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Impact of clock-associated Arabidopsis pseudo-response regulators in metabolic coordination 査読有り

Atsushi Fukushima, Miyako Kusano, Norihito Nakamichi, Makoto Kobayashi, Naomi Hayashi, Hitoshi Sakakibara, Takeshi Mizuno, Kazuki Saito

Proc Natl Acad Sci U S A 106 巻頁： 7251-7256 2009年

記述言語：英語掲載種別：研究論文（学術雑誌）

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.

Involvement of Arabidopsis clock-associated pseudo-response regulators in diurnal oscillations of gene expression in the presence of environmental time cues 査読有り

Takafumi Yamashino, Shogo Ito, Yosuke Niwa, Atsushi Kunihiro, Norihito Nakamichi, Takeshi Mizuno

Plant Cell Physiol 49 巻頁： 1839-1850 2008年

記述言語：英語掲載種別：研究論文（学術雑誌）

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.

Insight into missing genetic links between two evening-expressed pseudo-response regulator genes TOC1 and PRR5 in the circadian clock-controlled circuitry in Arabidopsis thaliana 査読有り

Shogo Ito, Yosuke Niwa, Norihito Nakamichi, Hideaki Kawamura, Takafumi Yamashino, Takeshi Mizuno

Plant Cell Physiol 49 巻頁： 201-213 2008年

記述言語：英語掲載種別：研究論文（学術雑誌）

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.

The function of the clock-associated transcriptional regulator CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) in Arabidopsis thaliana 査読有り

Masakazu Kawamura, Shogo Ito, Nakamichi Nakamichi, Takafumi Yamashino, Takeshi Mizuno

Biosci Biotechnol Biochem 72 巻頁： 1307-1316 2008年

記述言語：英語掲載種別：研究論文（学術雑誌）

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.

The common function of a novel subfamily of B-Box zinc finger proteins with reference to circadian-associated events in Arabidopsis thaliana 査読有り

Takeshi Kumagai, Shogo Ito, Norihito Nakamichi, Yosuke Niwa, Masaya Murakami, Takafumi Yamashino, Takeshi Mizuno

Biosci Biotechnol Biochem 72 巻頁： 1539-1549 2008年

記述言語：英語掲載種別：研究論文（学術雑誌）

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.

Characterization of genetic links between two clock-associated genes, GI and PRR5 in the current clock model of Arabidopsis thaliana 査読有り

Hideaki Kawamura, Shogo Ito, Takafumi Yamashino, Yosuke Niwa, Norihito Nakamichi, Takeshi Mizuno

Biosci Biotechnol Biochem 72 巻頁： 2770-2774 2008年

記述言語：英語掲載種別：研究論文（学術雑誌）

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.

Synchronization of Plant Circadian Oscillators with a Phase Delay Effect of Vein Network 査読有り

Hirokazu Fukuda, Norihito Nakamichi, Mihoe Hisatsune, Haruhiko Murase, Takeshi Mizuno

Physical Review Letters 99 巻頁： 098102 2007年

記述言語：英語掲載種別：研究論文（学術雑誌）

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.

Characterization of Circadian-associated pseudo-response regulators: I. Comparative studies on a series of transgenic lines misexpressing five distinctive PRR Genes in Arabidopsis thaliana 査読有り

Akinori Matsushika, Masaya Murakami, Shogo Ito, Norihito Nakamichi, Takafumi Yamashino, Takeshi Mizuno

Biosci Biotechnol Biochem 71 巻頁： 527-534 2007年

記述言語：英語掲載種別：研究論文（学術雑誌）

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.

Mutants of circadian-associated PRR genes display a novel and visible phenotype as to light responses during de-etiolation of Arabidopsis thaliana seedlings 査読有り

Takahiko Kato, Masaya Murakami, Yuko Nakamura, Shogo Ito, Norihito Nakamichi, Takafumi Yamashino, Takeshi Mizuno

Biosci Biotechnol Biochem 71 巻頁： 834-839 2007年

記述言語：英語掲載種別：研究論文（学術雑誌）

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.

Arabidopsis Clock-Associated Pseudo-Response Regulators PRR9, PRR7 and PRR5 Coordinately and Positively Regulate Flowering Time through the Canonical CONSTANS-Dependent Photoperiodic Pathway 査読有り

Norihito Nakamichi, Masanori Kita, Kanae Niinuma, Shogo Ito, Takafumi Yamashino, Tsuyoshi Mizuguchi, Takeshi Mizuno

Plant Cell Physiol 48 巻頁： 822-832 2007年

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）

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.

