Faculty Profiles - YAMASHINO, Takafumi

Papers - YAMASHINO, Takafumi

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Circadian-controlled basic/helix-loop-helix factor, PIL6, implicated in light-signal transduction in Arabidopsis thaliana Reviewed

Fujimori T, Yamashino T, Kato T, Mizuno T

Plant Cell Physiol. Vol. 45 ( 8 ) page： 1078-1086 2004.8

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

PHYTOCHROME-INTERACTING FACTOR-LIKE 6 (PIL6) is a member of the large family of basic/helix-loop-helix (bHLH) factors in Arabidopsis thaliana. This circadian-controlled transcription factor was previously suggested to interact with the clock component, TIMING OF CAB EXPRESSION 1 (TOC1). In this study, we isolated a loss-of-function mutant of PIL6, together with a transgenic line aberrantly expressing PIL6 in a manner independent of circadian rhythm. These mutant plants were simultaneously examined with special reference to circadian rhythm and light-signal transduction. The results suggested that PIL6 appears to be not directly involved in the clock function per se. However, the loss-of-function mutant (pil6-1) showed a remarkable phenotype in that it is hypersensitive to red light in seedling de-etiolation. This phenotype was similar to that observed for transgenic lines overexpressing TOC1 (or APRR1). Conversely, transgenic plants overexpressing PIL6 (PIL6-ox) are hyposensitive to red light under the same conditions. This phenotype was very similar to that observed for phyB mutants. The developmental morphologies of PIL6-ox, including the phenotype of early flowering, were also similar to those of phyB mutants. We propose that PIL6 acts as a negative regulator for a red light-mediated morphogenic response (e.g., elongation of hypocotyls in de-etiolation). Taken together, PIL6 might function at an interface between the circadian clock and red light-signal transduction pathways.

A Genome-wide view of the Escherichia coli BasS-BasR two-component system implicated in iron-responses Reviewed

Hagiwara D, Yamashino T, Mizuno T

Biosci. Biotechnol. Biochem. Vol. 68 ( 8 ) page： 1758-1767 2004.8

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

Histidine-to-aspartate (His-->Asp) phosphorelay (or two-component) systems are very common signal transduction mechanisms that are implicated in a wide variety of cellular responses to environmental stimuli in both prokaryotes and eukaryotes. Determination of the entire genomic sequence of Escherichia coli revealed that this gram-negative bacterium has 29 His-kinases and 32 response regulators. Of the 29 His-kinases, 23 have already been experimentally characterized at least to some extent in terms of their physiological functions. No physiological stimulus has yet been identified for each of the remaining 6 His-kinases (BasS, CreC, RstB, YfhK, YehU, and YpdA). Here we characterized the BasS-BasR two-component system with reference to its physiological function, taking genetic approaches together with genome-wide transcriptome profiling. First we showed that the hypothetical yfbE operon that appears to be implicated in the modification of lipopolysaccharides is regulated at the level of transcription in response to external iron, and then we showed that the BasS-BasR system is essential for this iron-dependent induction of yfbE. Another PhoQ-PhoP two-component system was also implicated in the full induction of yfbE in response to iron, but it was not essential. To gain more insight into the BasS-BasR system, we conducted genome-wide transcriptome analysis by microarray, finding that many of the uncovered putative iron-induced and BasS-BasR-dependent genes are somehow associated with acidic and/or anaerobic growth conditions. In this respect, it was found that mutant cells defective in the BasS-BasR system were sensitive to mild-acid growth conditions in the presence of a relatively high concentration of iron. These results are discussed with regard to a comprehensive picture of the His-->Asp phosphorelay signaling network in E. coli.

