記述言語：英語   掲載種別：研究論文（学術雑誌）  

To explore a possible recessive selective marker for future DNA-free genome editing by direct delivery of a CRISPR/Cas9-single guide RNA (sgRNA) ribonucleoprotein complex, we knocked out homologs of the ArabidopsisMulti-Antibiotic Resistance 1 (MAR1)/RTS3 gene, mutations of which confer aminoglycoside resistance, in tobacco plants by an efficient Agrobacterium-mediated gene transfer. A Cas9 gene was introduced into Nicotiana tabacum and Nicotiana sylvestris together with an sgRNA gene for one of three different target sequences designed to perfectly match sequences in both S- and T-genome copies of N. tabacumMAR1 homologs (NtMAR1hs). All three sgRNAs directed the introduction of InDels into NtMAR1hs, as demonstrated by CAPS and amplicon sequencing analyses, albeit with varying efficiency. Leaves of regenerated transformant shoots were evaluated for aminoglycoside resistance on shoot-induction media containing different aminoglycoside antibiotics. All transformants tested were as sensitive to those antibiotics as non-transformed control plants, regardless of the mutation rates in NtMAR1hs. The NtMAR1hs-knockout seedlings of the T1 generation showed limited aminoglycoside resistance but failed to form shoots when cultured on shoot-induction media containing kanamycin. The results suggest that, like Arabidopsis MAR1, NtMAR1hs have a role in plants' sensitivity to aminoglycoside antibiotics, and that tobacco has some additional functional homologs.

DOI： 10.3390/ijms23042006

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The CRISPR/Cas9 system is now commonly employed for genome editing in various plants such as Arabidopsis, rice and tobacco. In general, in genome editing of the Arabidopsis genome, the SpCas9 and guide RNA genes are introduced into the genome by the floral dip method. Mutations induced in the target sequence by SpCas9 are confirmed after selecting transformants by screening the T1 seed population. The advantage of this method is that genome-edited plants can be isolated easily. However, mutation efficiency in Arabidopsis using SpCas9 is not as high as that achieved in rice and tobacco, which are subjected to a tissue culture step. In this study, we compared four promoters and found that the parsley UBIQITIN promoter is highly active in Arabidopsis meristem tissue. Furthermore, we examined whether a simple heat treatment could improve mutation efficiency in Arabidopsis. Just one heat treatment at 37°C for 24 h increased the mutation efficiency at all four target sites from 3 to 42%, 43 to 62%, 54 to 75% and 89 to 91%, without detectable off-target mutations. We recommend heat treatment of plate-grown plants at 37°C for 24 h as a simple method to increase the efficiency of CRISPR/Cas9-mediated mutagenesis in Arabidopsis.

DOI： 10.1093/pcp/pcab123

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掲載種別：研究論文（学術雑誌）  

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. The authors wish to make the following corrections to this paper [1]: There was a misunderstanding in the annotation of a single gene. The gene, ICS1, has been mentioned several times in the main text and graph labels, but the gene should have been annotated to be SARD1, a regulator of ICS1. Although this mistake does not affect the conclusion of our paper, it is very misleading to any readers of the article. Therefore, the authors would like to publish this correction. The correction includes a change in the labels for the horizontal axis in Figure 1c and Supplementary Figure S1; several changes in the main text in the results, the discussion, and the materials and methods sections; and changes in Supplementary Table S2 and the entirety of Supplementary Tables S4, S5, S8, S9, and S10, in which relationships within the columns had been lost during sorting. The authors would like to apologize for any inconvenience caused to the readers by these changes.

DOI： 10.3390/ijms21228461

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

In the present study, we have shown the transcriptional changes in a chlorosis model transgenic tobacco plant, i-amiCHLI, in which an artificial micro RNA is expressed in a chemically inducible manner to silence the expression of CHLI genes encoding a subunit of a chlorophyll biosynthetic enzyme. Comparison to the inducer-treated and untreated control non-transformants and untreated i-amiCHLI revealed that 3568 and 3582 genes were up- and down-regulated, respectively, in the inducer-treated i-amiCHLI plants. Gene Ontology enrichment analysis of these differentially expressed genes indicated the upregulation of the genes related to innate immune responses, and cell death pathways, and the downregulation of genes for photosynthesis, plastid organization, and primary and secondary metabolic pathways in the inducer-treated i-amiCHLI plants. The cell death in the chlorotic tissues with a preceding H2O2 production was observed in the inducer-treated i-amiCHLI plants, confirming the activation of the immune response. The involvement of activated innate immune response in the chlorosis development was supported by the comparative expression analysis between the two transgenic chlorosis model systems, i-amiCHLI and i-hpHSP90C, in which nuclear genes encoding different chloroplast proteins were similarly silenced.

