Takeshi Mizuno

Publications (119)373.84 Total impact

• Article: Clock-Controlled and FLOWERING LOCUS T (FT)-Dependent Photoperiodic Pathway in Lotus japonicus I: Verification of the Flowering-Associated Function of an FT Homolog.

  Takafumi Yamashino, Saori Yamawaki, Emi Hagui, Hanayo Ueoka-Nakanishi, Norihito Nakamichi, Shogo Ito, Takeshi Mizuno
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  ABSTRACT: During the last decade, significant research progress in the study of Arabidopsis thaliana has been made in defining the molecular mechanism by which the plant circadian clock regulates flowering time in response to changes in photoperiod. It is generally accepted that the clock-controlled CONSTANS (CO)-FLOWERING LOCUS T (FT)-mediated external coincidence mechanism underlying the photoperiodic control of flowering time is conserved in higher plants, including A. thaliana and Oryza sativa. However, it is also assumed that the mechanism differs considerably in detail among species. Here we characterized the clock-controlled CO-FT pathway in Lotus japonicus (a model legume) in comparison with that of A. thaliana. L. japonicus has at least one FT orthologous gene (named LjFTa), which is induced specifically in short-days and complements the mutational lesion of the Arabidopsis FT gene. However, it was speculated that this legume might lack the upstream positive regulator CO. By employing L. japonicus phyB mutant plants, we showed that the photoreceptor mutant displays a phenotype of early flowering due to enhanced expression of LjFTa, suggesting that LjFTa is invovled in the promotion of flowering in L. japonicus. These results are discussed in the context of current knowledge of the flowering in crop legumes such as soybean and garden pea.
  Bioscience Biotechnology and Biochemistry 04/2013; · 1.28 Impact Factor
• Article: Verification at the protein level of the PIF4-mediated external coincidence model for the temperature-adaptive photoperiodic control of plant growth in Arabidopsis thaliana.

  Takafumi Yamashino, Yuichi Nomoto, Séverine Lorrain, Miki Miyachi, Shogo Ito, Norihito Nakamichi, Christian Fankhauser, Takeshi Mizuno
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  ABSTRACT: Plant circadian clock controls a wide variety of physiological and developmental events, which include the short-days (SDs)-specific promotion of the elongation of hypocotyls during de-etiolation and also the elongation of petioles during vegetative growth. In A. thaliana, the PIF4 gene encoding a phytochrome-interacting basic helix-loop-helix (bHLH) transcription factor plays crucial roles in this photoperiodic control of plant growth. According to the proposed external coincidence model, the PIF4 gene is transcribed precociously at the end of night specifically in SDs, under which conditions the protein product is stably accumulated, while PIF4 is expressed exclusively during the daytime in long days (LDs), under which conditions the protein product is degraded by the light-activated phyB and also the residual proteins are inactivated by the DELLA family of proteins. A number of previous reports provided solid evidence to support this coincidence model mainly at the transcriptional level of the PIF 4 and PIF4-traget genes. Nevertheless, the diurnal oscillation profiles of PIF4 proteins, which were postulated to be dependent on photoperiod and ambient temperature, have not yet been demonstrated. Here we present such crucial evidence on PIF4 protein level to further support the external coincidence model underlying the temperature-adaptive photoperiodic control of plant growth in A. thaliana.
  Plant signaling & behavior 01/2013; 8(3).
• Article: Molecular Mechanisms of Circadian Rhythm in Lotus japonicus and Arabidopsis thaliana Are Sufficiently Compatible to Regulate Heterologous Core Clock Genes Robustly.

