein (RPA-YFP) followed by staining the double-stranded DNA regions with YOYO-1. The local denaturation of λDNA and SV40ori-λDNA under a negative supercoil state was observed as single bright spots at the single-stranded regions. When negative supercoil densities were gradually increased to 0, -0.045, and -0.095 for λDNA and 0, -0.047, and -0.1 for SV40ori-λDNA, single bright spots at the single-stranded regions were frequently induced under higher negative supercoil densities of -0.095 for λDNA and -0.1 for SV40ori-λDNA. However, single bright spots of the single-stranded regions were rarely observed below a negative supercoil density of -0.045 and -0.047 for λDNA and SV40ori-λDNA, respectively. The probability of occurrence of the local denaturation increased with negative superhelicity for both λDNA and SV40ori-λDNA.

Insight into the mechanism of end-of-day far-red light (EODFR)-induced shade avoidance responses in Arabidopsis thaliana.

Shade avoidance responses are changes in plant architecture to reduce the part of a body that is in the shade in natural habitats. The most common warning signal that induces shade avoidance responses is reduction of red/far-red light ratio perceived by phytochromes. A pair of basic helix-loop-helix transcription factors, named PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) and PIF5, is crucially involved in the shade avoidance-induced hypocotyl elongation in Arabidopsis thaliana. It has been recently reported that PIF7 also plays a role in this event. Here, we examined the involvement of these PIFs in end-of-day far-red light (EODFR) responses under light and dark cycle conditions. It was shown that PIF7 played a predominant role in the EODFR-dependent hypocotyl elongation. We propose the mechanism by which PIF7 together with PIF4 and PIF5 coordinately transcribes a set of downstream genes to promote elongation of hypocotyls in response to the EODFR treatment.

Transcriptional repressor PRR5 directly regulates clock-output pathways.

The circadian clock is an endogenous time-keeping mechanism that enables organisms to adapt to external daily cycles. The clock coordinates biological activities with these cycles, mainly through genome-wide gene expression. However, the exact mechanism underlying regulation of circadian gene expression is poorly understood. Here we demonstrated that an Arabidopsis PSEUDO-RESPONSE REGULATOR 5 (PRR5), which acts in the clock genetic circuit, directly regulates expression timing of key transcription factors involved in clock-output pathways. A transient expression assay and ChIP-quantitative PCR assay using mutated PRR5 indicated that PRR5 associates with target DNA through binding at the CCT motif in vivo. ChIP followed by deep sequencing coupled with genome-wide expression profiling revealed the direct-target genes of PRR5. PRR5 direct-targets include genes encoding transcription factors involved in flowering-time regulation, hypocotyl elongation, and cold-stress responses. PRR5-target gene expression followed a circadian rhythm pattern with low, basal expression from noon until midnight, when PRR9, PRR7, and PRR5 were expressed. ChIP-quantitative PCR assays indicated that PRR7 and PRR9 bind to the direct-targets of PRR5. Genome-wide expression profiling using a prr9 prr7 prr5 triple mutant suggests that PRR5, PRR7, and PRR9 repress these targets. Taken together, our results illustrate a genetic network in which PRR5, PRR7, and PRR9 directly regulate expression timing of key transcription factors to coordinate physiological processes with daily cycles.

Implantation study of a tissue-engineered self-expanding aortic stent graft (bio stent graft) in a beagle model.

The use of stent grafts for endovascular aortic repair has become an important treatment option for aortic aneurysms requiring surgery. This treatment has achieved excellent outcomes; however, problems like type 1 endoleaks and stent graft migration remain. Bio stent grafts (BSGs), which are self-expanding stents covered with connective tissue, were previously developed using "in-body tissue architecture" technology. We assessed their early adaptation to the aorta after transcatheter implantation in a beagle model. BSGs were prepared by subcutaneous embedding of acryl rods mounted with self-expanding nitinol stents in three beagles for 4 weeks (n = 3/dog). The BSGs were implanted as allografts into infrarenal abdominal aortas via the femoral artery of three other beagles. After 1 month of implantation, aortography revealed no stenosis or aneurysmal changes. The luminal surface of the BSGs was completely covered with neointimal tissue, including endothelialization, without any thrombus formation. The cover tissue could fuse the luminal surface of the native aorta with tight conjunctions even at both ends of the stents, resulting in complete impregnation of the strut into the reconstructed vascular wall, which is expected to prevent endoleaks and migration in clinical applications.