DOI： 10.3390/ijms21197044

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掲載種別：研究論文（学術雑誌）  

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. RNA-seq analysis of a transgenic tobacco plant, i-hpHSP90C, in which chloroplast HSP90C genes can be silenced in an artificially inducible manner resulting in the development of chlorosis, revealed the up-and downregulation of 2746 and 3490 genes, respectively. Gene ontology analysis of these differentially expressed genes indicated the upregulation of ROS-responsive genes; the activation of the innate immunity and cell death pathways; and the downregulation of genes involved in photosynthesis, plastid organization, and cell cycle. Cell death was confirmed by trypan blue staining and electrolyte leakage assay, and the H2 O2 production was confirmed by diaminobenzidine staining. The results collectively suggest that the reduced levels of HSP90C chaperone lead the plant to develop chlorosis primarily through the global downregulation of chloroplast-and photosynthesis-related genes and additionally through the light-dependent production of ROS, followed by the activation of immune responses, including cell death.

DOI： 10.3390/ijms21124202

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記述言語：英語  

DOI： 10.1111/pbi.13120

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1111/tpj.14212

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1242/dev.171504

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Nature America, Inc  

DOI： 10.1038/s41477-018-0321-8

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© 2018 Taylor & Francis Group, LLC Unrepaired DNA damage hinders the maintenance of genome integrity because it blocks the catalytic activity of replicase. The stalled replication fork can be processed through either translesion synthesis (TLS) with specific polymerases, or replication using the undamaged template. To investigate how TLS activities are regulated and how the stalled replication fork is processed in plants, reversion frequencies and homologous recombination (HR) frequencies were analyzed using GUS-based substrates. The HR frequencies in plants deficient in DNA polymerase ζ (Pol ζ) or Rev1 were higher than that in wildtype plants under normal conditions, and were significantly increased by ultraviolet light irradiation. Heat shock protein (HSP) 90 is known to be involved in various stress responses. To examine the role of HSP90 in the regulation of damage tolerance, we analyzed reversion frequencies and HR frequencies in plants grown in the presence of a HSP inhibitor, geldanamycin (GDA). Reversion frequency was lower in GDA-treated plants than in mock-treated plants. Though the HR frequency was higher in GDA-treated wildtype plants than in mock-treated plants, no significant difference was detected in Rev1-deficient plants. In yeast, TLS polymerases interacted with each other or with a replication clump component, proliferating cell nuclear antigen (PCNA). HSP90 interacted with REV1 or REV7 in Nicotiana benthamiana cells. These results suggest that HSP90 interacts with TLS polymerase(s), which promotes error-prone TLS in plants.

DOI： 10.1080/15592324.2018.1483673

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS  

Split-protein methods-where a protein is split into two inactive fragments that must re-assemble to form an active protein-can be used to regulate the activity of a given protein and reduce the size of gene transcription units. Here, we show that a Staphylococcus aureus Cas9 (SaCas9) can be split, and that split-SaCas9 expressed from Agrobacterium can induce targeted mutagenesis in Nicotiana benthamiana. Since SaCas9 is smaller than the more commonly used Cas9 derived from Streptococcus pyogenes, the split-SaCas9 provides the smallest tool yet for clustered regularly interspaced short pal-indromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) plant genome editing. Both sets of split-SaCas9 (_430N/431C and _739N/740C) exhibited genome-editing activity, and the activity of split-SaCas9_739N/740C was almost the same as that of full-length SaCas9. This result indicates that split-SaCas9_739N/740C is suitable for use in targeted mutagenesis. We also show that the split-SaCas9 fragment expressed from Tomato mosaic virus could induce targeted mutagenesis together with another fragment expressed from Agrobacterium, suggesting that a split-SaCas9 system using a plant virus vector is a promising tool for integration-free plant genome editing. Split-SaCas9 has the potential to regulate CRISPR/Cas9-mediated genome editing activity in plant cells both temporally and spatially.

DOI： 10.1093/pcp/pcx034

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：FRONTIERS MEDIA SA  

Genome editing in plants becomes popular since the advent of sequence-specific nucleases (SSNs) that are simple to set up and efficient in various plant species. Although transcription activator-like effector nucleases (TALENs) are one of the most prevalent SSNs and have a potential to provide higher target specificity by their dimeric property, TALENs are sensitive to methylated cytosines that are present not only in transposons but also in active genes in plants. In mammalian cells, the methylation sensitivity of TALENs could be overcome by using a base-recognition module (N*) that has a higher affinity to methylated cytosine. In contrast to mammals, plants carry DNA methylation at all cytosine contexts (CG, CHG, and CHH, where H represents A, C, or T) with various degrees and effectiveness of N* module in genome editing in plants has not been explored. In this study, we designed sets of TALENs with or without N* modules and examined their efficiency in genome editing of methylated regions in rice. Although improvement in genome editing efficiency was observed with N*-TALENs designed to a stably methylated target, another target carrying cytosines with various levels of methylation showed resistance to both normal and N*-TALENs. The results suggest that variability of cytosine methylation in target regions is an additional factor affecting the genome editing efficiency of TALENs.