  Hanayo Ueoka-Nakanishi, Takafumi Yamashino, Kai Ishida, Mari Kamioka, Norihito Nakamichi, Takeshi Mizuno
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  ABSTRACT: Recent intensive studies of the model plant Arabidopsis thaliana have revealed the molecular mechanisms underlying circadian rhythms in detail. Results of phylogenetic analyses indicated that some of core clock genes are widely conserved throughout the plant kingdom. For another model plant the legume Lotus japonicus, we have reported that it has a set of putative clock genes highly homologous to A. thaliana. Taking advantage of the L. japonicus hairy root transformation system, in this study we characterized the promoter activity of A. thaliana core clock genes CCA1 and PRR5 in heterologous L. japonicus cells and found that the molecular mechanism of circadian rhythm in L. japonicus is compatible with that of A. thaliana.
  Bioscience Biotechnology and Biochemistry 12/2012; · 1.28 Impact Factor
• Article: Circadian clock and PIF4-mediated external coincidence mechanism coordinately integrates both of the cues from seasonal changes in photoperiod and temperature to regulate plant growth in Arabidopsis thaliana.

  Yuichi Nomoto, Saori Kubozono, Miki Miyachi, Norihito Nakamichi, Takeshi Mizuno, Takafumi Yamashino
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  ABSTRACT: In Arabidopsis thaliana, the circadian clock regulates the photoperiodic plant growth including the elongation of hypocotyls in a short-days (SDs)-specific manner. The clock-controlled PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) gene encoding a basic helix-loop-helix (bHLH) transcription factor plays crucial roles in this regulation. The SDs-specific elongation of hypocotyls is best explained by accumulation of the active PIF4 proteins at the end of night specifically in SDs due to coincidence between internal (circadian clock) and external (photoperiod) cues. However, this external coincidence model was challenged with the recent finding that the elongation of hypocotyls is markedly promoted at high growth temperature (28 ˚C) even in long-days (LDs), implying that the model to explain the photoperiodic response of plant architecture appears to be conditional on ambient temperature. With regard to this problem, the results of this and previous studies showed that the model holds under a wide range of ambient temperature conditions (16 ˚C to 28 ˚C). We propose that the circadian clock and PIF4-mediated external coincidence mechanism coordinately integrates both of the cues from seasonal changes in photoperiod and temperature to regulate plant growth in natural habitats.
  Plant signaling & behavior 11/2012; 8(2).
• Article: Circadian clock and PIF4-controlled plant growth: A coincidence mechanism directly integrates a hormones-signaling network into the photoperiodic control of plant architectures in Arabidopsis thaliana.

  Yuichi Nomoto, Saori Kubozono, Takafumi Yamashino, Norihito Nakamichi, Takeshi Mizuno
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  ABSTRACT: Plant circadian clock generates rhythms with a period close to 24 h, and it controls a wide variety of physiological and developmental events, enabling plants to adapt to ever-changing environmental light conditions. In Arabidopsis thaliana, the clock regulates the diurnal and photoperiodic plant growth including the elongation of hypocotyls and petioles in a time-of-day-specific and short-days (SDs)-specific manner. In this mechanism, the clock-regulated PHYTOCHROME-INTERACTING FACTOR 4 gene encoding a basic helix-loop-helix transcription factor, together with phytochromes (mainly phyB), plays crucial roles. This diurnal and photoperiodic control of plant growth is best explained by the accumulation of the PIF4 protein at the end of nighttime specifically under SDs, due to coincidence between the internal (circadian rhythm) and external (photoperiod) cues. In this model, however, the PIF4-controlled downstream factors are not fully identified, although it has been generally proposed that the auxin-mediated signal transduction is crucially implicated. Here, we identified a set of hormone-associated genes as the specific PIF4-targets implicated in the photoperiodic control of plant growth. They include not only auxin-associated genes (GH3.5, IAA19, IAA29), but also ones associated with other growth-regulating hormones such as brassinosteroids (BR6ox2), gibberellic acids (GAI), ethylene (ACS8), and cytokinin (CKX5). The dawn- and SDs-specific expression profiles of these genes are modified in a set of phyB and clock mutants, both of which compromise the coincidence mechanism. The results of this study suggest that the circadian clock orchestrates a variety of hormone-signaling pathways to regulate the photoperiod-dependent morphogenesis in A. thaliana.
  Plant and Cell Physiology 10/2012; · 4.70 Impact Factor
• Article: A circadian clock and PIF4-mediated double coincidence mechanism is implicated in the thermo-sensitive photoperiodic control of plant architectures in Arabidopsis thaliana.

