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Nozomu Koizumi

Publications and source records attributed to Nozomu Koizumi.

At least 19 recordsLinked to original sources
PubMed2006

Induction of BiP by sugar independent of a cis-element for the unfolded protein response in Arabidopsis thaliana.

BiP is a molecular chaperone induced in the unfolded protein response (UPR). In mammalian cells, BiP is induced by glucose starvation when it is called glucose-regulated protein 78 (GRP78). In Arabidopsis thaliana, however, we demonstrated that BiP transcripts decreased with sugar depletion and increased with sugar addition. Transcripts for beta-glucuronidase (GUS) driven by BiP promoter respond to tunicamycin and sugar, being similar with endogenous BiP transcripts in transgenic A. thaliana. When GUS was regulated by P-UPRE, a cis-element responsible for the UPR identified in BiP promoter, GUS transcripts were accumulated by sugar starvation. Subsequently, transgenic A. thaliana harboring luciferase (LUC) gene regulated by P-UPRE was analyzed. Sugar depletion also increased LUC activity. It is concluded that BiP is induced by sugar independent of the cis-element responsible for the UPR.

Arabidopsis↗
PubMed2005

An Arabidopsis transcription factor, AtbZIP60, regulates the endoplasmic reticulum stress response in a manner unique to plants.

Analysis of transcripts of 75 genes encoding putative basic leucine zipper (bZIP) transcription factors in the Arabidopsis genome identified AtbZIP60, which was induced by tunicamycin. AtbZIP60 encodes a predicted protein of 295 aa with a putative transmembrane domain near its C terminus after a bZIP domain. A truncated form of AtbZIP60 without a transmembrane domain (AtbZIP60 delta C) fused with GFP localized to the nucleus, suggesting translocation of native protein to the nucleus by release from the membrane. AtbZIP60 was also induced by DTT and azetidine-2-carboxylate, which induce the endoplasmic reticulum (ER) stress response (also called the unfolded protein response). Expression of AtbZIP60 delta C clearly activated any of three BiP and two calnexin promoters in a dual luciferase assay using protoplasts of cultured cells. The induction was considered to be through cis-elements plant-specific unfolded protein response element and ER stress-response element. Interestingly, AtbZIP60 delta C also appeared to induce the expression of AtbZIP60 through an ER stress-response element-like sequence in the promoter of AtbZIP60. These characteristics of AtbZIP60 imply a signal transduction pathway of the ER stress response unique to plants.

Amino Acid Sequence↗
PubMed2005

Unfolded protein response followed by induction of cell death in cultured tobacco cells treated with tunicamycin.

When correct folding of protein in the endoplasmic reticulum (ER) is prevented, cells respond to overcome the accumulation of unfolded proteins. This cellular response, which includes the induction of ER chaperones, is called an unfolded protein response (UPR). Although a link between the UPR and apoptosis has been reported in mammalian cells, little is known about this mechanism in plant cells. Asparagine (N)-linked glycosylation of proteins is critical for protein folding in the ER; and tunicamycin, a potent inhibitor of N-linked glycosylation, induces UPR. Growth arrest was observed in cultured tobacco cells treated with tunicamycin. Cell death and induction of Hsr203J, a marker for programmed cell death, were observed in the 24-h period after addition of tunicamycin, following UPR that started within 2 h. These results indicate a strong link between UPR and programmed cell death in plant cells.

Apoptosis↗
PubMed2005

Sugar responsible and tissue specific expression of a gene encoding AtCIPK14, an Arabidopsis CBL-interacting protein kinase.

Expression of AtCIPK14 (an Arabidopsis CBL-interacting protein kinase 14) was induced by metabolic sugars. Two A/T-rich sequences similar to elements involved in sugar-inducible expression of other genes were found within the -183 bp 5' region of the AtCIPK14 promoter that was responsible for the sugar induction. Histochemical analysis using a reporter gene indicated vascular-specific expression of AtCIPK14.

Arabidopsis↗
PubMed2003

Methylated DNA-binding proteins from Arabidopsis.

