Country：Japan

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41598-019-39720-2

Web of Science

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1111/nph.19323

Web of Science

PubMed

DOI： 10.1371/journal.pone.0285241

Web of Science

PubMed

DOI： 10.1093/pcp/pcad107

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PubMed

DOI： 10.5059/yukigoseikyokaishi.81.718

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Language：Japanese  

DOI： 10.1093/bbb/zbac160

Web of Science

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/pcp/pcac127

Web of Science

Authorship：Last author,　Corresponding author   Language：English  

DOI： 10.1093/pcp/pcac011

Web of Science

Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41467-021-21167-7

Web of Science

Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1080/09168451.2020.1719822

Web of Science

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/pld3.172

Web of Science

Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41598-019-46440-0

Web of Science

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/pcp/pcu214

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

In Arabidopsis thaliana, central circadian clock genes constitute several feedback loops. These interlocking loops generate an ~24-h oscillation that enables plants to anticipate the daily diurnal environment. The identification of additional clock proteins can help dissect the complex nature of the circadian clock. Previously, LIGHT-REGULATED WD1 (LWD1) and LWD2 were identified as two clock proteins regulating circadian period length and photoperiodic flowering. Here, we systematically studied the function of LWD1/2 in the Arabidopsis circadian clock. Analysis of the lwd1 lwd2 double mutant revealed that LWD1/2 plays dual functions in the light input pathway and the regulation of the central oscillator. Promoter:luciferase fusion studies showed that activities of LWD1/2 promoters are rhythmic and depend on functional PSEUDO-RESPONSE REGULATOR9 (PRR9) and PRR7. LWD1/2 is also needed for the expression of PRR9, PRR7, and PRR5. LWD1 is preferentially localized within the nucleus and associates with promoters of PRR9, PRR5, and TOC1 in vivo. Our results support the existence of a positive feedback loop within the Arabidopsis circadian clock. Further mechanistic studies of this positive feedback loop and its regulatory effects on the other clock components will further elucidate the complex nature of the Arabidopsis circadian clock.

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

In Arabidopsis thaliana, a consistent multiloop clock model has been widely adopted in many recent publications. This tentative model consists of three interactive feedback loops, namely the core CCA1/LHY-TOC1/X loop, the morning CCA1/LHY-PRR9/PRR7 loop and the evening Y-TOC1 loop, in which the undefined Y gene might be GI. The model in its current form provides us with a basis on which to address a number of fundamental issues for a better understanding of the molecular mechanism by which the central oscillator generates circadian rhythms. We have been conducting a series of genetic studies through the establishment of a set of combinatorial mutants. We have already characterized a prr9 prr7 double loss-of-function mutant that has lost the morning loop, and a cca1 lhy toc1 triple mutant that lacks the core loop. Extension of this line of study required characterization of a gi toc1 double loss-of-function mutant, which is expected to have no evening loop, and a prr9 prr7 toc1 triple mutant, lacking both the morning and evening loops. Genetic analysis of both these lines is reported here. From the results, we have clarified the genetic linkages between GI and TOC1 and those between PRR9/PRR7 and TOC1 with reference to the circadian clock-associated phenotypes, including: (i) length of hypocotyls during early photomor-phogenesis; (ii) photoperiodic control of flowering time; and (iii) expression profiles of CCA1 and LHY under free-running conditions. These results indicate that GI is not sufficient to fulfill the Y role, but plays more complicated clock-associated roles and, interestingly, that no epistatic interaction between PRR9/PRR7 and TOC1 was observed. Furthermore, these clock-defective mutants could still generate robust, free-running rhythms at the level of transcription. Therefore, we speculate that an as yet undefined oscillator (or loop) continues to generate rhythms within the plants lacking GI/TOC1 or PRR9/PRR7/TOC

Language：English   Publishing type：Research paper (scientific journal)  

The transcriptional/translational feedback loop is thought to play a central role in the circadian clock in Arabidopsis. The loop includes close paralogs of MYB transcription factors CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION 1 (TOC1, PSEUDO RESPONSE REGULATOR 1[PRR1]), PRR9, PRR7 and PRR5. The prr9 prr7 prr5 triple mutants (d975) and overexpression line of CCA1 (CCA1-ox) are arrhythmic under continuous light conditions followed by light and dark cycles. We recently demonstrated a tight link between the circadian clock and the tricarboxylic acid (TCA) cycle, from the metabolome analysis of d975. The levels of metabolites belonging to TCA cycle, such as 2-oxoglutarate, succinate, fumarate, citrate and malate increased in d975, whereas those of arginine and ornithine decreased. In this addendum, we further demonstrate that metabolism belonging to TCA cycle in CCA1-ox was partly similar to that of d975. This profiling also supported the linkage between circadian clock and metabolism associated to TCA cycle.

Language：English   Publishing type：Research paper (scientific journal)  

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.