DOI： 10.3389/fpls.2017.00302

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

CRISPR/Cas9 systems are nowadays applied extensively to effect genome editing in various organisms including plants. CRISPR from Prevotella and Francisella 1 (Cpf1) is a newly characterized RNA-guided endonuclease that has two distinct features as compared to Cas9. First, Cpf1 utilizes a thymidine-rich protospacer adjacent motif (PAM) while Cas9 prefers a guanidine-rich PAM. Cpf1 could be used as a sequence-specific nuclease to target AT-rich regions of a genome that Cas9 had difficulty accessing. Second, Cpf1 generates DNA ends with a 5' overhang, whereas Cas9 creates blunt DNA ends after cleavage. "Sticky" DNA ends should increase the efficiency of insertion of a desired DNA fragment into the Cpf1-cleaved site using complementary DNA ends. Therefore, Cpf1 could be a potent tool for precise genome engineering. To evaluate whether Cpf1 can be applied to plant genome editing, we selected Cpf1 from Francisella novicida (FnCpf1), which recognizes a shorter PAM (TTN) within known Cpf1 proteins, and applied it to targeted mutagenesis in tobacco and rice. Our results show that targeted mutagenesis had occurred in transgenic plants expressing FnCpf1 with crRNA. Deletions of the targeted region were the most frequently observed mutations. Our results demonstrate that FnCpf1 can be applied successfully to genome engineering in plants.

DOI： 10.1038/srep38169

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

The CRISPR/Cas9 system is an efficient and convenient tool for genome editing in plants. Cas9 nuclease derived from Streptococcus pyogenes (Sp) is commonly used in this system. Recently, Staphylococcus aureus Cas9 (SaCas9)-mediated genome editing was reported in human cells and Arabidopsis. Because SaCas9 (1053 a.a.) is smaller than SpCas9 (1368 a.a.), SaCas9 could have substantial advantages for delivering and expressing Cas9 protein, especially using virus vectors. Since the protospacer adjacent motif (PAM) sequence of SaCas9 (5'-NNGRRT-3') differs from that of SpCas9 (5'-NGG-3'), the use of this alternative Cas9 nuclease could expand the selectivity at potential cleavage target sites of the CRISPR/Cas9 system. Here we show that SaCas9 can mutagenize target sequences in tobacco and rice with efficiencies similar to those of SpCas9. We also analyzed the base preference for 'T' at the 6th position of the SaCas9 PAM. Targeted mutagenesis efficiencies in target sequences with non-canonical PAMs (5'-NNGRRV-3') were much lower than those with a canonical PAM (5'-NNGRRT-3'). The length of target sequence recognized by SaCas9 is one or two nucleotides longer than that recognized by SpCas9. Taken together, our results demonstrate that SaCas9 has higher sequence recognition capacity than SpCas9 and is useful for reducing off-target mutations in crop.

DOI： 10.1038/srep26871

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

In many flowering plants, the transition to flowering is primarily affected by seasonal changes in day length (photoperiod). An inductive photoperiod promotes flowering via synthesis of a floral stimulus, called florigen. In Arabidopsis thaliana, the FLOWERING LOCUS T (FT) protein is an essential component of florigen, which is synthesized in leaf phloem companion cells and is transported through phloem tissue to the shoot apical meristem where floral morphogenesis is initiated. However, the molecular mechanism involved in the long-distance transport of FT protein remains elusive. In this study, we characterized the classic Arabidopsis mutant fe, which is involved in the photoperiodic induction of flowering, and showed that FE encodes a phloem-specific Myb-related protein that was previously reported as ALTERED PHLOEM DEVELOPMENT. Phenotypic analyses of the fe mutant showed that FT expression is reduced in leaf phloem companion cells. In addition, the transport of FT protein from leaves to the shoot apex is impaired in the fe mutant. Expression analyses further demonstrated that FE is also required for transcriptional activation of FLOWERING LOCUS T INTERACTING PROTEIN 1 (FTIP1), an essential regulator for selective trafficking of the FT protein from companion cells to sieve elements. These findings indicate that FE plays a dual role in the photoperiodic induction of flowering: as a transcriptional activator of FT on the one hand, and its transport machinery component, FTIP1, on the other hand. Thus, FE is likely to play a role in regulating FT by coordinating FT synthesis and FT transport in phloem companion cells.

