Biomedical subjects

Norihito Nakamichi

Publications and source records attributed to Norihito Nakamichi.

12 recordsLinked to original sources

PubMed2005

PSEUDO-RESPONSE REGULATORS, PRR9, PRR7 and PRR5, together play essential roles close to the circadian clock of Arabidopsis thaliana.

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.

Arabidopsis↗

PubMed2005

Pseudo-Response Regulators (PRRs) or True Oscillator Components (TOCs).

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).

Arabidopsis↗

PubMed2005

The Arabidopsis pseudo-response regulators, PRR5 and PRR7, coordinately play essential roles for circadian clock function.

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.

Arabidopsis↗

PubMed2005

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

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.

Arabidopsis↗

PubMed2004

Characterization of plant circadian rhythms by employing Arabidopsis cultured cells with bioluminescence reporters.

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.

Arabidopsis↗

PubMed2004

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

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.

Arabidopsis↗

PubMed2003

Cell autonomous circadian waves of the APRR1/TOC1 quintet in an established cell line of Arabidopsis thaliana.

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.

Arabidopsis↗

PubMed2003

Characterization of the Prr1 response regulator with special reference to sexual development in Schizosaccharomyces pombe.

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.

Amino Acid Substitution↗

PubMed2002

The APRR3 component of the clock-associated APRR1/TOC1 quintet is phosphorylated by a novel protein kinase belonging to the WNK family, the gene for which is also transcribed rhythmically in Arabidopsis thaliana.

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.

Amino Acid Sequence↗

PubMed2002

Aberrant expression of the Arabidopsis circadian-regulated APRR5 gene belonging to the APRR1/TOC1 quintet results in early flowering and hypersensitiveness to light in early photomorphogenesis.

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.

Arabidopsis↗

PubMed2002

Compilation and characterization of a novel WNK family of protein kinases in Arabiodpsis thaliana with reference to circadian rhythms.

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.

Amino Acid Sequence↗

PubMed2002

His-to-Asp phosphorelay circuitry for regulation of sexual development in Schizosaccharomyces pombe.

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.

Anaerobiosis↗