Language：English   Publishing type：Research paper (scientific journal)  

In plants, the circadian clock is implicated in the biological system that generates diurnal oscillations in cellular and physiological activities. The circadian clock must be synchronized (or entrained) to local time by environmental time cues, such as light/dark and/or hot/cold cycles. In Arabidopsis thaliana, although a number of clock-associated components have been uncovered over the last decade, the clock-associated elements that are involved in entrainment to environmental time cues are largely unknown. In this regard, we have been characterizing one core group of clock components that together control the pace of the central oscillator, including PSEUDO-RESPONSE REGULATOR9 (PRR9), PRR7, PRR5 and TIMING OF CAB2 EXPRESSION 1 (TOC1; or PRR1). The primary aim of this study is to clarify whether these PRR members are implicated in entrainment of the circadian clock to environmental time cues. For this purpose, the diurnal oscillation profiles of clock-controlled genes in the presence of environmental time cues were determined in a set of prr mutants, including a prr9 prr7 prr5 toc1 quadruple mutant. As an extreme phenotype, the prr9-10 prr7-11 prr5-11 toc1-2 quadruple mutant showed an arrhythmia phenotype even under light/dark and hot/cold cycles. In contrast, a cca1-1 lhy-11 toc1-2 triple mutant maintained robust oscillations in the presence of these environmental time cues, although their phases were markedly affected. Based on these results, we propose that the clock components PRR9, PRR7 and PRR5 together might represent elements necessary for the circadian clock to entrain properly to local time in response to light/dark and hot/cold cycles in natural habitats.

Language：English   Publishing type：Research paper (scientific journal)  

In Arabidopsis thaliana, many circadian clock-associated genes have been identified. Among them, the evening-expressed TOC1 (TIMING OF CAB EXPRESSION 1) gene plays a role by forming a transcriptional feedback core loop together with the morning-expressed CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) gene and its homologous LHY (LATE ELONGATED HYPOCOTYL) gene. TOC1 encodes a member of the PSEUDO-RESPONSE REGULATOR (PRR) family, including PRR9, PRR7, PRR5, PRR3,and PRR1/TOC1. The PRR genes other than TOC1 (or PRR1) also appear to be crucial for certain circadian-associated events. To clarify missing genetic linkages amongst these PRR genes, here we constructed a toc1 prr5 double knockdown mutant. In free-running circadian rhythms, the resulting toc1-2 prr5-11 mutant plants showed an extremely short period and reduced amplitude phenotype, which was more severe than that of the toc1-2 single mutant plant, suggesting a non-linear genetic interaction between TOC1 and PRR5. Surprisingly, the hallmark early flowering phenotype of toc1-2 in the short-day conditions had been converted to a markedly late flowering phenotype in the long-day conditions, when combined with the prr5-11 allele, which itself showed a subtle flowering phenotype. This unexpected genetic result (i.e. phenotypic sign conversion) suggested that the TOC1 and PRR5 genes are coordinately implicated in a non-linear and closed genetic circuitry. In the toc1-2 prr5-11 double mutant, the diurnal expression profile of CDF1 (CYCLING DOF FACTOR 1) was markedly de-repressed in the evening in the long-day conditions. These and other results of this study led us to propose the novel view that TOC1 might play bipartite roles in the control of flowering time within a closed circuitry; the one is a GI (GIGANTEA)-dependent negative role through CCA1/LHY, and the other is a CDF1-dependent positive role through cooperating closely with PRR5.

Language：English   Publishing type：Research paper (scientific journal)  

In the model higher plant Arabidopsis thaliana, the CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) gene plays important circadian clock-associated roles. The CCA1 protein is a member of a small subfamily of single MYB-related transcription factors. This family consists of several homologous CCA1-like transcription factors, including the closest homolog LHY (LATE ELONGATED HYPOCOTYL). To gain insight into the molecular function of CCA1 and its homologs, here we took a unique genetic approach that was recently developed for Arabidopsis thaliana. Through this strategy, referred to as CRES-T (Chimeric REpressor Silencing Technology), a transgenic plant was constructed to produce a dominant negative transcriptional repressor (designated CCA1-SRDX). By employing the resulting transgenic lines, together with previously established cca1 lhy double mutant and CCA1-ox (over-expressing) plants, their circadian clock-associated phenotypes were examined and compared with each other. The observed clock-associated phenotypes of the CCA1-SRDX plants were very similar to those of CCA1-ox, but not to those of cca1 lhy, suggesting that CCA1 acts predominantly as a transcriptional repressor in nature. However, the developmental morphology (or architecture) of adult CCA1-SRDX plants were quite different from that of CCA1-ox, suggesting that CCA1 might also be implicated, directly or indirectly, in an as yet unknown circadian-associated output pathway at a late developmental stage.