The 5-methylcytosines (m5C) play a critical role in epigenetic control, often being recognized by proteins containing a methyl-CpG-binding domain (MBD). Database screening has identified at least 12 putative methyl-CpG-binding proteins from Arabidopsis; we have isolated corresponding cDNAs for seven of them. Despite variation in size and amino acid sequence, all seven proteins exclusively migrate into the nucleus as revealed by green fluorescent protein fusion protein assay, suggesting a relationship with chromatin structure. However, DNA-binding assays using bacterially expressed proteins and synthetic oligonucleotides containing m5C in CpGs showed only one to specifically bind, designated AtMBD5. Further analysis showed that AtMBD5 efficiently binds to m5C in CpNpN (N is A, T, or C) but not in CpNpG sequences, both frequently found in plant DNA. The other six proteins showed either nonspecific DNA binding or no ability to recognize m5C. RNA-blot hybridization and immunoblot analysis indicated AtMBD5 to be present essentially in all tissues. Using green fluorescent protein driven by the authentic promoter, AtMBD5 was found to be actively expressed in pistils and root tips. Because m5Cs in CpG and CpNpN are considered to function in gene expression and gene silencing, respectively, the present results suggest that AtMBD5 may have distinct functions in regulation and/or self defense of genes in actively proliferating cells.

Amino Acid Sequence↗
PubMed2003

The STT3a subunit isoform of the Arabidopsis oligosaccharyltransferase controls adaptive responses to salt/osmotic stress.

Arabidopsis stt3a-1 and stt3a-2 mutations cause NaCl/osmotic sensitivity that is characterized by reduced cell division in the root meristem. Sequence comparison of the STT3a gene identified a yeast ortholog, STT3, which encodes an essential subunit of the oligosaccharyltransferase complex that is involved in protein N-glycosylation. NaCl induces the unfolded protein response in the endoplasmic reticulum (ER) and cell cycle arrest in root tip cells of stt3a seedlings, as determined by expression profiling of ER stress-responsive chaperone (BiP-GUS) and cell division (CycB1;1-GUS) genes, respectively. Together, these results indicate that plant salt stress adaptation involves ER stress signal regulation of cell cycle progression. Interestingly, a mutation (stt3b-1) in another Arabidopsis STT3 isogene (STT3b) does not cause NaCl sensitivity. However, the stt3a-1 stt3b-1 double mutation is gametophytic lethal. Apparently, STT3a and STT3b have overlapping and essential functions in plant growth and developmental processes, but the pivotal and specific protein glycosylation that is a necessary for recovery from the unfolded protein response and for cell cycle progression during salt/osmotic stress recovery is associated uniquely with the function of the STT3a isoform.

Adaptation, Physiological↗
PubMed2003

Preferential de novo methylation of cytosine residues in non-CpG sequences by a domains rearranged DNA methyltransferase from tobacco plants.

In plant DNA, cytosines in symmetric CpG and CpNpG (N is A, T, or C) are thought to be methylated by DNA methyltransferases, MET1 and CMT3, respectively. Cytosines in asymmetric CpNpN are also methylated, and genetic analysis has suggested the responsible enzyme to be domains rearranged methyltransferase (DRM). We cloned a tobacco cDNA, encoding a novel protein consisting of 608 amino acids, that resembled DRMs found in maize and Arabidopsis and designated this as NtDRM1. The protein could be shown to be localized exclusively in the nucleus and exhibit methylation activity toward unmethylated synthetic as well as native DNA samples upon expression in Sf9 insect cells. It also methylated hemimethylated DNA, but the activity was lower than that for unmethylated substrates. Methylation mapping of a 962-bp DNA, treated with NtDRM1 in vitro, directly demonstrated methylation of approximately 70% of the cytosines in methylatable CpNpN and CpNpG sequences but only 10% in CpG. Further analyses indicated that the enzyme apparently non-selectively methylates any cytosines except in CpG, regardless of the adjacent nucleotide at both 5' and 3' ends. Transcripts of NtDRM1 ubiquitously accumulated in all tissues and during the cell cycle in tobacco cultured BY2 cells. These results indicate that NtDRM1 is a de novo cytosine methyltransferase, which actively excludes CpG substrate.