DOI： 10.1111/tpj.12951

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS INC  

Reactive oxygen species (ROS) accumulate at the tip of growing pollen tubes. In Arabidopsis, NADPH oxidases RbohH and RbohJ are localized at the plasma membrane of pollen tube tip and produce ROS in a Ca2+-dependent manner. The ROS produced by Rbohs and Ca2+ presumably play a critical role in the positive feedback regulation that maintains the tip growth. Ultrastructural cytochemical analysis revealed ROS accumulation in the apoplast/cell wall of the pollen grains on the stigmatic papillae in the wild type, but not in the rbohH rbohJ double mutant, suggesting that apoplastic ROS derived from RbohH and RbohJ are involved in pollen tube elongation into the stigmatic papillae by affecting the cell wall metabolism.

DOI： 10.4161/15592324.2014.989050

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC PLANT BIOLOGISTS  

In flowering plants, pollen germinates on the stigma and pollen tubes grow through the style to fertilize the ovules. Enzymatic production of reactive oxygen species (ROS) has been suggested to be involved in pollen tube tip growth. Here, we characterized the function and regulation of the NADPH oxidases RbohH and RbohJ (Respiratory burst oxidase homolog H and J) in pollen tubes in Arabidopsis thaliana. In the rbohH and rbohJ single mutants, pollen tube tip growth was comparable to that of the wild type; however, tip growth was severely impaired in the double mutant. In vivo imaging showed that ROS accumulation in the pollen tube was impaired in the double mutant. Both RbohH and RbohJ, which contain Ca2+ binding EF-hand motifs, possessed Ca2+-induced ROS-producing activity and localized at the plasma membrane of the pollen tube tip. Point mutations in the EF-hand motifs impaired Ca2+-induced ROS production and complementation of the double mutant phenotype. We also showed that a protein phosphatase inhibitor enhanced the Ca2+-induced ROS-producing activity of RbohH and RbohJ, suggesting their synergistic activation by protein phosphorylation and Ca2+. Our results suggest that ROS production by RbohH and RbohJ is essential for proper pollen tube tip growth, and furthermore, that Ca2+-induced ROS positive feedback regulation is conserved in the polarized cell growth to shape the long tubular cell.

DOI： 10.1105/tpc.113.120642

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DOI： 10.1016/j.bbamcr.2013.06.024

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

Reactive oxygen species (ROS) produced by NADPH oxidases play critical roles in plant environmental responses. Arabidopsis thaliana NADPH oxidase AtRbohF-mediated ROS-production is involved in abiotic stress responses. Because overproduction of ROS is highly toxic to cells, the activity of AtRbohF needs to be tightly regulated in response to diverse stimuli. The ROS-producing activity of AtRbohF is activated by Ca2+ and protein phosphorylation, but other regulatory factors for AtRbohF are mostly unknown. In this study, we screened for proteins that interact with the N-terminal cytosolic region of AtRbohF by a yeast two-hybrid screen, and isolated AtSRC2, an A. thaliana homolog of SRC2 (soybean gene regulated by cold-2). A co-immunoprecipitation assay revealed that AtSRC2 interacts with the N-terminal region of AtRbohF in plant cells. Intracellular localization of GFP-tagged AtSRC2 was partially overlapped with that of GFP-tagged AtRbohF at the cell periphery. Co-expression of AtSRC2 enhanced the Ca2+ -dependent ROS-producing activity of AtRbohF in HEK293T cells, but did not affect its phosphorylation-dependent activation. Low-temperature treatment induced expression of the AtSRC2 gene in Arabidopsis roots in proportion to levels of ROS production that was partially dependent on AtRbohF. Our findings suggest that AtSRC2 is a novel activator of Ca2+-dependent AtRbohF-mediated ROS production and may play a role in cold responses. (c) 2013 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbamcr.2013.06.024

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS  

The plant NADPH oxidases, known as respiratory burst oxidase homologues (Rbohs), play an indispensable role in a wide array of cellular and developmental processes. Arabidopsis thaliana RbohF (AtRbohF)-mediated production of reactive oxygen species (ROS) is involved in biotic and abiotic stress responses. Because of the toxicity of excess amount of ROS, the ROS-producing activity of Rbohs is speculated to be negatively regulated. However, its mechanism is mostly unknown to date. Here, we report the identification of calcineurin B-like protein-interacting protein kinase 26 (CIPK26) as a novel regulatory factor of AtRbohF. We isolated CIPK26 as an AtRbohF-interacting partner by a yeast two-hybrid screen. Our co-immunoprecipitation assay revealed that the CIPK26 protein interacts with the N-terminal region of AtRbohF in Nicotiana benthamiana cell extracts. The fluorescence of both GFP-tagged CIPK26 and AtRbohF was predominantly observed at the cell periphery. We also showed that co-expression of CIPK26 decreases the ROS-producing activity of AtRbohF in HEK293T cells. Together, these results suggest that the direct binding of CIPK26 to AtRbohF negatively modulates ROS production and play a role in the regulation of ROS signalling in plants.