Language：English   Publishing type：Research paper (scientific journal)  

Over 1,600 genes encoding putative transcription factors have been identified in the Arabidopsis genome sequence, however, their physiological functions are not yet fully understood. In this study, a small subfamily of double B-box zinc finger (DBB, DOUBLE B-BOX) genes, encoding eight putative transcription factors, were characterized with reference to the circadian rhythm and the early photomorphogenic regulation of hypocotyl elongation in response to light signals. Among these, it was found that the transcriptions of five DBB genes were under the control of circadian rhythm. To gain insight into the physiological roles of these putative transcription factors, forward and reverse genetic studies were carried out. The results suggested that they are commonly implicated in light signal transduction during early photomorphogenesis, however, their functions are not totally redundant, as judged by the fact that their circadian-expression profiles (or phases) were distinctive from each other, and by the fact that some DBBs (named DBB1a, DBB1b, STO, and STH) were apparently implicated in light signal transduction in a negative manner, whereas another (named DBB3) was implicated in a positive manner with regard to light-induced inhibition of elongation of hypocotyls. We also found that homologous B-box zinc finger genes are widely conserved in higher plants (e.g., Oryza sativa). Taking this altogether, it is probable that in addition to previously characterized bZIP-type transcription factors (e.g., HY5 and HYH) and bHLH-type transcription factors (e.g., PIF4 and PIF5/PIL6), a set of B-box zinc finger transcription factors should also be taken into consideration for a better understanding of the complex molecular mechanisms underlying the early photomorphogenic development of Arabidopsis thaliana.

Language：English   Publishing type：Research paper (scientific journal)  

The GI (GIGANTEA) and PRR5 (PSEUDO-RESPONSE REGULATOR 5) genes are crucially implicated in the Arabidopsis circadian clock. We characterized a gi prr5 double loss-of-function mutant for the first time with reference to circadian clock-associated phenotypes. The results of this study revealed the genetic linkages between GI and PRR5 in the control of free running circadian oscillation of gene expression, early photomorphogenesis and flowering time. A mathematical clock model consisting of three interactive transcriptional feedback loops has recently been proposed. The model includes the hypothetical evening Y-TOC1 feedback loop, in which "Y" is suspected to be GI and/or PRR5. This issue was also addressed in this study; perhaps GI and PRR5 are not sufficient to fulfill the Y role.

Language：English   Publishing type：Research paper (scientific journal)  

Synchronization phenomena in coupled circadian oscillators of plant leaves were investigated experimentally using bioluminescence technology for a clock gene. Analyzing the phase of circadian oscillation, the phase-wave propagations and the phase delay caused by the vein network were observed. We describe these phase dynamics using a two-layer model with coupled Stuart-Landau equations. Global synchronization of circadian oscillators in the leaf is also investigated.

Language：English   Publishing type：Research paper (scientific journal)  

Every member of a small family of Pseudo-Response Regulator (PRR) genes, including Timing of Cab Expression 1 (TOC1 [or PRR1]), are believed to play roles close to the circadian clock in the model higher plant Arabidopsis thaliana. In this study we established a transgenic line that misexpresses (or overexpresses) the PRR7 gene. As compared with wild-type plants, the resulting PRR7-misexpressing plants (designated PRR7-ox) showed characteristic phenotypes as to hallmarked circadian-associated biological events: (i) early flowering in a manner independent of photoperiodicity, (ii) hypersensitive response to red light during early photomorphogenesis, and (iii) altered free-running rhythms with long period of clock-associated genes. Finally, a series of all transgenic lines (PRR1-ox, PRR3-ox, PRR5-ox, PRR7-ox, and PRR9-ox) were characterized comparatively with regard to their clock-associated roles. The results suggested that the five homologous PRR factors play coordinate roles, distinctively from one another, and closely to the circadian clock in higher plants.

Language：English   Publishing type：Research paper (scientific journal)  

In Arabidopsis thaliana, it is currently accepted that certain mutants with lesions in clock-associated genes commonly display hallmarked phenotypes with regard to three characteristic biological events: (i) altered rhythmic expression of circadian-controlled genes, (ii) changes in flowering time, and (iii) altered sensitivity to red light in elongation of hypocotyls. During the course of examination of the clock-associated mutants of PSEUDO-RESPONSE REGULATORS, PRRs, including TOC1 (PRR1), we found that they commonly show another visible phenotype of anomalous greening responses upon the onset to light exposure of etiolated seedlings. These findings are indicative of a novel link between circadian rhythms and chloroplast development.

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Photoperiodism allows organisms to measure daylength, or external photoperiod, and to anticipate coming seasons. Daylength measurement requires the integration of light signal and temporal information by the circadian clock. In the long-day plant Arabidopsis thaliana, CONSTANS (CO) plays a crucial role in integrating the circadian rhythm and environmental light signals into the photoperiodic flowering pathway. Nevertheless, the molecular mechanism by which the circadian clock modulates the cyclic expression profile of CO is poorly understood. Here, we first showed that the clock-associated genes PSEUDO-RESPONSE REGULATOR (PRR) PRR9, PRR7 and PRR5 are involved in activation of CO expression during the daytime. Then, extensive genetic studies using CIRCADIAN CLOCK-ASSOCIATED1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY) double mutants (cca1/lhy) and prr7/prr5 were conducted. The results suggested that PRR genes act coordinately in a manner parallel with and antagonistic to CCA/LHY, upstream of the canonical CO-FLOWERING LOCUS T (FT) photoperiodic flowering pathway. Finally, we provided evidence to propose a model, in which CCA1/LHY repress CO through GIGANTEA (GI), while PRR9, PRR7 and PRR5 activate CO predominantly by repressing CYCLING DOF FACTOR1 (CDF1) encoding a DNA-binding transcriptional repressor.