Characterization of circadian-associated APRR3 pseudo-response regulator belonging to the APRR1/TOC1 quintet in Arabidopsis thaliana Reviewed

Murakami M, Yamashino T, Mizuno T

Vol. 45 ( 5 ) page： 645-650 2004.5

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

In higher plants, there are wide ranges of biological processes that are controlled through the circadian clock. In this connection, we have been characterizing a small family of proteins, designated as ARABIDOPSIS PSEUDO-RESPONSE REGULATORS (APRR1, APRR3, APRR5, APRR7, and APRR9), among which APRR1 is identical to TOC1 (TIMING OF CAB EXPRESSION1) that is believed to be a component of the central oscillator. Through previous genetic studies, several lines of evidence have already been provided to support the view that, not only APRR1/TOC1, but also other APRR1/TOC1 quintet members are important for a better understanding of the molecular links between circadian rhythm, control of flowering time, and also photomorphogenesis. However, the least characterized one was APRR3 in that no genetic study has been conducted to see if APRR3 also plays an important role in the circadian-associated biological events. Here we show that APRR3-overexpressing transgenic plants (APRR3-ox) exhibited: (i). a phenotype of longer period (and/or delayed phase) of rhythms of certain circadian-controlled genes under continuous white light, (ii). a phenotype of late flowering under long-day photoperiod conditions, (iii). a phenotype of hypo-sensitiveness to red light during early photomorphogenesis of de-etiolated seedlings, supporting the current idea as to the APRR1/TOC1 quintet described above.

Genome-wide comparison of the His-to-Asp phosphorelay signaling components of three symbiotic genera of Rhizobia Reviewed

Hagiwara D, Yamashino T, Mizuno T

DNA Res. Vol. 11 ( 1 ) page： 57-65 2004.2

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

Histidine-to-aspartate (His-Asp) phosphorelay (or two-component) systems are very common signal transduction mechanisms that are implicated in a wide variety of cellular responses to environmental stimuli. The His-Asp phosphorelay components include "sensor histidine kinase (HK)", "phosphotransfer intermediate (HPt)", and "response regulator (RR)". With special reference to three bacterial species (Mesorhizobium loti, Bradyrhizobium japonicum, Sinorhizobium meliloti), each of which belongs to a different genera of Rhizobia, here we attempted to compile all of the His-Asp phosphorelay components in order to reveal a comparative genome-wide overview as to the His-Asp phosphorelay. It was revealed that M. loti has 47 HKs, 1 HPts, and 58 RRs; B. japonicum has 80 HKs, 3 HPts, and 91 RRs; whereas S. meliloti has 40 HKs, 1 HPt, and 58 RRs. These His-Asp phosphorelay components were extensively compiled and characterized. The resulting overview as to the His-Asp phosphorelay of Rhizobia will provide us with a basis for understanding of the fundamental mechanisms underlying interactions between plants and microorganisms (including symbiosis), as well as nitrogen fixation.

Comparative studies of the AHP histidine-containing phosphotransmitters implicated in His-to-Asp phosphorelay in Arabidopsis thaliana Reviewed

Tanaka Y, Suzuki T, Yamashino T, Mizuno T

Biosci. Biotechnol. Biochem. Vol. 68 ( 2 ) page： 462-465 2004.2

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

The evolutionarily-conserved histidine to aspartate (His-to-Asp) phosphorelay signal transduction is common in both prokaryotes and eukaryotes. Such a phosphorelay system is generally made up of 'a histidine (His)-kinase', 'a histidine-containing phosphotransmitter (HPt)', and 'a phospho-accepting response regulator (RR)'. In general, an HPt factor acts as an intermediate in a given multistep His-to-Asp phosphorelay. In Arabidopsis thaliana, this model higher plant has five genes (named AHP1 to AHP5), each of which seems to encode an HPt factor. Recent studies suggested that the His-to-Asp phosphorelay involving the AHP factors is at least partly implicated in signal transduction in response to cytokinin (a plant hormone). Nevertheless, the properties of AHPs have not yet been fully clarified. Here we did comparative studies of all the AHP factors, in terms of (i) expression profiles in plants, (ii) intracellular localization, (iii) ability to acquire a phosphoryl group in vitro, and (iv) ability to interact with the downstream components, ARRs (Arabidopsis response regulators). The results of this study provided us with a comprehensive view at the molecular level for understanding the functions of the AHP phosphotransmitters in the His-to-Asp phosphorelay.

Characterization of plant circadian rhythms by employing Arabidopsis</> cultured cells with bioluminescence reporters Reviewed

Nakamichi N, Ito S, Oyama T, Yamashino T, Kondo T, Mizuno T

Plant Cell Physiol. Vol. 45 ( 1 ) page： 57-67 2004.1

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.