Genetic Linkages between Circadian Clock-Associated Components and Phytochrome-Dependent Red Light-Signal Transduction in Arabidopsis thaliana 査読有り

Shogo Ito, Norihito Nakamichi, Yuko Nakamura, Yosuke Niwa, Takahuko Kato, Masaya Murakami, Masanori Kita, Tsuyoshi Mizoguchi, Kanae Niinuma, Takafumi Yamashino, Takeshi Mizuno

Plant Cell Physiol 48 巻頁： 971-983 2007年

記述言語：英語掲載種別：研究論文（学術雑誌）

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.

Genetic linkages of the circadian clock-associated genes, TOC1, CCA1 and LHY, in the photoperiodic control of flowering time in Arabidopsis thaliana 査読有り

Yosuke Niwa, Shogo Ito, Norihito Nakamichi, Tsuyoshi Mizoguchi, Kanae Niinuma, Takafumi Yamashino, Takeshi Mizuno

Plant Cell Physiol 48 巻頁： 925-937 2007年

記述言語：英語掲載種別：研究論文（学術雑誌）

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.

Rhythmic and light-inducible appearance of clock-associated pseudo-response regulator protein PRR9 through programmed degradation in the dark in Arabidopsis thaliana 査読有り

Shogo Ito, Norihito Nakamichi, Takatoshi Kiba, Takafumi Yamashino, Takeshi Mizuno

Plant Cell Physiol 48 巻頁： 1644-1651 2007年

記述言語：英語掲載種別：研究論文（学術雑誌）

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.

Psuedo-Pesponse Regulators(PRRs) or True Oscillator Components(TOCs) 査読有り

Takeshi Mizuno, Norihito Nakamichi

Plant Cell Physiol 46 巻頁： 677-685 2005年

記述言語：英語

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).

Three Psudo-Resoponse Regulators, PRR9, PRR7, and PRR5 Together Play an Essential Role Close to the Circadian Clock of Alabidopsis thaliana 査読有り

Norihito Nakamichi, Masanori Kita, Shogo Ito, Takafumi Yamashino, Takeshi Mizuno

Plant Cell Physiol 46 巻頁： 686-698 2005年

記述言語：英語掲載種別：研究論文（学術雑誌）

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.

The Arabidopsis Pseudo-Response Regulators, PRR5 and PRR7, Coordinately Play Essential Roles for Circadian Clock 査読有り

Norihito Nakamichi, Masanori Kita, Shogo Ito, Eriko Sato, Takafumi Yamashino, Takeshi Mizuno

Plant Cell Physiol 46 巻頁： 609-619 2005年

記述言語：英語掲載種別：研究論文（学術雑誌）

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.

Molecular dissection of the promoter of the light-induced and circadian-controlled APRR9 gene encoding a clock associated component of Arabidopsis thaliana 査読有り

Shogo Ito, Norihito Nakamichi, Akinori Matsushika, Toru Fujimori, Takafumi Yamashino, Takeshi Mizuno

Biosci.Biotechnol.Biochem 69 巻頁： 382-390 2005年

記述言語：英語掲載種別：研究論文（学術雑誌）

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.

Rapid response of Arabidopsis T87 cultured cells to cytokinin through His-to-Asp phosphorelay signal transduction 査読有り

Hisami Yamada, Nozomu Koizumi, Norihito Nakamichi, Takatoshi Kiba, Takafumi Yamashino, Takeshi Mizuno

Biosci Biotechnol Biochem 68 巻頁： 1966-1976 2004年

記述言語：英語掲載種別：研究論文（学術雑誌）

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.

Characterization of Plant Circadian Rhythms by Employing Arabidopsis Cultured Cells with Bioluminescence Reporters 査読有り

Norihito Nakamichi, Shogo Ito, Tokitaka Oyama, Takafumi Yamashino, Takao Kondo, Takeshi Mizuno

Plant Cell Physiol 45 巻頁： 57-67 2004年

記述言語：英語掲載種別：研究論文（学術雑誌）

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.

Comparative Genetic Studies on the APRR5 and APRR7 Genes Belonging to the APRR1/TOC1 Quintet Implicated in Circadian Rhythm, Control of Flowering, and Early Photomorphogenesis 査読有り

Yoko Yamamoto, Eriko Sato, Tomo Shimizu, Norihito Nakamichi, Shusei Sato, Tomohiko Kato, Satoshi Tabata, Akira Nagatani, Takafumi Yamashino, Takeshi Mizuno

Plant Cell Physiol 44 巻頁： 1119-1130 2003年

記述言語：英語掲載種別：研究論文（学術雑誌）

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.

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