  Yuichi Nomoto, Saori Kubozono, Miki Miyachi, Takafumi Yamashino, Norihito Nakamichi, Takeshi Mizuno
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  ABSTRACT: In Alabidopsis thaliana, the circadian clock regulates the diurnal and photoperiodic plant growth including the elongation of hypocotyls in a time-of-day-specific and short-days (SDs)-specific manner. The clock-controlled PHYTOCHROME-INTERACTING FACTOR 4 encoding a basic helix-loop-helix (bHLH) transcription factor plays crucial roles in this regulation. PIF4 is transcribed precociously at the end of night in SDs, under which conditions the protein product is stably accumulated, while PIF4 is expressed exclusively during the daytime in long days (LDs), under which conditions the protein product is degraded by the light-activated phyB. The dawn- and SDs-specific elongation of hypocotyls is best explained by the coincident accumulation of the active PIF4 protein during the nighttime before dawn specifically in SDs. However, this coincidence model was challenged with the recent finding that the elongation of hypocotyls is markedly promoted at high growth temperature (i.e., 28 °C) even under LDs in a PIF4-dependent manner. Here, we reconciled the apparently conflicting facts by showing that the transcription of PIF4 occurs precociously at the end of nighttime at 28 °C in LDs, similarly as in SDs. Both the events resulted in the same consequence that a set of PIF4-target genes (ATHB2, GH3.5, IAA19, IAA29, BRox2, GAI, ACS8, and CKX5) was induced accordingly in a time-of-day-specific manner. Taken together, we propose an extended double coincidence mechanism, by which the two environmental cues (i.e., photoperiods and temperatures), both of which vary on a season-to-season basis, are integrated into the same clock and PIF4-mediated output pathway that regulate a hormone-signaling network to fit plant architectures properly to domestic habitats.
  Plant and Cell Physiology 10/2012; · 4.70 Impact Factor
• Source

  Available from: Norihito Nakamichi

  Article: Transcriptional repressor PRR5 directly regulates clock-output pathways.

  Norihito Nakamichi, Takatoshi Kiba, Mari Kamioka, Takamasa Suzuki, Takafumi Yamashino, Tetsuya Higashiyama, Hitoshi Sakakibara, Takeshi Mizuno
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  ABSTRACT: The circadian clock is an endogenous time-keeping mechanism that enables organisms to adapt to external daily cycles. The clock coordinates biological activities with these cycles, mainly through genome-wide gene expression. However, the exact mechanism underlying regulation of circadian gene expression is poorly understood. Here we demonstrated that an Arabidopsis PSEUDO-RESPONSE REGULATOR 5 (PRR5), which acts in the clock genetic circuit, directly regulates expression timing of key transcription factors involved in clock-output pathways. A transient expression assay and ChIP-quantitative PCR assay using mutated PRR5 indicated that PRR5 associates with target DNA through binding at the CCT motif in vivo. ChIP followed by deep sequencing coupled with genome-wide expression profiling revealed the direct-target genes of PRR5. PRR5 direct-targets include genes encoding transcription factors involved in flowering-time regulation, hypocotyl elongation, and cold-stress responses. PRR5-target gene expression followed a circadian rhythm pattern with low, basal expression from noon until midnight, when PRR9, PRR7, and PRR5 were expressed. ChIP-quantitative PCR assays indicated that PRR7 and PRR9 bind to the direct-targets of PRR5. Genome-wide expression profiling using a prr9 prr7 prr5 triple mutant suggests that PRR5, PRR7, and PRR9 repress these targets. Taken together, our results illustrate a genetic network in which PRR5, PRR7, and PRR9 directly regulate expression timing of key transcription factors to coordinate physiological processes with daily cycles.
  Proceedings of the National Academy of Sciences 10/2012; 109(42):17123-8. · 9.68 Impact Factor
• Article: Characterization of shade avoidance responses in Lotus japonicus.