DOI： 10.1093/jb/mvs132

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS  

Reactive oxygen species (ROS) produced by plant NADPH oxidases (NOXes) are important in plant innate immunity. The Oryza sativa respiratory burst oxidase homologue B (OsRbohB) gene encodes a NOX the regulatory mechanisms of which are largely unknown. Here, we used a heterologous expression system to demonstrate that OsRbohB shows ROS-producing activity. Treatment with ionomycin, a Ca2+ ionophore, and calyculin A, a protein phosphatase inhibitor, activated ROS-producing activity; it was thus OsRbohB activated by both Ca2+ and protein phosphorylation. Mutation analyses revealed that not only the first EF-hand motif but also the upstream amino-terminal region were necessary for Ca2+-dependent activation, while these regions are not required for phosphorylation-induced ROS production.

DOI： 10.1093/jb/mvs044

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記述言語：日本語  

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DOI： 10.1016/j.bbamcr.2011.09.011

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

Reactive oxygen species (ROS) produced by NADPH oxidases play critical roles in signalling and development. Given the high toxicity of ROS, their production is tightly regulated. In Arabidopsis, respiratory burst oxidase homologue F (AtrbohF) encodes NADPH oxidase. Here we characterised the activation of AtRbohF using a heterologous expression system. AtRbohF exhibited ROS-producing activity that was synergistically activated by protein phosphorylation and Ca2+. The two EF-hand motifs of AtRbohF in the N-terminal cytosolic region were crucial for its Ca2+-dependent activation. AtrbohD and AtrbohF are involved in stress responses. Although the activation mechanisms for AtRbohD and AtRbohF were similar, AtRbohD had significantly greater ROS-producing activity than AtRbohF, which may reflect their functional diversity, at least in part. We further characterised the interrelationship between Ca2+ and phosphorylation regarding activation and found that protein phosphorylation-induced activation was independent of Ca2+. In contrast, K-252a, a protein kinase inhibitor, inhibited the Ca2+-dependent ROS-producing activity of AtRbohD and AtRbohF in a dose-dependent manner, suggesting that protein phosphorylation is a prerequisite for the Ca2+-dependent activation of Rboh. Positive feedback regulation of Ca2+ and ROS through AtRbohC has been proposed to play a critical role in root hair tip growth. Our findings suggest that Rboh phosphorylation is the initial trigger for the plant Ca2+-ROS signalling network (C) 2011 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbamcr.2011.09.011

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Plant respiratory burst oxidase homolog ( rboh) proteins, which are homologous to the mammalian 91-kDa glycoprotein subunit of the phagocyte oxidase ( gp91(phox)) or NADPH oxidase 2 ( NOX2), have been implicated in the production of reactive oxygen species ( ROS) both in stress responses and during development. Unlike mammalian gp91(phox)/NOX2 protein, plant rboh proteins have hydrophilic N-terminal regions containing two EF-hand motifs, suggesting that their activation is dependent on Ca2+. However, the significance of Ca2+ binding to the EF-hand motifs on ROS production has been unclear. By employing a heterologous expression system, we showed that ROS production by Arabidopsis thaliana rbohD ( AtrbohD) was induced by ionomycin, which is a Ca2+ ionophore that induces Ca2+ influx into the cell. This activation required a conformational change in the EF-hand region, as a result of Ca2+ binding to the EF-hand motifs. We also showed that AtrbohD was directly phosphorylated in vivo, and that this was enhanced by the protein phosphatase inhibitor calyculin A ( CA). Moreover, CA itself induced ROS production and dramatically enhanced the ionomycin-induced ROS production of AtrbohD. Our results suggest that Ca2+ binding and phosphorylation synergistically activate the ROS-producing enzyme activity of AtrbohD.

DOI： 10.1074/jbc.M708106200

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER ASSOC ADVANCEMENT SCIENCE  

The specification and maintenance of growth sites are tightly regulated during cell morphogenesis in all organisms. ROOT HAIR DEFECTIVE 2 reduced nicotinamide adenine dinucleotide phosphate ( RHD2 NADPH) oxidase - derived reactive oxygen species ( ROS) stimulate a Ca2+ influx into the cytoplasm that is required for root hair growth in Arabidopsis thaliana. We found that Ca2+, in turn, activated the RHD2 NADPH oxidase to produce ROS at the growing point in the root hair. Together, these components could establish a means of positive feedback regulation that maintains an active growth site in expanding root hair cells. Because the location and stability of growth sites predict the ultimate form of a plant cell, our findings demonstrate how a positive feedback mechanism involving RHD2, ROS, and Ca2+ can determine cell shape.