Base Sequence↗
PubMed2003

The crystal structure of the novel calcium-binding protein AtCBL2 from Arabidopsis thaliana.

Arabidopsis thaliana calcineurin B-like protein (AtCBL2) is a member of a recently identified family of calcineurin B-like calcium-binding proteins in A. thaliana. The crystal structure of AtCBL2 has been determined at 2.1 A resolution. The protein forms a compact alpha-helical structure with two pairs of EF-hand motifs. The structure is similar in overall folding topology to the structures of calcineurin B and neuronal calcium sensor 1, but differs significantly in local conformation. The two calcium ions are coordinated in the first and fourth EF-hand motifs, whereas the second and third EF-hand motifs are maintained in the open form by internal hydrogen bonding without coordination of calcium ions. Both a possible site and a possible mechanism for the target binding to AtCBL2 are discussed based on the three-dimensional structure.

Amino Acid Motifs↗
PubMed2003

Crystallization and preliminary X-ray characterization of a novel calcium-binding protein AtCBL2 from Arabidopsis thaliana.

A new family of calcineurin B-like calcium-binding proteins has recently been identified in Arabidopsis thaliana. AtCBL2, a member of this family, has been crystallized in the presence of calcium ions using polyethylene glycol as a precipitant at 293 K. The crystals belong to space group C222(1), with unit-cell parameters a = 83.9, b = 118.1, c = 49.1 A. The asymmetric unit contains one molecule, with a V(M) of 2.36 A(3) Da(-1) and a solvent content of 48%. Native diffraction data to 2.1 A resolution have been collected using synchrotron radiation at SPring-8.

Arabidopsis↗
PubMed2003

Conservation between animals and plants of the cis-acting element involved in the unfolded protein response.

Using Arabidopsis thaliana, we identified the cis-element involved in the plant unfolded protein response (UPR). In transgenic plants, tunicamycin stimulated expression of a reporter gene under the control of the BiP promoter and promoter analysis identified a 24 bp sequence crucial to this induction. When fused with a minimal promoter, a hexamer of this sequence was sufficient for induction of a reporter gene in protoplasts treated with tunicamycin or dithiothreitol. Induction rate equivalent to original promoter was observed when the assay was conducted in transgenic plants. This 24 bp sequence contained two elements also responsible for the UPR in animals. Either of these elements was sufficient for the plant UPR, indicating conservation between animals and plants of cis-elements involved in the UPR.

Animals↗
PubMed2003

Transcriptional activation of phosphoenolpyruvate carboxylase by phosphorus deficiency in tobacco.

Phosphoenolpyruvate carboxylase (PEPC), which catalyses the carboxylation of phosphoenolpyruvate using HCO(3)(-) to generate oxaloacetic acid, is an important enzyme in the primary metabolism of plants. Although the PEPC genes (ppc) comprise only a small gene family, the function of each gene is not clear, except for roles in C(4) photosynthesis and CAM. Three PEPC genes (Nsppc1-3) from the C(3) plant Nicotiana sylvestris were used to investigate their roles and regulation in a C(3) plant, and their regulation by phosphorus depletion in particular. First, the induction of PEPC by phosphorus depletion was confirmed. Next, Nsppc1 was determined to be mainly responsive to phosphorus deficiency at the transcriptional level. Further studies using transgenic tobacco harbouring a chimeric gene consisting of the 2.0 kb promoter region of Nsppc1 and the beta-glucuronidase (GUS) reporter showed that PEPC is transcriptionally induced. It was also found that sucrose had a synergistic effect on the induction of PEPC by phosphorus deficiency. A series of transgenic tobacco containing 5'-deletion mutants of Nsppc1 promoter::GUS fusion revealed that the -539 to -442 bp Nsppc1 promoter region, relative to the translation start site, was necessary for the response to phosphorus deficiency. Gain-of-function analysis using a construct containing three tandem repeats of the -539 to -442 bp region confirmed that this region was sufficient to induce the phosphorus-deficiency response in tobacco.

Base Sequence↗
PubMed2003

Osmotic stress tolerance of transgenic tobacco expressing a gene encoding a membrane-located receptor-like protein from tobacco plants.