  Hanayo Ueoka-Nakanishi, Nanako Hori, Kai Ishida, Natsuko Ono, Takafumi Yamashino, Norihito Nakamichi, Takeshi Mizuno
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  ABSTRACT: Sessile plants must continuously adjust their growth and development to optimize photosynthetic activity under ever-fluctuating light conditions. Among such light responses in plants, one of the best-characterized events is the so-called shade avoidance, for which a low ratio of the red (R):far-red (FR) light intensities is the most prominent stimulus. Such shade avoidance responses enable plants to overtop their neighbors, thereby enhancing fitness and competitiveness in their natural habitat. Considerable progress has been achieved during the last decade in understanding the molecular mechanisms underlying the shade avoidance responses in the model rosette plant, Arabidopsis thaliana. We characterize here the fundamental aspects of the shade avoidance responses in the model legume, Lotus japonicus, based on the fact that its phyllotaxis (or morphological architecture) is quite different from that of A. thaliana. It was found that L. japonicus displays the characteristic shade avoidance syndrome (SAS) under defined laboratory conditions (a low R:FR ratio, low light intensity, and low blue light intensity) that mimic the natural canopy. In particular, the outgrowth of axillary buds (i.e., both aerial and cotyledonary shoot branching) was severely inhibited in L. japonicus grown in the shade. These results are discussed with special emphasis on the unique aspects of SAS observed with this legume.
  Bioscience Biotechnology and Biochemistry 11/2011; 75(11):2148-54. · 1.28 Impact Factor
• Article: Functional characterization of HY5 homolog genes involved in early light-signaling in Physcomitrella patens.

  Saori Yamawaki, Takafumi Yamashino, Hanayo Nakanishi, Takeshi Mizuno
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  ABSTRACT: The developmental programs of Physcomitrella patens, a basal lineage of land plants, are regulated by phytohormones and light-signaling responses. In this study, our attention was focused on the HY5-family of transcription factors, which are known to play important roles immediately downstream of photoreceptors during the early photomorphogenesis of Arabidopsis thaliana. We retrieved two HY5-homologs, named PpHY5a and PpHY5b, from the whole genome sequence database of P. patens. Arabidopsis transgenic plants overproducing the basic leucine zipper (bZIP) domain of PpHY5a exhibited a phenotype of short hypocotyls, suggesting a functional relationship between PpHY5 and Arabidopsis HY5. A loss-of-function Δhy5a Δhy5b double mutant was defective in the vigorous protrusion of caulonema cells from the protonema networks of P. patens under light and dark conditions. These results suggest that the function of HY5-homologs in P. patens is evolutionarily conserved, and is implicated in a process of caulonema development.
  Bioscience Biotechnology and Biochemistry 08/2011; 75(8):1533-9. · 1.28 Impact Factor
• Source

  Available from: Norihito Nakamichi

  Article: Phytochrome-interacting factor 4 and 5 (PIF4 and PIF5) activate the homeobox ATHB2 and auxin-inducible IAA29 genes in the coincidence mechanism underlying photoperiodic control of plant growth of Arabidopsis thaliana.

  Atsushi Kunihiro, Takafumi Yamashino, Norihito Nakamichi, Yusuke Niwa, Hanayo Nakanishi, Takeshi Mizuno
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  ABSTRACT: The plant circadian clock generates rhythms with a period close to 24 h, and it controls a wide variety of physiological and developmental events. Among clock-controlled developmental events, the best characterized is the photoperiodic control of flowering time, which is mediated through the CONSTANS (CO)-FLOWERING LOCUS T (FT) pathway in Arabidopsis thaliana. The clock also regulates the diurnal plant growth including the elongation of hypocotyls in a short day (SDs)-specific manner. In this mechanism, phytochromes (mainly phyB) and the PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5, encoding phytochrome-interacting basic helix-loop-helix (bHLH) transcription factors, play crucial roles. The time of day-specific and photoperiodic control of hypocotyl elongation is best explained by the accumulation of the PIF4 and PIF5 proteins during night-time before dawn, especially under SDs, due to coincidence between the internal (circadian rhythm) and external (photoperiod) time cues. However, the PIF4- and/or PIF5-controlled downstream factors have not yet been identified. Here, we provide evidence that ARABIDOPSIS THALIANA HOMEOBOX PROTEIN2 (ATHB2), together with auxin-inducible IAA29, is diurnally expressed with a peak at dawn under the control of PIF4 and PIF5 specifically in SDs. This coincidentally expressed transcription factor serves as a positive regulator for the elongation of hypocotyls. The expression profiles of ATHB2 were markedly altered in certain clock and phytochrome mutants, all of which show anomalous phenotypes with regard to the photoperiodic control of hypocotyl elongation. Taken together, we propose that an external coincidence model involving the clock-controlled PIF4/PIF5-ATHB2 pathway is crucial for the diurnal and photoperiodic control of plant growth in A. thaliana.
  Plant and Cell Physiology 06/2011; 52(8):1315-29. · 4.70 Impact Factor
• Article: Erratum to: Characterization of the conserved phosphorylation site in the Aspergillus nidulans response regulator SrrA.

