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

A novel gain-of-function mutant showing dwarfism and bushy inflorescences was isolated from Arabidopsis activation-tagging lines. Transformed plants, in which the gene responsible for the phenotype of the mutant was overexpressed, exhibited phenotypes similar to constitutive systematic acquired resistance (SAR)-like defense responses including simulation of lesions in leaves and a highly activated PATHOGENESIS-RELATED 1 gene. We, therefore, designated the gene as DWARF AND LESION FORMATION 1 (DLE1) and showed it encodes a DUF 247 protein. Microarray and gene ontology analyses revealed that defense response genes, especially to biotic stimulus, were enriched in DLE1 overexpressing plants. Fusion proteins of the N-terminal fragment of DLE1 and GFP were likely to be localized in the endoplasmic reticulum (ER) in tobacco BY-2 cells. DLE1 may function in the plant defense system responding to environmental stimuli including biotic stresses in the ER. © 2013 The Japanese Society for Plant Cell and Molecular Biology.

DOI： 10.5511/plantbiotechnology.13.0605a

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

In the cyanobacterium Synechococcus elongatus PCC 7942, the circadian clock entrains to a daily light/dark cycle. The transcription factor Pex is abundant under dark conditions and represses kaiA transcription to fine-tune the KaiC-based core circadian oscillator. The transcription of pex also increases during exposure to darkness; however, its mechanism is unknown. We performed a molecular genetic study by constructing a pex expression bioluminescent reporter and screening for brightly luminescent mutants by random insertion of a drug resistance gene cassette in the reporter genome. One mutant contained an insertion of an antibiotic resistance cassette in the cmpR locus, a transcriptional regulator of inorganic carbon concentration. Insertions of the cassette in the remaining two mutant genomes were in the genes encoding flavodoxin and a putative partner of an ABC transporter with unknown function (ycf22). We further analyzed the cmpR mutant to examine whether CmpR directly or indirectly targeted pex expression. In the cmpR mutant, the pex mRNA level was 1.8-fold that of the wild type, and its circadian peak phase in bioluminescence rhythm occurred 5 h later. Moreover, a high-light stress phenotype was present in the colony. The abnormalities were complemented by ectopic induction of the native gene. However, the cmpR/pex double mutation partly suppressed the phase abnormality (2.5 h). In vitro DNA binding analysis of CmpR showed positive binding to the psbAII promoter, but not to any pex DNA. We postulate that the phenotypes of cmpR-deficient cells were attributable mainly to a feeble metabolic and/or redox status.

DOI： 10.1093/pcp/pcs095

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Language：English   Publishing type：Part of collection (book)   Publisher：Nova Science Publishers, Inc.  

In cyanobacteria, bioluminescence reporters have been applied to the measurement of physiological phenomenon, such as in the study of circadian clock and nitrite, ferric, and light responses. Cyanobacterial researchers have so far used several types of bioluminescence reporter systems-consisting of luminescence genes, genetically tractable host cells, and a monitoring device-because their studies require a method that offers gene expression data with high fidelity, high resolution for time, and enough dynamic range in data collection. In addition, no extraction of the products of the reporter gene from the culture is required to measure the luminescence, even in the living cell. In this chapter, applications using the bioluminescence genes luxAB (and luxCDE for substrate production) and insect genes are introduced. For measurement and imaging, general apparatuses, such as a luminometer and a luminoimager, have been used with several methods of substrate administration. Automated bioluminescence monitoring apparatuses were also newly developed. The initial machine was similar to that used to measure the native circadian rhythms in bioluminescence of the marine dinoflagellate Gonyaulax polyedra. Then, the machine with a cooled CCD camera which was automatically operated by a computer was used to screen mutant colonies representing abnormal bioluminescence profile or level from a mutagen-treated cyanobacterial cell with a luxAB reporter. Recently, different two promoter activities could be examined in the same cell culture and with the same timing by using railroad-worm luciferase genes. © 2009 Nova Science Publishers, Inc. All rights reserved.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

In the cyanobacterium Synechococcus sp. strain PCC 7942, a circadian clock-related gene, pex, was identified as the gene prolonging the period of the clock. A PadR domain, which is a newly classified transcription factor domain, and the X-ray crystal structure of the Pex protein suggest a role for Pex in transcriptional regulation in the circadian system. However, the regulatory target of the Pex protein is unknown. To determine the role of Pex, we monitored bioluminescence rhythms that reported the expression activity of the kaiA gene or the kaiBC operon in pex deficiency, pex constitutive expression, and the wild-type genotype. The expression of kaiA in the pex-deficient or constitutive expression genotype was 7 or 1/7 times that of the wild type, respectively, suggesting that kaiA is the target of negative regulation by Pex. In contrast, the expression of the kaiBC gene in the two pex-related genotypes was the same as that in the wild type, suggesting that Pex specifically regulates kaiA expression. We used primer extension analysis to map the transcription start site for the kaiA gene 66 bp upstream of the translation start codon. Mapping with deletion and base pair substitution of the kaiA upstream region revealed that a 5-bp sequence in this region was essential for the regulation of kaiA. The repression or constitutive expression of the kaiA transgene caused the prolongation or shortening of the circadian period, respectively, suggesting that the Pex protein changes the period via the negative regulation of kaiA.