Comparative studies on the type-B response regulators revealing their distinctive properties in the His-to-Asp phosphorelay signal transduction of Arabidopsis thaliana Reviewed

Tajima Y, Imamura A, Kiba T, Amano Y, Yamashino T, Mizuno T

Plant Cell Physiol. Vol. 45 ( 1 ) page： 28-39 2004.1

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

In Arabidopsis thaliana, a Histidine-to-Aspartate (His-->Asp) phosphorelay is involved in the signal transduction for propagation of certain stimuli, such as plant hormones. Through the phosphorelay, the type-B phospho-accepting response regulator (ARR) family members serve as DNA-binding transcriptional regulators, whose activities are most likely regulated by phosphorylation/dephosphorylation. Based on the fact that this higher plant has 11 type-B ARR family genes, we clarified the expression profiles for all of their transcripts in plants. We constructed and characterized a series of transgenic lines, each carrying a given ARR-promoter::GUS transgene. Transcripts of some type-B ARR family genes were detected more or less ubiquitously in many organs tested, while others were expressed predominantly in reproductive organs. These ARR family members were phylogenetically classified into three sub-families, the largest of which includes the well-characterized ARR1, ARR2, and ARR11. Comparative studies were conducted focusing on ARR20 and ARR21, each of which is a representative member of an uncharacterized minor sub-family. A set of transgenic lines was constructed, in each of which a C-terminal DNA-binding domain lacking the N-terminal phospho-accepting receiver of a given ARR was aberrantly overexpressed. These resulting transgenic lines, including ARR14-C-ox, ARR20-C-ox, and ARR21-C-ox, showed characteristic anomalies during development. These results are discussed with special reference to the His-->Asp phosphorelay signal transduction in A. thaliana.

Characterization of the APRR9 pseudo-response regulator belonging to the APRR1/TOC1 quintet in Arabidopsis thaliana Reviewed

Ito S, Matsushika A, Yamada H, Sato S, Kato T, Tabata S, Yamashino T, Mizuno T.

Plant Cell Physiol. Vol. 44 ( 11 ) page： 1237-1245 2003.11

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

In Arabidopsis thaliana, a number of circadian-associated factors have been identified, including TOC1 (TIMING OF CAB EXPRESSION1) that 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). As demonstrated previously, these APRR1/TOC1 quintet members are crucial for a better understanding of the molecular links between circadian rhythms and photosensory signal transduction. Here we focused on the light-induced quintet member, APRR9, and three critical issues with regard to this member were simultaneously addressed: (i) clarification of the mechanism underlying the light-dependent acute response of APRR9, (ii) clarification of the phenotype of a null mutant of APRR9, (iii) identification of protein(s) that interacts with APRR9. In this study, we present the results that support the following views. (i) A phytochrome-mediated signaling pathway(s) activates the transcription of APRR9, leading to the acute light response of APRR9. (ii) The severe mutational lesion of APRR9 singly, if not directly, affects the period (and/or phase) of free-running rhythms, in continuous light, of every circadian-controlled gene tested, including the clock genes, APRR1/TOC1, CCA1, and LHY. (iii) The APRR9 protein is capable of interacting with APRR1/TOC1, suggesting a hetrodimer formation between these cognate family members. These results are discussed within the context of a current consistent model of the Arabidopsis circadian oscillator.

Comparative genetic studies on the APRR5 and APRR7 genes belonging to the APRR1/TOC1 quintet implicated in circadian rhythm, control of flowering time, and early photomorphogenesis Reviewed

Yamamoto Y, Sato E, Shimizu T, Nakamich N, Sato S, Kato T, Tabata S, Nagatani A, Yamashino T, Mizuno T

Plant Cell Physiol. Vol. 44 ( 11 ) page： 1119-1130 2003.11

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.