The EC night-time repressor plays a crucial role in modulating circadian clock transcriptional circuitry by conservatively double-checking both warm-night and night-time-light signals in a synergistic manner in Arabidopsis thaliana.

During the last decade, significant research progress has been made in Arabidopsis thaliana in defining the molecular mechanisms behind the plant circadian clock. The circadian clock must have the ability to integrate both external light and ambient temperature signals into its transcriptional circuitry to regulate its function properly. We previously showed that transcription of a set of clock genes including LUX (LUX ARRHYTHMO), GI (GIGANTEA), LNK1 (NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED GENE 1), PRR9 (PSEUDO-RESPONSE REGULATOR 9) and PRR7 is commonly regulated through the evening complex (EC) night-time repressor in response to both moderate changes in temperature (Δ6°C) and differences in steady-state growth-compatible temperature (16-28°C). Here, we further show that a night-time-light signal also feeds into the circadian clock transcriptional circuitry through the EC night-time repressor, so that the same set of EC target genes is up-regulated in response to a night-time-light pulse. This light-induced event is dependent on phytochromes, but not cryptochromes. Interestingly, both the warm-night and night-time-light signals negatively modulate the activity of the EC night-time repressor in a synergistic manner. In other words, an exponential burst of transcription of the EC target genes is observed only when these signals are simultaneously fed into the repressor. Taken together, we propose that the EC night-time repressor plays a crucial role in modulating the clock transcriptional circuitry to keep track properly of seasonal changes in photo- and thermal cycles by conservatively double-checking the external light and ambient temperature signals.

Ambient temperature signal feeds into the circadian clock transcriptional circuitry through the EC night-time repressor in Arabidopsis thaliana.

An interlocking multiloop model has been generally accepted to describe the transcriptional circuitry of core clock genes, through which robust circadian rhythms are generated in Arabidopsis thaliana. The circadian clock must have the ability to integrate ambient temperature signals into the clock transcriptional circuitry to regulate clock function properly. Clarification of the underlying mechanism is a longstanding subject in the field. Here, we provide evidence that temperature signals feed into the clock transcriptional circuitry through the evening complex (EC) night-time repressor consisting of EARLY FLOWERING 3 (ELF3, ELF4) and LUX ARRHYTHMO (LUX; also known as PCL1). Chromatin immunoprecipitation assays showed that PSEUDO-RESPONSE REGULATOR7 (PRR7), GIGANTEA (GI) and LUX are direct targets of the night-time repressor. Consequently, transcription of PRR9/PRR7, GI and LUX is commonly regulated through the night-time repressor in response to both moderate changes in temperature (Δ6°C) and differences in the steady-state growth-compatible temperature (16-28°C). A warmer temperature inhibits EC function more, whereas a cooler temperature stimulates it more. Consequently, the expression of these target genes is up-regulated in response to a warm temperature specifically during the dark period, whereas they are reversibly down-regulated in response to a cool temperature. Transcription of another EC target, the PIF4 (PHYTOCHROME-INTERACTING FACTOR 4) gene, is modulated through the same thermoregulatory mechanism. The last finding revealed the sophisticated physiological mechanism underlying the clock-controlled output pathway, which leads to the PIF4-mediated temperature-adaptive regulation of hypocotyl elongation.

A new direct single-molecule observation method for DNA synthesis reaction using fluorescent replication protein A.

Using a single-stranded region tracing system, single-molecule DNA synthesis reactions were directly observed in microflow channels. The direct single-molecule observations of DNA synthesis were labeled with a fusion protein consisting of the ssDNA-binding domain of a 70-kDa subunit of replication protein A and enhanced yellow fluorescent protein (RPA-YFP). Our method was suitable for measurement of DNA synthesis reaction rates with control of the ssλDNA form as stretched ssλDNA (+flow) and random coiled ssλDNA (-flow) via buffer flow. Sequentially captured photographs demonstrated that the synthesized region of an ssλDNA molecule monotonously increased with the reaction time. The DNA synthesis reaction rate of random coiled ssλDNA (-flow) was nearly the same as that measured in a previous ensemble molecule experiment (52 vs. 50 bases/s). This suggested that the random coiled form of DNA (-flow) reflected the DNA form in the bulk experiment in the case of DNA synthesis reactions. In addition, the DNA synthesis reaction rate of stretched ssλDNA (+flow) was approximately 75% higher than that of random coiled ssλDNA (-flow) (91 vs. 52 bases/s). The DNA synthesis reaction rate of the Klenow fragment (3'-5'exo-) was promoted by DNA stretching with buffer flow.