Language：English   Publishing type：Research paper (scientific journal)  

The current best candidates for Arabidopsis thaliana clock components are CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) and its homolog LHY (LATE ELONGATED HYPOCOTYL). In addition, five members of a small family, PSEUDO-RESPONSE REGULATORS (including PRR1, PRR3, PRR5, PRR7 and PRR9), are believed to be another type of clock component. The originally described member of PRRs is TOC1 (or PRR1) (TIMING OF CAB EXPRESSION 1). Interestingly, seedlings of A. thaliana carrying a certain lesion (i.e. loss-of-function or misexpression) of a given clock-associated gene commonly display a characteristic phenotype of light response during early photomorphogenesis. For instance, cca1 lhy double mutant seedlings show a shorter hypocotyl length than the wild type under a given fluence rate of red light (i.e. hypersensitivity to red light). In contrast, both toc1 single and prr7 prr5 double mutant seedlings with longer hypocotyls are hyposensitive under the same conditions. These phenotypes are indicative of linkage between the circadian clock and red light signal transduction mechanisms. Here this issue was addressed by conducting combinatorial genetic and epistasis analyses with a large number of mutants and transgenic lines carrying lesions in clock-associated genes, including a cca1 lhy toc1 triple mutant and a cca1 lhy prr7 prr5 quadruple mutant. Taking these results together, we propose a genetic model for clock-associated red light signaling, in which CCA1 and LHY function upstream of TOC1 (PRR1) in a negative manner, in turn, TOC1 (PRR1) serves as a positive regulator. PRR7 and PRR5 also act as positive regulators, but independently from TOC1 (PRR1). It is further suggested that these signaling pathways are coordinately integrated into the phytochrome-mediated red light signal transduction pathway, in which PIF3 (PHYTOCHROME-INTERACTING FACTOR 3) functions as a negative regulator immediately downstream of phyB.

Language：English   Publishing type：Research paper (scientific journal)  

In Arabidopsis thaliana, the flowering time is regulated through the circadian clock that measures day-length and modulates the photoperiodic CO-FT output pathway in accordance with the external coincidence model. Nevertheless, the genetic linkages between the major clock-associated TOC1, CCA1 and LHY genes and the canonical CO-FT flowering pathway are less clear. By employing a set of mutants including an extremely early flowering toc1 cca1 lhy triple mutant, here we showed that CCA1 and LHY act redundantly as negative regulators of the photoperiodic flowering pathway. The partly redundant CCA1/LHY functions are largely, but not absolutely, dependent on the upstream TOC1 gene that serves as an activator. The results of examination with reference to the expression profiles of CO and FT in the mutants indicated that this clock circuitry is indeed linked to the CO-FT output pathway, if not exclusively. For this linkage, the phase control of certain flowering-associated genes, GI, CDF1 and FKF1, appears to be crucial. Furthermore, the genetic linkage between TOC1 and CCA1/LHY is compatible with the negative and positive feedback loop, which is currently believed to be a core of the circadian clock. The results of this study suggested that the circadian clock might open an exit for a photoperiodic output pathway during the daytime. In the context of the current clock model, these results will be discussed in connection with the previous finding that the same clock might open an exit for the early photomorphogenic output pathway during the night-time.

Language：English   Publishing type：Research paper (scientific journal)  

In Arabidopsis thaliana, it is currently believed that the members of a small family of PSEUDO-RESPONSE REGULATOR (PRR) proteins, including TOC1 (TIMING OF CAB EXPRESSION 1), coordinately play roles close to the circadian clock. Among these PRR members, the PRR9 gene is unique in that not only does its transcription oscillate diurnally, but it is also rapidly induced by light in a manner dependent on phytochromes. These events at the level of transcription must be crucial for the clock-associated functions of PRR9. Nonetheless, little is known about the expression of the PRR9 protein product itself in plant cells. Here, we show that PRR9 polypeptides themselves oscillate diurnally, and that they accumulate rapidly in response to light. Our work further suggests that the presence of PRR9 polypeptides is controlled through proteasome-mediated programmed degradation in the dark.

Language：English  

In Arabidopsis thaliana, AUTHENTIC RESPONSE REGULATORS (ARRs) act as downstream components of the His-to-Asp phosphorelay (two-component) signaling pathway that is propagated primarily by the cytokinin receptor kinases, AUTHENTIC HIS-KINASES (AHK2, AHK3 and AHK4/CRE1). Thus, this bacterial type of signaling system is essential for responses to a class of hormones in plants. Interestingly, this higher plant has also evolved its own atypical (or unique) variants of two-component signal transducers, PSEUDO-RESPONSE REGULATORS (PRRs). Several lines of recent results suggest that the functions of PRRs are closely relevant to the plant clock (oscillator) that is central to circadian rhythms, the underlying mechanisms of which have long been the subject of debate. Through an overview of recent results, the main issue addressed here is whether or not the pseudo-response regulators (PRRs) are true oscillator components (TOCs).