DOI： 10.1126/science.1152505

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

Chromatin assembly factor 1 (CAF-1) is involved in nucleo some assembly following DNA replication and nucleotide excision repair. In Arabidopsis thaliana, the three CAF-1 subunits are encoded by FAS1, FAS2 and, most likely, MSI1, respectively. In this study, we asked whether genomic stability is altered in fas1 and fas2 mutants that are lacking CAF-1 activity. Depletion of either subunit increased the frequency of somatic homologous recombination (HR) in planta similar to 40-fold. The frequency of transferred DNA (T-DNA) integration was also elevated. A delay in loading histones onto newly replicated or repaired DNA might make these DNA stretches more accessible, both to repair enzymes and to foreign DNA. Furthermore, fas mutants exhibited increased levels of DNA double-strand breaks, a G2-phase retardation that accelerates endoreduplication, and elevated levels of mRNAs coding for proteins involved in HR-all factors that could also contribute to upregulation of HR frequency in fas mutants.

DOI： 10.1038/sj.emboj.7601434

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BLACKWELL PUBLISHING  

Newly synthesized DNA is rapidly assembled into mature nucleosomes by the deposition of pre-existing and nascent histones, and some parts of this process are facilitated by chromatin assembly factor 1 (CAF-1). Loss-of-function mutants of CAF-1 in Arabidopsis, fasciata (fas), show a variety of morphological abnormalities and unique defects in gene expression in the meristems. In order to clarify the implications of CAF-1 in the maintenance of chromatin states in higher eukaryotes, we investigated transcriptional gene silencing (TGS) of various genes in fas mutants. Here, we show that TGS of endogenous CACTA transposons was released in a stochastic manner in fas. Other endogenous silent genes, a transposon AtMu1 and a hypothetical gene T5L23.26 at a heterochromatin knob, were also transcriptionally activated, and the activation of the three different silent loci at different chromosomal sites occurred non-concomitantly with each other. Furthermore, TGS of the silent beta-glucuronidase (GUS) transgene was also de-repressed randomly in fas. We conclude that CAF-1 ensures the stable inheritance of epigenetic states through growth and development in Arabidopsis.

DOI： 10.1111/j.1365-2443.2006.00928.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Plants are always exposed to the menace of oxidative stress and protect themselves by activating a variety of defense responses. However, molecular mechanisms for oxidative stress-induced gene expression are largely unknown. We here studied the roles of the oxidative stress-responsive putative voltage-dependent Ca2+ permeable channels, NtTPC1A and NtTPC1B, and cell cycle in H2O2-induced expression of antioxidant enzymes, glutathione peroxidase (GPX) and ascorbate peroxidase (APX) in tobacco BY-2 cells. H2O2-induced [Ca2+](cyt) rise and expression of GPX and APX were inhibited by the cosuppression of NtTPC1A/B as well as Al ion, a specific blocker for NtTPC1s, and enhanced by overexpression of AtTPC1, suggesting that NtTPC1s are the major Ca2+-permeable channels activated by H2O2 and that Ca2+ influx via NtTPC1s is involved in induction of H2O2-triggered gene expression. Oxidative stress-induced signal transduction mechanisms were highly dependent on the phases of the cell cycle; H2O2-induced [Ca2+](cyt) rise and expression of GPX and APX as well as the level of NtTPC1s transcripts correlated with each other and were maximal at G1 phase. In contrast, the cell cycledependence of hypoosmotic shock-induced [Ca2+](cyt) rise that is known to be independent of NtTPC Is was almost reverse and maximal at S phase. These results suggest that the cell cycle-dependent regulation of oxidative stress-induced [Ca2+](cyt) rise and expression of NtTPC1s contribute to the cell cycle dependence of H2O2-induced expression of peroxidases. Various Ca2+-mediated signal transduction pathways are differentially regulated by the cell cycle. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2005.09.004

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT  

DNA repair associated with DNA replication is important for the conservation of genomic sequence information, whereas reconstitution of chromatin after replication sustains epigenetic information. We have isolated and characterized mutations in the BRU1 gene of Arabidopsis that suggest a novel link between these underlying maintenance mechanisms. Brul plants are highly sensitive to genotoxic stress and show stochastic release of transcriptional gene silencing. They also show increased intrachromosomal homologous recombination and constitutively activated expression of poly (ADP-ribose) polymerase-2 (AtPARP-2), the induction of which is associated with elevated DNA damage. Brul mutations affect the stability of heterochromatin organization but do not interfere with genome-wide DNA methylation. BRU1 encodes a novel nuclear protein with two predicted protein-protein interaction domains. The developmental abnormalities characteristic of brul mutant plants resemble those triggered by mutations in genes encoding subunits of chromatin assembly factor (CAF-1), the condensin complex, or MRE11. Comparison of brul with these mutants indicates cooperative roles in the replication and stabilization of chromatin structure, providing a novel link between chromatin replication, epigenetic inheritance, S-phase DNA damage checkpoints, and the regulation of meristem development.