The evolutionarily conserved OsPRR quintet: rice pseudo-response regulators implicated in circadian rhythm Reviewed

Murakami M, Ashikari M, Miura K, Yamashino T, Mizuno T

Plant Cell Physiol. Vol. 44 ( 11 ) page： 1229-1236 2003.11

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

In Arabidopsis thaliana, a number of circadian-associated factors have been identified, including TOC1 (TIMING OF CAB EXPRESSION 1) that 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). As demonstrated previously, these APRR1/TOC1 quintet members are crucial for a better understanding of the molecular links between circadian rhythms, control of flowering time through photoperiodic pathways, and also photosensory signal transduction in this dicotyledonous plant. In this respect, both the dicotyledonous (e.g. A. thaliana) and monocotyledonous (e.g. Oryza sativa) plants might share the evolutionarily conserved molecular mechanism underlying the circadian rhythm. Based on such an assumption, and as the main objective of this study, we asked the question of whether rice also has a set of pseudo-response regulators, and if so, whether or not they are associated with the circadian rhythm. Here we showed that rice has five members of the OsPRR family (Oryza sativa Pseudo-Response Regulator), and also that the expressions of these OsPRR genes are under the control of circadian rhythm. They are expressed in a diurnal and sequential manner in the order of OsPRR73 (OsPRR37)-->OsPRR95 (OsPRR59)-->OsPRR1, which is reminiscent of the circadian waves of the APRR1/TOC1 quintet in A. thaliana. These and other results of this study suggested that the OsPRR quintet, including the ortholog of APRR1/TOC1, might play important roles within, or close to, the circadian clock of rice.

Genome-wide analyses revealing a signaling network of the RcsC-YojN-RcsB phosphorelay system in Escherichia coli Reviewed

Hagiwara D, Sugiura M, Oshima T, Mori H, Aiba H, Yamashino T, Mizuno T

J. Bacteriol. Vol. 185 ( 19 ) page： 5735-5746 2003.10

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

In Escherichia coli, capsular colanic acid polysaccharide synthesis is regulated through the multistep RcsC-->YojN-->RcsB phosphorelay. By monitoring a hallmarked cps::lacZ reporter gene, we first searched for physiological stimuli that propagate the Rcs signaling system. The expression of cps::lacZ was activated when cells were grown at a low temperature (20 degrees C) in the presence of glucose as a carbon source and in the presence of a relatively high concentration of external zinc (1 mM ZnCl(2)). In this Rcs signaling system, the rcsF gene product (a putative outer membrane-located lipoprotein) was also an essential signaling component. Based on the defined signaling pathway and physiological stimuli for the Rcs signaling system, we conducted genome-wide analyses with microarrays to clarify the Rcs transcriptome (i.e., Rcs regulon). Thirty-two genes were identified as putative Rcs regulon members; these genes included 15 new genes in addition to 17 of the previously described cps genes. Using a set of 37 two-component system mutants, we performed alternative genome-wide analyses. The results showed that the propagation of the zinc-responsive Rcs signaling system was largely dependent on another two-component system, PhoQ/P. Considering the fact that the PhoQ/P signaling system responds to external magnesium, we obtained evidence which supports the view that there is a signaling network that connects the Rcs system with the PhoQ/P system, which coordinately regulates extracellular polysaccharide synthesis in response to the external concentrations of divalent cations.

The type-A response regulator, ARR15, acts as a negative regulator in the cytokinin-mediated signal transduction in Arabidopsis thaliana Reviewed

Kiba T, Yamada H, Sato S, Kato T, Tabata S, Yamashino T, Mizuno T

Plant Cell Physiol. Vol. 44 ( 8 ) page： 868-874 2003.8

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

The Arabidopsis thaliana AHK4 histidine kinase (also known as CRE1 or WOL) acts as a cytokinin signal transducer, presumably, in concert with downstream components, such as histidine-containing phosphotransfer factors (AHPs) and response regulators (ARRs), through the histidine-to-aspartate (His-->Asp) phosphorelay. Among 10 members of the type-A ARR family, the cytokinin-induced expression of ARR15 in roots is selectively impaired in the cre1-1 mutant, which carries a mutation in the AHK4 gene, suggesting a link between this type-A response regulator and the AHK4-mediated cytokinin signal transduction in roots. To address this issue further, we characterized a T-DNA insertion mutant of ARR15, and also constructed transgenic lines (referred to as ARR15-ox) that overexpress the ARR15 gene in a manner independent of cytokinin. While the T-DNA insertion mutant (arr15-1) showed no apparent phenotype, the cytokinin-independent overexpression of ARR15 in ARR15-ox plants resulted in a reduced sensitivity toward exogenously applied cytokinin, not only in elongation of roots in plants, but also in green callus formation (or shoot formation) in explants. Cytokinin-induced expressions of certain type-A ARRs were also down-regulated in ARR15-ox plants. These results support the view that ARR15 acts as a repressor that mediates a negative feedback loop in the cytokinin and AHK4-mediated His-->Asp phosphorelay.