Language：English   Publishing type：Research paper (scientific journal)  

In Arabidopsis thaliana, a number of clock-associated protein components have been identified. Among them, CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1)/LHY (LATE ELONGATED HYPOCOTYL) and TOC1 (TIMING OF CAB EXPRESSION 1) are believed to be the essential components of the central oscillator. CCA1 and LHY are homologous and partially redundant Myb-related DNA-binding proteins, whereas TOC1 is a member of a small family of proteins, designated as PSEUDO-RESPONSE REGULATOR. It is also believed that these two different types of clock components form an autoregulatory positive/negative feedback loop at the levels of transcription/translation that generates intrinsic rhythms. Nonetheless, it was not yet certain whether or not other PRR family members (PRR9, PRR7, PRR5 and PRR3) are implicated in clock function per se. Employing a set of prr9, prr7 and prr5 mutant alleles, here we established all possible single, double and triple prr mutants. They were examined extensively by comparing them with each other with regard to their phenotypes of circadian rhythms, photoperiodicity-dependent control of flowering time and photomorphogenic responses to red light during de-etiolation. Notably, the prr9 prr7 prr5 triple lesions in plants resulted in severe phenotypes: (i) arrhythmia in the continuous light conditions, and an anomalous phasing of diurnal oscillation of certain circadian-controlled genes even in the entrained light/dark cycle conditions; (ii) late flowering that was no longer sensitive to the photoperiodicity; and (iii) hyposensitivity (or blind) to red light in the photomorphogenic responses. The phenotypes of the single and double mutants were also characterized extensively, showing that they exhibited circadian-associated phenotypes characteristic for each. These results are discussed from the viewpoint that PRR9/PRR7/PRR5 together act as period-controlling factors, and they play overlapping and distinctive roles close to (or within) the central oscillator in which the relative, PRR1/TOC1, plays an essential role.

Language：English   Publishing type：Research paper (scientific journal)  

In Arabidopsis thaliana, a number of clock-associated protein factors have been identified. Among them, TOC1 (TIMING OF CAB EXPRESSION 1) is believed to be a component of the central oscillator. TOC1 is a member of a small family of proteins, designated as ARABIDOPSIS PSEUDO-RESPONSE REGULATOR, including PRR1/TOC1, PRR3, PRR5, PRR7 and PRR9. It has not been certain whether or not other PRR family members are also implicated in clock function per se. To clarify this problem, here we constructed a double mutant line, which is assumed to have severe lesions in both the PRR5 and PRR7 genes. Resulting homozygous prr5-11 prr7-11 young seedlings showed a marked phenotype of hyposensitivity to red light during de-etiolation. In addition, they displayed a phenotype of extremely late flowering under long-day photoperiod conditions, but not short-day conditions. The rhythms at the level of transcription of certain clock-controlled genes were severely perturbed in the double mutant plants when they were released into continuous light (LL) and darkness (DD). The observed phenotype was best interpreted as 'arrhythmic in both LL and DD' and/or 'very short period with markedly reduced amplitude'. Even under the light entrainment (LD) conditions, the mutant plants showed anomalous diurnal oscillation profiles with altered amplitude and/or phase with regard to certain clock-controlled genes, including the clock component CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) gene. Such events were observed even under temperature entrainment conditions, suggesting that the prr5-11 prr7-11 lesions cannot simply be attributed to a defect in the light signal input pathway. These pleiotropic circadian-associated phenotypes of the double mutant were very remarkable, as compared with those observed previously for each single mutant. Taking these results together, we propose for the first time that PRR5 and PRR7 coordinately (or synergistically) play essential clock-associated roles close to the central oscillator.

Language：English   Publishing type：Research paper (scientific journal)  

In the model higher plant Arabidopsis thaliana, a number of circadian clock-associated protein components have recently been identified. Among them, a small family of ARABIDOPSIS PSEUDO-RESPONSE REGULATORS (APRR1/TOC1, APRR3, APRR5, APRR7, and APRR9) is interesting because the most probable clock component TIMING OF CAB EXPRESSION 1 (TOC1) belongs to this family. Several lines of evidence have already been accumulated to support the view that not only APRR1/TOC1 but also other APRR family members are crucial for a better understanding of the molecular link between circadian rhythm and light-signal transduction. Among the APRR1/TOC1 family members, the circadian-controlled APRR9 gene is unique in that its expression is rapidly induced by light at the level of transcription. In this study we dissected the regulatory cis-elements of the light-induced and/or circadian-controlled APRR9 promoter by employing not only a mutant plant carrying a T-DNA insertion in the APRR9 promoter, but also a series of APRR9-promoter::LUC (luciferase) reporters that were introduced into an Arabidopsis cultured cell line (T87 cells). Taking the results of these approaches together, we provide several lines of evidence that the APRR9 promoter contains at least two distinctive and separable regulatory cis-elements: an "L element" responsible for the light-induced expression, followed by an "R element" necessary for the fundamental rhythmic expression of APRR9. Furthermore, APRR1/TOC1 was implicated in the L-element-mediated light response of APRR9, directly or indirectly.