PSEUDO-RESPONSE REGULATORS 9, 7, and 5 are transcriptional repressors in the Arabidopsis circadian clock.

An interlocking transcriptional-translational feedback loop of clock-associated genes is thought to be the central oscillator of the circadian clock in plants. TIMING OF CAB EXPRESSION1 (also called PSEUDO-RESPONSE REGULATOR1 [PRR1]) and two MYB transcription factors, CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), play pivotal roles in the loop. Genetic studies have suggested that PRR9, PRR7, and PRR5 also act within or close to the loop; however, their molecular functions remain unknown. Here, we demonstrate that PRR9, PRR7, and PRR5 act as transcriptional repressors of CCA1 and LHY. PRR9, PRR7, and PRR5 each suppress CCA1 and LHY promoter activities and confer transcriptional repressor activity to a heterologous DNA binding protein in a transient reporter assay. Using a glucocorticoid-induced PRR5-GR (glucorticoid receptor) construct, we found that PRR5 directly downregulates CCA1 and LHY expression. Furthermore, PRR9, PRR7, and PRR5 associate with the CCA1 and LHY promoters in vivo, coincident with the timing of decreased CCA1 and LHY expression. These results suggest that the repressor activities of PRR9, PRR7, and PRR5 on the CCA1 and LHY promoter regions constitute the molecular mechanism that accounts for the role of these proteins in the feedback loop of the circadian clock.

Direct observation of fluorescently labeled single-stranded λDNA molecules in a micro-flow channel.

We developed two labeling methods for the direct observation of single-stranded DNA (ssDNA), using a ssDNA binding protein and a ssDNA recognition peptide. The first approach involved protein fusion between the 70-kDa ssDNA-binding domain of replication protein A and enhanced yellow fluorescent protein (RPA-YFP). The second method used the ssDNA binding peptide of Escherichia coli RecA labeled with Atto488 (ssBP-488; Atto488-IRMKIGVMFGNPETTTGGNALKFY). The labeled ssλDNA molecules were visualized over time in micro-flow channels. We report substantially different dynamics between these two labeling methods. When ssλDNA molecules were labeled with RPA-YFP, terminally bound fusion proteins were sheared from the free ends of the ssλDNA molecules unless 25-mer oligonucleotides were annealed to the free ends. RPA-YFP-ssλDNA complexes were dissociated by the addition of 0.2 M NaCl, although complex reassembly was possible with injection of additional RPA-YFP. In contrast to the flexible dynamics of RPA-YFP-ssλDNA complexes, the ssBP-488-ssλDNA complexes behaved as rigid rods and were not dissociated even in 2 M NaCl.

Transcription of ST2A encoding a sulfotransferase family protein that is involved in jasmonic acid metabolism is controlled according to the circadian clock- and PIF4/PIF5-mediated external coincidence mechanism in Arabidopsis thaliana.

Plant elongation growth on a day-to-day basis is enhanced under specific photoperiod and temperature conditions. Circadian clock is involved in the temperature adaptive photoperiodic control of plant architecture, including hypocotyl elongation in Arabidopsis thaliana. In this regulation, phytochrome interacting transcriptional factors, PIF4 and PIF5, are activated at the end of night under short photoperiod or high temperature conditions, due to the coincidence between internal (circadian rhythm of the transcripts) and external (length of dark period) time cues. It is previously found that biosynthesis or metabolism of phytohormones including auxin, and their signal transduction-related genes are downstream targets of circadian clock and PIF4/PIF5 mediated external coincidence mechanism. Brassinosteroid and gibberellic acid played a positive role in the hypocotyl elongation of seedlings under light and dark cycle conditions. On the other hand, cytokinin and jasmonic acid played an opposite role. In this study, diurnal expression profile of a gene encoding a sulfotransferase family protein that is involved in the jasmonic acid metabolism, ST2A, was examined. It was found that transcription of ST2A is induced at the end of night under LD/22 °C and SD/28 °C conditions according to the external coincidence mechanism. The results of this study support the idea that the circadian clock orchestrates a variety of hormone-signaling pathways to regulate the photoperiod and temperature-dependent morphogenesis in A. thaliana.