  Daisuke Hagiwara, Takeshi Mizuno, Keietsu Abe
  Current Genetics 06/2011; 57(3):223-4. · 2.56 Impact Factor
• Article: Heterologous expression and functional characterization of a Physcomitrella Pseudo response regulator homolog, PpPRR2, in Arabidopsis.

  Santosh B Satbhai, Takafumi Yamashino, Takeshi Mizuno, Setsuyuki Aoki
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  ABSTRACT: Physcomitrella patens has four homologs of the pseudo-response regulator involved in the circadian clock mechanism in seed plants. To gain insight into their function, Arabidopsis transgenic lines misexpressing PpPRR2 were constructed. Phenotypic analysis of the transformants with reference to clock-related gene expression and photoperiodic responses revealed that heterologous expression of the moss PpPRR2 gene modifies the intrinsic mechanism underlying the circadian clock in Arabidopsis, suggesting that PpPRR2 serves as a clock component in P. patens.
  Bioscience Biotechnology and Biochemistry 05/2011; 75(4):786-9. · 1.28 Impact Factor
• Article: Light-responsive double B-box containing transcription factors are conserved in Physcomitrella patens.

  Saori Yamawaki, Takafumi Yamashino, Norihito Nakamichi, Hanayo Nakanishi, Takeshi Mizuno
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  ABSTRACT: In the model seed plant Arabidopsis thaliana, a sub-family of B-box containing transcriptional factors (BBXs), which is classified in the BBX-IV group based on the domain structure, contains two tandem B-box domains and plays crucial roles in early photomorphogenesis under the control of blue light receptors, cry1 and cry2. The results of an examination of light responsiveness of representative Physcomitrella BBX-IV genes and their heterologous expression in Arabidopsis suggested that the light signaling-related characteristics of the BBX-IV subfamily are evolutionarily conserved in a moss, which is a basal lineage of land plants.
  Bioscience Biotechnology and Biochemistry 01/2011; 75(10):2037-41. · 1.28 Impact Factor
• Source

  Available from: Daisuke Hagiwara

  Article: Characterization of the conserved phosphorylation site in the Aspergillus nidulans response regulator SrrA.

  Daisuke Hagiwara, Takeshi Mizuno, Keietsu Abe
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  ABSTRACT: Ssk1- and Skn7-type response regulators are widely conserved in fungal His-Asp phosphorelay (two-component) signaling systems. SrrA, a Skn7-type RR of Aspergillus nidulans, is implicated not only in oxidative stress responses but also in osmotic adaptation, conidia production (asexual development), inhibition by fungicides, and cell wall stress resistance. Here, we characterized SrrA, focusing on the role of the conserved aspartate residue in the receiver domain, which is essential for phosphorelay function. We constructed strains carrying an SrrA protein in which aspartate residue D385 was replaced with either asparagine (N) or alanine (A). These mutants exhibited normal conidiation and partial oxidative stress resistance. In osmotic adaptation, mutants with substitution at SrrA D385 showed as much sensitivity as ΔsrrA strains, suggesting that SrrA plays a role in osmotic stress adaptation in a phosphorelay-dependent manner. The SrrA D385 substitution mutants showed significant resistance to fungicides and cell wall stresses. These results together led us to conclude that the conserved aspartate residue has a substantial impact on SrrA function, and that SrrA plays a role in several aspects of cellular function via His-Asp phosphorelay circuitry in Aspergillus nidulans.
  Current Genetics 01/2011; 57(2):103-14. · 2.56 Impact Factor
• Source

  Available from: PubMed Central

  Article: Pseudo-response regulator (PRR) homologues of the moss Physcomitrella patens: insights into the evolution of the PRR family in land plants.