A Link between circadian-controlled bHLH factors and the APRR1/TOC1 quintet in Arabidopsis thaliana Reviewed

Yamashino T, Matsushika A, Fujimori T, Sato S, Kato T, Tabata S, Mizuno T

Plant Cell Physiol. Vol. 44 ( 6 ) page： 619-629 2003.6

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

APRR1 (ARABIDPSIS PSUEDO-RESPONSE REGULATOR 1) (or TOC1, TIMING OF CAB EXPRESSION 1) is believed to be a crucial component of biological clocks of Arabidopsis thaliana. Nevertheless, its molecular function remains to be fully elucidated. Based on the results of yeast two-hybrid and in vitro binding assays, we previously showed that APRR1/TOC1 interacts with certain bHLH factors (i.e. PIF3 and PIL1, which are PHYTOCHROME INTERACTING FACTOR 3 and its homolog (PIF3-LIKE 1), respectively). To critically examine the relevance of PIL1 with reference to the function of APRR1/TOC1, T-DNA insertion mutants were isolated for PIL1. No phenotype was observed for such homozygous pil1 mutants, in terms of circadian-associated events in plants. We then examined more extensively a certain set of bHLH factors, which are considerably similar to PIL1 in their structural designs. The results of extensive analyses of such bHLH factors (namely, HFR1, PIL2, PIF4, PIL5 and PIL6) in wild-type and APRR1-overexressing (APRR1-ox) transgenic lines provided us with several new insights into a link between APRR1/TOC1 and these bHLH factors. In yeast two-hybrid assays, APRR1/TOC1 showed the ability to interact with these proteins (except for HFR1), as well as PIL1 and PIF3. Among them, it was found that the expressions of PIF4 and PIL6 were regulated in a circadian-dependent manner, exhibiting free-running robust rhythms. The expressions of PIF4 and PIL6 were regulated also by light in a manner that their transcripts were rapidly accumulated upon exposure of etiolated seedlings to light. The light-induced expressions of PIF4 and PIL6 were severely impaired in APRR1-ox transgenic lines. Taken together, here we propose the novel view that these bHLH factors (PIF4 and PIL6) might play roles, in concert with APRR1/TOC1, in the integration of light-signals to control both circadian and photomorphogenic processes.

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

Nakamichi N, Matsushika A, Yamashino T, Mizuno T

Plant Cell Physiol. Vol. 44 ( 3 ) page： 360-365 2003.3

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.

In vivo and in vitro characterization of the ARR11 response regulator implicated in the His-to-Asp phosphorelay signal transduction in Arabidopsis thaliana Reviewed

Imamura A, Kiba T, Tajima Y, Yamashino T, Mizuno T

Plant Cell Physiol. Vol. 44 ( 2 ) page： 122-131 2003.2

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

In Arabidopsis thaliana, Histidine-to-Aspartate (His--> Asp) phosphorelay is a paradigm of a signaling system that is considered to be involved in response to plant hormones, including ethylene and cytokinin. In the current framework of His-->Asp phosphorelay in this higher plant, the type-B ARR (response regulator) family members appear to act as DNA-binding transcriptional regulators. Although Arabidopsis thaliana has 11 type-B ARR family members, except for ARR1 and ARR2, no biological information is available with regard to others. As the main objective of this study, we characterized another example, ARR11, in terms of not only its in vitro biochemical properties, but also its biological activity in plants. In plants, the ARR11 gene was expressed predominantly in roots. In vitro, ARR11 showed the ability to acquire a phosphoryl group from a histidine-containing phosphotransfer intermediate (AHP), and also it showed the ability to recognize a specific nucleotide sequence, GGATT. These in vitro results supported the view that ARR11 is indeed a DNA-binding transcription factor, the ability of which is most likely modulated by phosphorylation in its receiver domain. In vivo, when a C-terminal DNA-binding domain lacking the N-terminal phospho-accepting (or receiver) domain was aberrantly expressed, the resulting transgenic plants showed characteristic anomalies during development of apical parts. The observed anomalies included "unusual proliferation of tissues in cotyledons" and "outgrowth of adventitious shoots near cotyledons". These results with regard to the functions of ARR11 are mainly discussed in comparison with those of the previously characterized type-B response regulators.