DOI： 10.1128/JB.00835-07

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1074/jbc.m608148200

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：SAGE Publications  

The time measurement system of the unicellular cyanobacterium Synechococcus elongatus PCC 7942 is analogous to the circadian clock of eukaryotic cells. Circadian clock-related genes have been identified in this strain. The clock-related gene pex is thought to maintain the normal clock period because constitutive transcription or deficiency of this gene causes respectively longer (~28 h) or shorter (~24 h) circadian periods than that of the wild type (~25 h). Here, the authors report other properties of pex in the circadian system. Levels of pex mRNA increased significantly in a 12-h exposure to darkness. Western blotting with a GST-Pex antibody revealed a 13.5-kDa protein band in wild-type cells that were incubated in the dark, while this protein was not detected in pex-deficient mutant cells. Therefore, the molecular weight of the Pex protein appears to be 13.5 kDa in vivo. The PadR domain, which is conserved among DNA-binding transcription factors in lactobacilli, was found in Pex. In the pex mutant, several 12-h light/12-h dark cycles reset the phase of the clock by 3 h earlier (phase advance) compared to wild-type cells. The degree of the advance in the pex mutant was proportional to the number of exposed light-dark cycles. In addition, ectopic induction of pex with an inducible Escherichia coli promoter, Ptrc, delayed the phase in the examined recombinant cells by 2.5 h (phase delay) compared to control cells. These results suggest that the dark-responsive gene expression of pex delays the circadian clock under daily light-dark cycles.

DOI： 10.1177/0748730406289400

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Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1111/j.1365-2958.2005.04781.x

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Publishing type：Research paper (scientific journal)   Publisher：Proceedings of the National Academy of Sciences  

A kaiABC clock gene cluster was previously identified from cyanobacterium Synechococcus elongatus PCC 7942, and the feedback regulation of kai genes was proposed as the core mechanism generating circadian oscillation. In this study, we confirmed that the Kai-based oscillator is the dominant circadian oscillator functioning in cyanobacteria. We probed the nature of this regulation and found that excess KaiC represses not only kaiBC but also the rhythmic components of all genes in the genome. This result strongly suggests that the KaiC protein primarily coordinates genomewide gene expression, including its own expression. We also found that a promoter derived from E. coli is feedback controlled by KaiC and restores the complete circadian rhythm in kaiBC -inactivated arrhythmic mutants, provided it can express kaiB and kaiC genes at an appropriate level. Unlike eukaryotic models, specific regulation of the kaiBC promoter is not essential for cyanobacterial circadian oscillations.

DOI： 10.1073/pnas.0307411100

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

We identified an open reading frame from a database of the entire genome of Synechococcus elongatus, the product of which was very similar to pixJ1, which was proposed as photoreceptor gene for phototaxis in Synechocystis sp. PCC6803 [Yoshihara et al. (2000) Plant Cell Physiol. 41: 1299]. The mRNA of S. elongatus pixJ (SepixJ) was expressed in vivo as a part of the product of an operon. SePixJ was detected exclusively in the membrane fraction after cell fractionation. Immunogold labeling of SePixJ in ultra-thin sections indicated that it existed only in both ends of the rod-shaped cell
probably bound with the cytoplasmic membrane.

DOI： 10.1093/pcp/pcf176

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Publishing type：Research paper (scientific journal)   Publisher：American Association for the Advancement of Science (AAAS)  

Cyanobacteria are the simplest organisms known to have a circadian clock. A circadian clock gene cluster kaiABC was cloned from the cyanobacterium Synechococcus. Nineteen clock mutations were mapped to the three kai genes. Promoter activities upstream of the kaiA and kaiB genes showed circadian rhythms of expression, and both kaiA and kaiBC messenger RNAs displayed circadian cycling. Inactivation of any single kai gene abolished these rhythms and reduced kaiBC -promoter activity. Continuous kaiC overexpression repressed the kaiBC promoter, whereas kaiA overexpression enhanced it. Temporal kaiC overexpression reset the phase of the rhythms. Thus, a negative feedback control of kaiC expression by KaiC generates a circadian oscillation in cyanobacteria, and KaiA sustains the oscillation by enhancing kaiC expression.

DOI： 10.1126/science.281.5382.1519

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Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

ABSTRACT

We cloned the pS1K1 plasmid in the process of apparently “complementing” a circadian clock mutant of cyanobacterium Synechococcus sp. strain PCC 7942, SP22, which has a 22-h period (T. Kondo, N. F. Tsinoremas, S. S. Golden, C. H. Johnson, S. Kutsuna, and M. Ishiura, Science 266:1233–1236, 1994). Sequence analysis revealed that SP22 did not have a mutation in the genomic DNA segment carried on pS1K1, and the sp22 mutation was later found in a recently cloned new clock gene, kaiC . Therefore, the period-extender gene pex that was carried on pS1K1 was a suppressor gene for the sp22 mutation. The pex gene encoded a protein of 148 amino acid residues. No meaningful homologs were found in DNA or protein databases including the Synechocystis genome database. The pex gene was transcribed from 129 and 164 bp upstream of the translation initiation codon as 0.6-kb transcripts. The Pex protein was detected as a fusion protein with a molecular mass of 15 kDa by the epitope tag fusion method using a c-Myc epitope tag. Disruption of the pex gene in wild-type cells shortened the period of the rhythms by 1 h, although it did not affect other properties of the rhythms, whereas its overexpression extended the period by 3 h with a concomitant reduction in the amplitude of the rhythms. In various clock mutants examined, overexpression caused arrhythmicity. Thus, Pex is likely to function as a modifier of the circadian clock in Synechococcus.

DOI： 10.1128/jb.180.8.2167-2174.1998

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Publishing type：Research paper (scientific journal)   Publisher：American Association for the Advancement of Science (AAAS)  

DOI： 10.1126/science.7973706

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Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Poster presentation  

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