Language：English   Publishing type：Research paper (scientific journal)  

According to the current consistent model for the higher plant Arabidopsis thaliana, the scheme for an immediate early response to the plant hormone cytokinin can be formulated as Arabidopsis histidine kinase (AHK) cytokinin receptor-mediated His --> Asp phosphorelay signal transduction. Nonetheless, clarification of the comprehensive picture of cytokinin-mediated signal transduction in this higher plant is at a very early stage. As a new approach to this end, we studied whether or not a certain Arabidopsis cell line (named T87) would be versatile for such work on cytokinin signal transduction. We show that T87 cells had the ability to respond to cytokinin, displaying the immediate early induction of type-A Arabidopsis response regulator (ARR) family genes (e.g., ARR6) at the transcriptional level. This event was further confirmed by employing the stable transgenic lines of T87 cells with a set of ARR::LUC reporter transgenes. We also show that T87 cells had the ability to respond to auxin when the expression of a set of AUX/IAA genes (e.g., IAA5) was examined. As postulated for intact plants, in T87 cells too, the induction of IAA5 by auxin was selectively inhibited in the presence of a proteasome inhibitor, while the induction of ARR6 by cytokinin was not significantly affected under the same conditions. Through transient expression assays with T87 protoplasts, it is shown that the intracellular localization profiles of the phosphorelay intermediate Arabidopsis histidine-containing phosphotransfer factor (AHPs; e.g., AHP1 and AHP4) were markedly affected in response to cytokinin, but those of type-A ARRs were not (e.g., ARR15 and ARR16). Taken together, we conclude that, in T87 cells, the AHK-dependent His --> Asp phosphorelay circuitry appears to be propagated in response to cytokinin, as in the case of plants, as far as the immediate early responses were concerned. This cultured cell system might therefore provide us with an alternative means to further characterize the mechanisms underlying cytokinin (and also auxin) responses at the molecular level.

Language：English   Publishing type：Research paper (scientific journal)  

Recent intensive studies have begun to shed light on the molecular mechanisms underlying the plant circadian clock in Arabidopsis thaliana. During the course of these previous studies, the most powerful technique, elegantly adopted, was a real-time bioluminescence monitoring system of circadian rhythms in intact plants carrying a luciferase (LUC) fusion transgene. We previously demonstrated that Arabidopsis cultured cells also retain an ability to generate circadian rhythms, at least partly. To further improve the cultured cell system for studies on circadian rhythms, here we adopted a bioluminescence monitoring system by establishing the cell lines carrying appropriate reporter genes, namely, CCA1::LUC and APRR1::LUC, with which CCA1 (CIRCADIAN CLOCK-ASSOCIATED1) and APRR1 (or TOC1) (ARABIDOPSIS PSEUDO-RESPONSE REGULATORS1 or TIMING OF CAB EXPRESSION1) are believed to be the components of the central oscillator. We report the results that consistently supported the view that the established cell lines, equipped with such bioluminescence reporters, might provide us with an advantageous means to characterize the plant circadian clock.

Language：English   Publishing type：Research paper (scientific journal)  

In Arabidopsis thaliana, a number of circadian-associated factors have been identified. Among those, TOC1 (TIMING OF CAB EXPRESSION 1) is believed to be a component of the central oscillator. TOC1 is a member of a small family of proteins, designated as Arabidopsis PSEUDO-RESPONSE REGULATORS (APRR1/TOC1, APRR3, APRR5, APRR7, and APRR9). Nonetheless, it is not very clear whether or not the APRR family members other than APRR1/TOC1 are also implicated in the mechanisms underlying the circadian rhythm. To address this issue further, here we characterized a set of T-DNA insertion mutants, each of which is assumed to have a severe lesion in each one of the quintet genes (i.e. APRR5 and APRR7). For each of these mutants (aprr5-11 and aprr7-11) we demonstrate that a given mutation singly, if not directly, affects the circadian-associated biological events simultaneously: (i) flowering time in the long-day photoperiod conditions, (ii) red light sensitivity of seedlings during the early photomorphogenesis, and (iii) the period of free-running rhythms of certain clock-controlled genes including CCA1 and APRR1/TOC1 in constant white light. These results suggest that, although the quintet members other than APRR1/TOC1 may not be directly integrated into the framework of the central oscillator, they are crucial for a better understanding of the molecular mechanisms underlying the Arabidopsis circadian clock.