DOI： 10.1101/gad.295404

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記述言語：日本語  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：CELL PRESS  

Postembryonic development of plants depends on the activity of apical meristems established during embryogenesis. The shoot apical meristem (SAM) and the root apical meristem (RAM) have similar but distinct cellular organization. Arabidopsis FASCIATA1 (FAS1) and FAS2 genes maintain the cellular and functional organization of both SAM and RAM, and FAS gene products are subunits of the Arabidopsis counterpart of chromatin assembly factor-1 (CAF-1). fas mutants are defective in maintenance of the expression states of WUSCHEL (WUS) in SAM and SCARECROW (SCR) in RAM. We suggest that CAF-1 plays a critical role in the organization of SAM and RAM during postembryonic development by facilitating stable maintenance of gene expression states.

DOI： 10.1016/S0092-8674(01)00197-0

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：JAPANESE SOC PLANT PHYSIOLOGISTS  

We isolated a T-DNA-tagged mutant named hosoba toge toge (hot) in which a pleiotropic phenotype was observed in both the shoot and root throughout the life cycle, The phenotype and allelism indicated that the mutant has a defect in both the FASCIATA1 (FAS1) gene and the FT gene located on the bottom arm of chromosome 1, Analysis of the junctions between the T-DNA ends and the plant genome suggested the presence of a 75.8-kbp deletion at the insertion site. In addition to FAS1 and FT, 13 genes were predicted to exist in the region corresponding to that deleted in hot. They include homologs of genes for type II inositol-1,4,5-triphosphate 5-phosphatase (IP5Pase), the beta-chain of N-acetyl-beta-glucosaminidase (NAGase), NADPH oxidoreductase of the zeta-crystallin family, polygalacturonase, and endo-1,4-beta-glucanase. Although most aspects of the hot phenotype can be explained by loss of FAS1 and FT functions, some novel phenotypic features which may represent aspects of a mutant phenotype due to loss-of-function of other gene(s) were observed. One "wild-type" ecotype and a previously reported T-DNA insertion line, neither of which has any obvious phenotypic abnormality, carry a possible loss-of-function mutation in the zeta-crystallin homolog and in the NAGase beta chain homolog, respectively.

DOI： 10.1093/pcp/pcd032

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Growth of a wall-less, L-form of Escherichia coli specifically requires calcium, and in its absence, cells ceased dividing, became spherical, swelled, developed large vacuoles, and eventually lysed. The key cell division protein, FtsZ, was present in the L-form at a concentration five times less than that in the parental strain. One interpretation of these results is that the L-form possesses an enzoskeleton partly regulated by calcium.

DOI： 10.1128/JB.182.5.1419-1422.2000

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER ASSOC ADVANCEMENT SCIENCE  

Flowering in Arabidopsis is promoted via several interacting pathways. A photoperiod-dependent pathway relays signals from photoreceptors to a transcription factor gene, CONSTANS (CO), which activates downstream meristem identity genes such as LEAFY (LFY). FT, together with LFY, promotes flowering and is positively regulated by CO. Loss of FT causes delay in flowering, whereas overexpression of FT results in precocious flowering independent of CO or photoperiod. FT acts in part downstream of CO and mediates signals for flowering in an antagonistic manner with its homologous gene, TERMINAL FLOWER1 (TFL1).

DOI： 10.1126/science.286.5446.1960

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BLACKWELL SCIENCE LTD  

Conservation of the Oct motif (CGCGGATC) is a remarkable feature of plant histone gene promoters. Many of the Oct motifs are paired with a distinct motif, Hex, TCA or CCAAT-box, constituting the type I element (CCACGTCANCGATCCGCG), type II element (TCACGCGGATC) and type III element (GATCCGCG-N14-ACCAATCA). To clarify the roles of these Oct-containing composite elements (OCEs) in cell cycle-dependent and tissue-specific expression, we performed gain-of-function experiments with transgenic tobacco cell lines and plants harboring a derivative of the 35S core promoter/ beta-glucuronidase fusion gene in which three or four copies of an OCE had been placed upstream. Although their activities were slightly different, results showed that each of the three types of OCEs could confer the ability to direct S phase-specific expression on a heterologous promoter. In transgenic plants, the type I and III elements exhibited a similar activity, directing expression in meristematic tissues, whereas the activity of the type II element appeared to be restricted to young cotyledons and maturating guard cells. Mutational analyses demonstrated that the co-operation of Oct with another module (Hex, TCA or CCAAT-box) was absolutely required for both temporal and spatial regulation. Thus, OCEs play a pivotal role in regulation of the expression of plant histone genes.