Clock-controlled and FLOWERING LOCUS T (FT)-dependent photoperiodic pathway in Lotus japonicus I: verification of the flowering-associated function of an FT homolog.

During the last decade, significant research progress in the study of Arabidopsis thaliana has been made in defining the molecular mechanism by which the plant circadian clock regulates flowering time in response to changes in photoperiod. It is generally accepted that the clock-controlled CONSTANS (CO)-FLOWERING LOCUS T (FT)-mediated external coincidence mechanism underlying the photoperiodic control of flowering time is conserved in higher plants, including A. thaliana and Oryza sativa. However, it is also assumed that the mechanism differs considerably in detail among species. Here we characterized the clock-controlled CO-FT pathway in Lotus japonicus (a model legume) in comparison with that of A. thaliana. L. japonicus has at least one FT orthologous gene (named LjFTa), which is induced specifically in long-days and complements the mutational lesion of the A. thaliana FT gene. However, it was speculated that this legume might lack the upstream positive regulator CO. By employing L. japonicus phyB mutant plants, we showed that the photoreceptor mutant displays a phenotype of early flowering due to enhanced expression of LjFTa, suggesting that LjFTa is invovled in the promotion of flowering in L. japonicus. These results are discussed in the context of current knowledge of the flowering in crop legumes such as soybean and garden pea.

Clock-controlled and FLOWERING LOCUS T (FT)-dependent photoperiodic pathway in Lotus japonicus II: characterization of a microRNA implicated in the control of flowering time.

Plant circadian clock generates rhythms with a period close to 24 h, and it controls a wide variety of physiological and developmental events, including the transition to reproductive growth (or flowering). During the last decade, significant research progress in Arabidopsis thaliana has been made in defining the molecular mechanism by which the circadian clock regulates flowering time in response to changes in photoperiod. In Lotus japonicus, we have found that LjFTa, which encodes a ortholog of the Arabidopsis FLOWERING LOCUS T (FT), plays an important role in the promotion of flowering, but it is not clear how the expression of LjFTa is regulated in L. japonicus. Based on current knowledge of photoperiodic control of flowering time in A. thaliana, here we examined whether a microRNA is involved in the activation of LjFTa in L. japonicus. Two putative L. japonicus genes that are responsible for the production of miR172 (designated LjmiR172a and LjmiR172b) were cloned. Overexpression of LjmiR172a/b in A. thaliana resulted in markedly accelerated flowering through enhancement of the expression of FT, concomitantly reducing the expression level of TARGET OF EARLY ACTIVATION TAGGED 1 (TOE1) transcripts, the protein product of which functions as a transcriptional repressor of FT. These results suggest that LjmiR172 genes play a positive role in the LjFTa-mediated promotion of flowering in L. japonicus.

Circadian clock- and PIF4-controlled plant growth: a coincidence mechanism directly integrates a hormone signaling network into the photoperiodic control of plant architectures in Arabidopsis thaliana.

The plant circadian clock generates rhythms with a period close to 24 h, and it controls a wide variety of physiological and developmental events, enabling plants to adapt to ever-changing environmental light conditions. In Arabidopsis thaliana, the clock regulates the diurnal and photoperiodic plant growth including the elongation of hypocotyls and petioles in a time-of-day-specific and short-day (SD)-specific manner. In this mechanism, the clock-regulated PHYTOCHROME-INTERACTING FACTOR 4 gene encoding a basic helix-loop-helix transcription factor, together with phytochromes (mainly phyB), plays crucial roles. This diurnal and photoperiodic control of plant growth is best explained by the accumulation of the PIF4 protein at the end of the night-time specifically under SDs, due to coincidence between the internal (circadian rhythm) and external (photoperiod) cues. In this model, however, the PIF4-controlled downstream factors are not fully identified, although it has been generally proposed that the auxin-mediated signal transduction is crucially implicated. Here, we identified a set of hormone-associated genes as the specific PIF4 targets implicated in the photoperiodic control of plant growth. They include not only auxin-associated genes (GH3.5, IAA19 and IAA29), but also genes associated with other growth-regulating hormones such as brassinosteroids (BR6ox2), gibberellic acids (GAI), ethylene (ACS8) and cytokinin (CKX5). The dawn- and SD-specific expression profiles of these genes are modified in a set of phyB and clock mutants, both of which compromise the coincidence mechanism. The results of this study suggest that the circadian clock orchestrates a variety of hormone signaling pathways to regulate the photoperiod-dependent morphogenesis in A. thaliana.