Language：English   Publishing type：Research paper (scientific journal)  

The histidine (His)-to-Aspartate (Asp) phosphorelay is a paradigm of intracellular signaling systems through protein phosphorylation in both prokaryotes and eukaryotes. The fission yeast Schizosaccharomyces pombe has three histidine kinases (Phk1/Mak2, Phk2/Mak3, and Phk3/Mak1), together with two response regulators (Mcs4 and Prr1). The results of recent extensive studies suggested that these His-to-Asp phosphorelay components are involved in oxidative stress responses through the transcriptional regulation of several scavenger genes for toxic free radicals. It was also suggested that they were somehow implicated in control of both the mitotic and meiotic cell proliferations. Among these S. pombe His-to-Asp phosphorelay components, however, the function of Prr1 is less clear. We here characterized a mutant, named prr1-D418N, specifying an altered Prr1 protein that presumably acts as a gain-of-function (or constitutive-active) mutant, with special reference to sexual development. The mutant cells showed a striking phenotype in that they underwent mating even in a nitrogen-sufficient medium, under which conditions the wild-type cells hardly did so. Furthermore, the mutant cells underwent mating very rapidly in a nitrogen-deficient medium. Under anaerobic (or micro-aerobic) growth conditions, the wild-type cells were not capable of undergoing sexual development even in a nitrogen-deficient medium. The prr1-D418N cells underwent mating efficiently under such anaerobic growth conditions. Taken these together, it was suggested that the function of Prr1 is closely linked to the well-characterized signaling pathways for induction of the sexual development, in a way that this response regulator regulates a critical step of the initiation of meiosis through activating the transcription of ste11+, mam2+, and mei2+, in S. pombe.

Language：English   Publishing type：Research paper (scientific journal)  

A small family of genes, named Arabidopsis Pseudo Response Regulator (APRR), are intriguing with special reference to circadian rhythms in plants, based on the fact that one of the members (APRR1) is identical to TOC1 (Timing of CAB Expression 1) that is believed to encode a clock component. In Arabidopsis plants, each transcript of the APRR1/TOC1 quintet genes starts accumulating after dawn rhythmically and one after another at intervals in the order of APRR9 --> APRR7 --> APRR5 --> APRR3 --> APRR1/TOC1. To characterize such intriguing circadian-associated events, we employed an established Arabidopsis cell line (named T87). When T87 cells were grown in an appropriate light and dark cycle, cell autonomous diurnal oscillations of the APRR1/TOC1 quintet genes were observed at the level of transcription, as seen in intact plants. After transfer to the conditions without any environmental time cues, particularly in constant dark, we further showed that free-running circadian rhythms persisted in the cultured cells, not only for the APRR1/TOC1 quintet genes, but also other typical circadian-controlled genes including CCA1 (Circadian Clock Associated 1), LHY (Late Elongated Hypocotyl) and CCR2 (Cold Circadian Rhythm RNA Binding 2). To our knowledge, this is the first indication of cell autonomous circadian rhythms in cultured cells in Arabidopsis thaliana, which will provide us with an alternative and advantageous means to characterize the plant biological clock.

Language：English   Publishing type：Research paper (scientific journal)  

In higher plants, clock-controlled circadian rhythms are a longstanding issue in physiology, and a newly emerging paradigm of molecular biology. In the model higher plant Arabidopsis thaliana, several genes have been proposed to encode potential clock-associated components, including a member (APRR1/TOC1) of the pseudo-response regulator family. We previously showed that transcripts of the APRR1/TOC1 family start accumulating after dawn rhythmically and sequentially at approximately 2 h intervals in the order of APRR9-->APRR7-->APRR5-->APRR3-->APRR1/ TOC1. This and other results led us to propose that this APRR1/TOC1 quintet might play coordinate roles in the mechanism underlying circadian rhythms in higher plants. To gain further insight as to such an idea, we here attempt to identify proteins that interact with one of the quintet members, APRR3. The identified component is a novel protein kinase, named WNK1, which is considerably similar to, but clearly distinct from, mitogen-activated protein kinases (MAPKs). It was found that APRR3 is a substrate of this novel protein kinase, the gene for which also shows a rhythmic transcription profile that is well coincident with the APRR3 rhythm. These findings give new insight into the mechanisms underlying the circadian rhythm in A. thaliana, providing a molecular link between the putative clock component, APRR3, and WNK1, a novel protein kinase that might be implicated as a signal transducer.

Language：English   Publishing type：Research paper (scientific journal)  

The fission yeast Schizosaccharomyces pombe has three histidine kinases (Phk1/Mak2, Phk2/Mak3, and Phk3/Mak1), and two response regulators (Mcs4 and Prr1). The results of recent extensive studies on the S. pombe His-to-Asp phosphorelay circuitry suggested that it is involved in oxidative stress responses through the transcriptional regulation of several scavenger genes for toxic free radicals. The functions of these histidine kinases have not yet been fully characterized. Here we characterize a homothallic (h90) mutant lacking the genes for all the histidine kinases, with special reference to sexual development. Homothallic phk1/2/3delta cells underwent mating precociously in a nitrogen-deficient medium. Surprisingly, the mutant cells underwent mating even in a nitrogen-sufficient medium, under which conditions wild-type cells did so rarely if at all. Under anaerobic (or microaerobic) growth conditions, wild-type cells did not undergo sexual development even in a nitrogen-deficient medium, but the homothallic phk1/2/3delta cells mated efficiently. Oxidative reagents such as H2O2 induced sexual development in wild-type cells grown anaerobically. On the basis of these results, we propose the novel view that the S. pombe His-to-Asp phosphorelay, initiated by the Phk histidine kinases, is crucial for regulation of sexual development. This Phk-mediated signaling pathway is linked to the well-documented canonical pathway for induction of the sexual development, in that both converge at the initiation of meiosis through activation of ste11+, mam2+, and mei2+ transcription.