  Santosh B Satbhai, Takafumi Yamashino, Ryo Okada, Yuji Nomoto, Takeshi Mizuno, Yuki Tezuka, Tomonori Itoh, Mitsuru Tomita, Susumu Otsuki, Setsuyuki Aoki
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  ABSTRACT: The pseudo-response regulators (PRRs) are the circadian clock component proteins in the model dicot Arabidopsis thaliana. They contain a receiver-like domain (RLD) similar to the receiver domains of the RRs in the His-Asp phosphorelay system, but the RLDs lack the phosphoacceptor aspartic acid residue invariably conserved in the receiver domains. To study the evolution of PRR genes in plants, here we characterize their homologue genes, PpPRR1, PpPRR2, PpPRR3 and PpPRR4, from the moss Physcomitrella patens. In the phylogenetic analysis, PpPRRs cluster together, sister to an angiosperm PRR gene subfamily, illustrating their close relationships with the angiosperm PRRs. However, distinct from the angiosperm sequences, the RLDs of PpPRR2/3/4 exhibit a potential phosphoacceptor aspartic acid-aspartic acid-lysine (DDK) motif. Consistently, the PpPRR2 RLD had phosphotransfer ability in vitro, suggesting that PpPRR2 functions as an RR. The PpPRR1 RLD, on the other hand, shows a partially diverged DDK motif, and it did not show phosphotransfer ability. All PpPRRs were expressed in a circadian and light-dependent manner, with differential regulation between PpPRR2/4 and PpPRR1/3. Altogether, our results illustrate that PRRs originated from an RR(s) and that there are intraspecific divergences among PpPRRs. Finally, we offer scenarios for the evolution of the PRR family in land plants.
  DNA Research 01/2011; 18(1):39-52. · 5.16 Impact Factor
• Article: Genomewide characterization of the light-responsive and clock-controlled output pathways in Lotus japonicus with special emphasis of its uniqueness.

  Natsuko Ono, Kai Ishida, Takafumi Yamashino, Hanayo Nakanishi, Shusei Sato, Satoshi Tabata, Takeshi Mizuno
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  ABSTRACT: During the last decade, tremendous progress has been made in understanding the molecular mechanisms underlying the plant circadian clock in Arabidopsis thaliana, mainly taking advantage of the availability of its entire genomic sequence. It is also well understood how the clock controls the photomorphogenesis of seedlings, including the shade avoidance response, and how the clock controls the photoperiodic flowering time in the spring annual long-days herb A. thaliana. Based on this, here we attempt to shed light on these clock-controlled fundamental and physiological events in Lotus japonicus, which is a perennial temperate legume with a morphological nature quite different from Arabidopsis. In the Lotus database, we first compiled as many clock-, light-, and flowering-associated coding sequences as possible, which appear to be orthologous or homologous to the Arabidopsis counterparts. Then we focused on the PHYTOCHROME INTERACTING FACTOR4 (PIF4)-mediated photomorphogenic pathway and the FLOWERING LOCUS T (FT)-mediated photoperiodic flowering pathway. It was shown in L. japonicus that the putative LjPIF4 homologue is expressed in a manner dependent on the circadian clock, and the putative LjFT orthologue is expressed coincidentally and especially in the long-days conditions, as in the case of A. thaliana. LjFT is capable of promoting flowering in A. thaliana, whereas the function of LjPIF4 seems to be divergent to a certain extent from that of AtPIF4. These results are discussed with emphasis on the intriguing differences between these model plant species.
  Plant and Cell Physiology 10/2010; 51(10):1800-14. · 4.70 Impact Factor
• Source