A circadian clock- and PIF4-mediated double coincidence mechanism is implicated in the thermosensitive photoperiodic control of plant architectures in Arabidopsis thaliana.

In Arabidopsis thaliana, the circadian clock regulates diurnal and photoperiodic plant growth including the elongation of hypocotyls in a time-of-day-specific and short-day (SD)-specific manner. The clock-controlled PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) encoding a basic helix-loop-helix (bHLH) transcription factor plays crucial roles in this regulation. PIF4 is transcribed precociously at the end of the night in SDs, under which conditions the protein product is stably accumulated, while PIF4 is expressed exclusively during the daytime in long days (LDs), under which conditions the protein product is degraded by light-activated phytochrome B. The dawn- and SD-specific elongation of hypocotyls is best explained by the coincident accumulation of the active PIF4 protein during the night-time before dawn specifically in SDs. However, this coincidence model was challenged with the recent finding that the elongation of hypocotyls is markedly promoted at high growth temperature (i.e. 28°C) even under LDs in a PIF4-dependent manner. Here, we reconciled these apparently conflicting facts by showing that the transcription of PIF4 occurs precociously at the end of the night-time at 28°C in LDs, similarly to in SDs. Both the events resulted in the same consequence, i.e. that a set of PIF4 target genes (ATHB2, GH3.5, IAA19, IAA29, BRox2, GAI, ACS8 and CKX5) was induced accordingly in a time-of-day-specific manner. Taken together, we propose an extended double coincidence mechanism, by which the two environmental cues (i.e. photoperiods and temperatures), both of which vary on a season to season basis, are integrated into the same clock- and PIF4-mediated output pathway and regulate a hormone signaling network to fit plant architectures properly to domestic habitats.

Molecular mechanisms of circadian rhythm in Lotus japonicus and Arabidopsis thaliana are sufficiently compatible to regulate heterologous core clock genes robustly.

Recent intensive studies of the model plant Arabidopsis thaliana have revealed the molecular mechanisms underlying circadian rhythms in detail. Results of phylogenetic analyses indicated that some of core clock genes are widely conserved throughout the plant kingdom. For another model plant the legume Lotus japonicus, we have reported that it has a set of putative clock genes highly homologous to A. thaliana. Taking advantage of the L. japonicus hairy root transformation system, in this study we characterized the promoter activity of A. thaliana core clock genes CCA1 and PRR5 in heterologous L. japonicus cells and found that the molecular mechanism of circadian rhythm in L. japonicus is compatible with that of A. thaliana.

Impact of clock-associated Arabidopsis pseudo-response regulators in metabolic coordination.

In higher plants, the circadian clock controls a wide range of cellular processes such as photosynthesis and stress responses. Understanding metabolic changes in arrhythmic plants and determining output-related function of clock genes would help in elucidating circadian-clock mechanisms underlying plant growth and development. In this work, we investigated physiological relevance of PSEUDO-RESPONSE REGULATORS (PRR 9, 7, and 5) in Arabidopsis thaliana by transcriptomic and metabolomic analyses. Metabolite profiling using gas chromatography-time-of-flight mass spectrometry demonstrated well-differentiated metabolite phenotypes of seven mutants, including two arrhythmic plants with similar morphology, a PRR 9, 7, and 5 triple mutant and a CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1)-overexpressor line. Despite different light and time conditions, the triple mutant exhibited a dramatic increase in intermediates in the tricarboxylic acid cycle. This suggests that proteins PRR 9, 7, and 5 are involved in maintaining mitochondrial homeostasis. Integrated analysis of transcriptomics and metabolomics revealed that PRR 9, 7, and 5 negatively regulate the biosynthetic pathways of chlorophyll, carotenoid and abscisic acid, and alpha-tocopherol, highlighting them as additional outputs of pseudo-response regulators. These findings indicated that mitochondrial functions are coupled with the circadian system in plants.