DOI： 10.1046/j.1365-313X.1999.00486.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BOTANICAL SOC JAPAN  

Transition from vegetative to reproductive development (flowering) is one of the most important decisions during the post-embryonic development of flowering plants. More than twenty loci are known to regulate this process in Arabidopsis. Some of these flowering-time genes may act at the shoot apical meristem to regulate its competence to respond to floral inductive signals and floral evocation. Genetic and phenotypic analyses of mutants suggest that the late-flowering gene FT may be a good candidate for such genes. To test this, we have cloned the FT gene using a FT-deficiency line associated with a T-DNA insertion. Cloned genes and loss-of-function mutants in hand, it is now possible to analyse the role of FT and other genes in flowering at the biochemical and cellular levels as well as at the genetic level. The deduced FT protein has homology with TFL1 and CEN proteins believed to be involved in regulation of inflorescence meristem identity. Phylogenetic analysis suggests that the FT group and the TFL1/CEN group of genes diverged before the diversification of major angiosperm clades. This raises the interesting question of the evolutionary relationship between the regulation of vegetative/reproductive switching in the shoot apical meristem and the regulation of inflorescence architecture in angiosperms.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BLACKWELL SCIENCE LTD  

We have previously shown that the proximal promoter region (-185 to +57) of the wheat histone H3 gene (TH012) is sufficient for regulating S phase-specific expression of a reporter GUS gene. To define the cis-acting element(s) responsible for S phase-specific expression, GUS fusion genes under the control of wild-type or variously mutated H3 promoters were stably introduced into cultured rice Oc cells and their temporal expression was analyzed during the cell cycle by quantitative S1 analysis. The S phase-specific expression of the full-sized promoter (-1716 to +52) was significantly impaired by short internal deletions disrupting the type I element from -175 to -158 (CCACGTCACCaATCCGCG), composed of the Hex (CCACGTCA) and reverse-oriented Oct (GATCCGCG) motifs. Moreover, the H3 proximal promoters (-184 to +52) harboring base-substitution mutations in either or both of the Hex and Oct motifs could no longer activate gene expression during the S phase. These results indicate that the type I element is the first cis-acting element identified responsible for the S phase-specific expression of plant histone genes. Results also suggested the presence of a redundant cis-acting element(s) responsible for S phase-specific expression in the H3 far-upstream region (-1716 to -185).

DOI： 10.1046/j.1365-313X.1997.11061219.x

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記述言語：日本語  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）  

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© 2014 by Nova Science Publishers, Inc. All rights reserved. Although reactive oxygen species (ROS) are highly toxic substances and are produced during aerobic respiration and photosynthesis, recent studies have demonstrated that ROS, such as superoxide anion (O2-) and hydrogen peroxide (H2O2), are deliberately produced as important signaling messengers playing key roles in regulating a broad range of physiological processes including cellular growth and development as well as adaptation to environmental changes in plants. Given the toxicity of ROS, the enzymatic ROS production needs to be tightly regulated both spatially and temporally. Respiratory burst oxidase homologues (Rboh) have been identified as ROS-producing NADPH oxidases, which act as key signaling nodes integrating multiple signal transduction pathways in plants. We here discuss the interrelationship among signaling pathways involving Ca2+, protein phosphorylation and Rboh-mediated ROS production, as well as physiological roles of the signaling networks.

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記述言語：日本語   出版者・発行元：一般社団法人情報処理学会  

本論文では,プロファイルHMMに基づくたんぱく質相同性検索システムであるHMMERの最新版であるHMMER3を複数ドメインに拡張したシステムについて述べ,その性能を評価するための実験結果を示す.

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DOI： 10.14841/jspp.2008.0.0761.0

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DOI： 10.14841/jspp.2008.0.0818.0

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記述言語：日本語   出版者・発行元：人工知能学会  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：OXFORD UNIV PRESS  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：OXFORD UNIV PRESS  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：OXFORD UNIV PRESS  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：OXFORD UNIV PRESS  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPAN SOC CELL BIOLOGY  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：OXFORD UNIV PRESS  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：OXFORD UNIV PRESS  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：OXFORD UNIV PRESS  

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記述言語：英語   出版者・発行元：Japanese Society of Plant Physiologists  

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その他リンク： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=184724

記述言語：英語   出版者・発行元：Japanese Society of Plant Physiologists  

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その他リンク： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=184715

記述言語：英語   出版者・発行元：Japanese Society of Plant Physiologists  

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その他リンク： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=184278

記述言語：英語   出版者・発行元：Japanese Society of Plant Physiologists  

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その他リンク： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=184303

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