Language：English   Publishing type：Research paper (scientific journal)  

The complete genome sequence of Arabidopsis thaliana revealed that this higher plant has a tremendous number of protein kinases. We recently isolated a novel type of protein kinase, named AtWNK1, which shows an in vitro ability to phosphorylate the APRR3 member of the APRR1/TOC1 quintet that has been implicated in a mechanism underlying circadian rhythms in Arabidopsis. We here address two issues, one general and one specific, as to this novel protein kinase. We first asked the general question of how many WNK family members are present in this higher plant, then whether or not other members are also relevant to circadian rhythms. The results of our analyses showed that Arabidopsis has at least 9 members of the WNK1 family of protein kinases (designated here as WNK1 to WNK9), the structural design of which is clearly distinct from those of other known protein kinases, such as receptor-like kinases and mitogen-activated protein kinases. They were examined with special reference to the circadian-related APRR1/TOC1 quintet. It was found that not only the transcription of the WNK1 gene, but also those of three other members (WNK2, WNK4, and WNK6) are under the control of circadian rhythms. These results suggested that certain members of the WNK family of protein kinases might play roles in a mechanism that generates circadian rhythms in Arabidopsis.

Language：English   Publishing type：Research paper (scientific journal)  

In Arabidopsis thaliana, the transcripts of the APRR1/TOC1 family genes each start accumulating after dawn rhythmically and one after another at intervals in the order of APRR9-->APRR7-->APRR5-->APRR3-->APRR1/TOC1 under continuous light. Except for the well-characterized APRR1/TOC1, however, no evidence has been provided that other APRR1/TOC1 family genes are indeed implicated in the mechanisms underlying circadian rhythms. We here attempted to provide such evidence by characterizing transgenic plants that constitutively express the APRR5 gene. The resulting APRR5-overexpressing (APRR5-ox) plants showed intriguing properties with regard to not only circadian rhythms, but also control of flowering time and light response. First, the aberrant expression of APRR5 in such transgenic plants resulted in a characteristic phenotype with regard to transcriptional events, in which free-running rhythms were considerably altered for certain circadian-regulated genes, including CCA1, LHY, APRR1/TOC1, other APRR1/TOC1 members, GI and CAB2, although each rhythm was clearly sustained even after plants were transferred to continuous light. With regard to biological events, APRR5-ox plants flowered much earlier than wild-type plants, more or less, in a manner independent of photoperiodicity (or under short-day conditions). Furthermore, APRR5-ox plants showed an SRL (short-hypocotyls under red light) phenotype that is indicative of hypersensitiveness to red light in early photomorphogenesis. Both APRR1-ox and APRR9-ox plants also showed the same phenotype. Therefore, APRR5 (together with APRR1/TOC1 and APRR9) must be taken into consideration for a better understanding of the molecular links between circadian rhythms, control of flowering time through the photoperiodic long-day pathway, and also light signaling-controlled plant development.

Language：English   Publishing type：Research paper (scientific journal)  

hos2 mutants of the fission yeast Schizosaccharomyces pombe showed the phenotype of high osmolarity sensitivity for growth. An S. pombe strain carrying the hos2-M10 allele cannot form colonies on agar plates containing 2 M glucose, but the parental strain can do so very well, as demonstrated previously. In this study, the hos2+ gene was identified as one that encodes a small protein of 94 amino acids, which shows no sequence similarity to any other proteins in the current databases. The hos2-M10 mutation resulted in Gln-62 to TAG-termination codon. A Hos2-defective (hos2delta) strain, which we then constructed, showed the phenotype of high osmolarity sensitivity, as in the case of the original hos2-M10 mutant. For this hos2delta mutant, three multicopy suppressor genes were isolated and one of which was identified as the pgk1+ gene, encoding a phosphoglycerate kinase.

Event date： 2018.11

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Edinburgh   Country：United Kingdom  

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Venue：鹿児島   Country：Japan  

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Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Country：Japan  

Event date： 2013.3

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Country：Japan  

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Country：Japan  

Event date： 2012.9

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Country：Japan  

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Event date： 2012.7

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Country：Austria  

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Language：English   Presentation type：Symposium, workshop panel (nominated)  

Country：Japan  

Event date： 2012.3

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Country：Japan  

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Country：Japan  

Event date： 2011.9

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Country：Japan  

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Country：Japan  

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Venue：熊本   Country：Japan  

Event date： 2009.7

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Country：Japan  

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Event date： 2008.3

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Venue：札幌   Country：Japan  

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Country：Japan  

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Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：東京   Country：Japan  

Event date： 2005.8

Language：English   Presentation type：Oral presentation (keynote)  

Country：Japan  

Author：Other 

Author：Other