  Available from: Norihito Nakamichi

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

  Norihito Nakamichi, Takatoshi Kiba, Rossana Henriques, Takeshi Mizuno, Nam-Hai Chua, Hitoshi Sakakibara
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  ABSTRACT: 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.
  The Plant Cell 03/2010; 22(3):594-605. · 8.99 Impact Factor
• Article: PHYTOCHROME-INTERACTING FACTORS PIF4 and PIF5 are implicated in the regulation of hypocotyl elongation in response to blue light in Arabidopsis thaliana.

  Atsushi Kunihiro, Takafumi Yamashino, Takeshi Mizuno
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  ABSTRACT: In Arabidopsis thaliana, plant growth, including elongation of hypocotyl is regulated in response to light conditions through circadian clock-controlled PHYTOCHROME-INTERACTING FACTORs PIF4 and PIF5. In this study, we found that these transcription factors were responsible not only for red light signaling through the phytochromes but also for blue light signaling in the photomorphogenic control of hypocotyl elongation. It is possible that plant hormone gibberellins play an important role in blue light signaling in part through PIF4/PIF5.
  Bioscience Biotechnology and Biochemistry 01/2010; 74(12):2538-41. · 1.28 Impact Factor
• Article: Biochemical characterization of plant hormone cytokinin-receptor histidine kinases using microorganisms.

  Takeshi Mizuno, Takafumi Yamashino
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  ABSTRACT: Results of recent studies on the model higher plant Arabidopsis thaliana have led us to learn about the generality and versatility of two-component systems (TCS) in eukaryotes. In the plant, TCS are crucially involved in certain signal transduction mechanisms underlying the regulation of plant development in response to a subset of plant hormones, namely, cytokinin and ethylene. Results of extensive plant genomics revealed that these hormone-responsive TCS are evolutionarily conserved in many other plants, including mosses, grasses, crops, and trees. In particular, the conserved cytokinin-responsive TCS is typical in the sense that the signaling pathway consists of cytokinin-receptor histidine kinases (HK), histidine-containing phosphotransfer (HPt) factors, and downstream phosphoaccepting response regulators (RR), which together act as His-to-Asp multistep phosphorelay components, and which together modulate the downstream network of cytokinin-responsive gene regulation. The ethylene-responsive TCS is atypical in that ethylene-receptor HKs appear to directly interact with the downstream mitogen-activated protein kinase (MAPK) cascade. The ethylene-responsive HKs have already been introduced in the previous edition of Methods in Enzymology [Schaller, G. E., and Binder, B. M. (2007). Biochemical characterization of plant ethylene receptors following transgenic expression in yeast. Methods Enzymol. 422, 270-287]. Hence, here we focus on the cytokinin-receptor HKs, which are capable of functioning in microorganisms, such as Escherichia coli and Saccharomyces cerevisiae. Some versatile protocols useful for analyzing plant TCS factors by employing these microorganisms will be introduced.
  Methods in enzymology 01/2010; 471:335-56. · 1.90 Impact Factor
• Article: Classification of the genes involved in the two-component system of the moss Physcomitrella patens.

  Kai Ishida, Takafumi Yamashino, Hanayo Nakanishi, Takeshi Mizuno
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  ABSTRACT: Physcomitrella patens, belonging to bryopsida, is a basal lineage of land plants. To gain insight into the diversification of the two-component system (TCS), which is widely conserved from prokaryotes to eukaryotes, we compiled TCS-associated genes by employing P. patens genome databases. The moss has a set of His-kinases (HKs), including homologs of the cytokinin- and ethylene-receptors in seed plants. In addition, it has a number of coding-sequences specifying unique HKs. We found evidence that a putative cytokinin-receptor HK in P. patans serves as a sensor for this hormone, and that the HK activity of a putative ethylene-receptor homolog is regulated by ethylene, as observed for Arabidopsis thaliana.
  Bioscience Biotechnology and Biochemistry 01/2010; 74(12):2542-5. · 1.28 Impact Factor