Expression of chaA, a sodium ion extrusion system of Escherichia coli, is regulated by osmolarity and pH. Reviewed

Shijuku T, Yamashino T, Ohashi H, Saito H, Kakegawa T, Ohta M, Kobayashi H

Biochim. Biophys. Acta. Vol. 1556 ( 2-3 ) page： 142-148 2002.12

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

ChaA, one of the sodium ion extrusion systems of Escherichia coli, was found to function at high pH [Biochim. Biophys. Acta 1363 (1998) 231]. A chaA-lacZ transcriptional fusion gene was constructed using chaA of E. coli O157:H7 and its expression was observed in strains derived from E. coli K12. The fusion gene was expressed at high pH and was induced by the addition of NaCl, KCl or sucrose. The amount of chaA mRNA measured by reverse transcription-polymerase chain reaction (RT-PCR) was increased by the addition of sucrose to alkaline growth medium. These results suggested that chaA expression was regulated by medium osmolarity and pH.

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

Nakamichi N, Murakami-Kojima M, Sato E, Kishi Y, Yamashino T, Mizuno T

Biosci. Biotechnol. Biochem. Vol. 66 ( 11 ) page： 2429-2439 2002.11

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.

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 Reviewed

Sato E, Nakamichi N, Yamashino T, Mizuno T

Plant Cell Physiol. Vol. 43 ( 11 ) page： 1374-1385 2002.11

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.

Aberrant expression of the light-inducible and circadian-regulated APRR9 gene belonging to the circadian-associated APRR1/TOC1 quintet results in the phenotype of early flowering in Arabidopsis thaliana Reviewed

Matsushika A, Imamura A, Yamashino T, Mizuno T

Plant Cell Physiol. Vol. 43 ( 8 ) page： 833-843 2002.8

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

Several Arabidopsis genes have been proposed to encode potential clock-associated components, including the Myb-related CCA1 and LHY transcription factors and a member (APRR1/TOC1) of the family of pseudo-response regulators. We previously showed that transcripts of the APRR1/TOC1 family genes each start accumulating after dawn rhythmically and sequentially at intervals in the order of APRR9-->APRR7-->APRR5-->APRR3-->APRR1/TOC1, under the conditions of continuous light. Nevertheless, no evidence has been provided that each member of the APRR1/TOC1 quintet, except for APRR1/TOC1, is indeed relevant to the mechanisms underlying circadian rhythms. Here we attempt to provide such evidence by characterizing transgenic plants that aberrantly (or constitutively) express the APRR9 gene in a manner independent of circadian rhythms. The resulting APRR9-ox plants showed intriguing phenotypes with regard to circadian rhythms, in two aspects. First, the aberrant expression of APRR9 resulted in a characteristic phenotype with regard to transcriptional events, in which short-period rhythms were commonly observed for certain circadian-regulated genes, including CCA1, LHY, APRR1/TOC1, other APRR1/TOC1 members, ELF3, and CAB2. With regard to biological consequences, such APRR9-ox plants flowered much earlier than wild-type plants, in a manner independent of photoperiodicity (or under short-day conditions). These results suggest that APRR9 (and perhaps other members of the APRR1/TOC1 quintet) must also be taken into consideration for a better understanding of the molecular mechanisms underlying circadian rhythms, and also underlying control of the flowering time through the photoperiodic long-day pathway.

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 Reviewed

Murakami-Kojima M, Nakamichi N, Yamashino T, Mizuno T

Plant Cell Physiol. Vol. 43 ( 6 ) page： 675-683 2002.6

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.

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