Tobacco (Nicotiana tabacum) genes regulated during the early stage of responses to wounding were screened by a modified fluorescence differential display method. Among 28 genes initially identified, a particular clone designated NtC7 was subjected to further analysis. Its transcripts were found to accumulate rapidly and transiently within 1 h upon treatments with not only wounding but also salt and osmotic stresses. However, jasmonic and abscisic acids and ethylene did not effectively induce NtC7 transcripts. Amino acid sequence analysis suggested NtC7 to be a new type of transmembrane protein that belongs to the receptor-like protein family, and a membrane location was confirmed in onion (Allium cepa) epidermis cells transiently expressing an NtC7-green fluorescent protein fusion protein. Seeds of transgenic tobacco overexpressing NtC7 normally germinated and grew in the presence of 500 mM mannitol, but not in the presence of 220 mM sodium chloride or 60 mM lithium chloride. Cuttings of mature transgenic leaf exhibited a marked tolerance upon treatment with 500 mM mannitol for 12 h, at which concentration wild-type counterparts were seriously damaged. These results suggested that NtC7 predominantly functions in maintenance of osmotic adjustment independently of ion homeostasis.

Adaptation, Physiological↗
PubMed2003

Molecular cloning and functional characterization of three distinct N-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants.

Caffeine is synthesized from xanthosine through N-methylation and ribose removal steps. In the present study, three types of cDNAs encoding N-methyltransferases were isolated from immature fruits of coffee (Coffea arabica) plants, and designated as CaXMT1, CaMXMT2, and CaDXMT1, respectively. The bacterially expressed encoded proteins were characterized for their catalytic properties. CaXMT1 catalyzed formation of 7-methylxanthosine from xanthosine with a K(m) value of 78 microM, CaMXMT2 catalyzed formation of 3,7-dimethylxanthine (theobromine) from 7-methylxanthine with a K(m) of 251 microM, and CaDXMT1 catalyzed formation of 1,3,7-trimethylxanthine (caffeine) from 3,7-dimethylxanthine with a K(m) of 1,222 microM. The crude extract of Escherichia coli was found to catalyze removal of the ribose moiety from 7-methylxanthosine, leading to the production of 7-methylxanthine. As a consequence, when all three recombinant proteins and E. coli extract were combined, xanthosine was successfully converted into caffeine in vitro. Transcripts for CaDXMT1 were predominantly found to accumulate in immature fruits, whereas those for CaXMT1 and CaMXMT2 were more broadly detected in sites encompassing the leaves, floral buds, and immature fruits. These results suggest that the presently identified three N-methyltransferases participate in caffeine biosynthesis in coffee plants and substantiate the proposed caffeine biosynthetic pathway: xanthosine --> 7-methylxanthosine --> 7-methylxanthine --> theobromine --> caffeine.

Amino Acid Sequence↗
PubMed2003

Variable interactions between sucrose non-fermented 1-related protein kinases and regulatory proteins in higher plants.

WPK4 is a sucrose non-fermented 1 (SNF1)-related wheat protein kinase, and was previously reported to interact with 14-3-3 proteins. We identified four Arabidopsis thaliana WPK4-like genes, and designated them AtWL1 through AtWL4. Yeast two-hybrid analysis, however, indicated that none of the AtWLs interacted with any of A. thaliana 14-3-3 (At14-3-3) proteins, although WPK4 itself interacted with six of them. Structurally, AtWLs were classified into a subfamiliy of AtCIPK, which generally interacts with calucineurin B-like proteins (CBL). This was also the case for AtWL1 and AtWL2, showing an efficient interaction with AtCBL2. In contrast, WPK4 interacted with none of the CBLs. In addition, to ascertain the possible interaction in vivo, expression of those genes was examined with a promoter-GUS assay. These results suggested that the interacting partner of SNF1-related protein kinases varies among plant species, and that, in the case of A. thaliana, it was CBLs, some of which were predicted to broadly regulate multiple CIPKs.

Amino Acid Sequence↗
PubMed2002

Periodic DNA methylation in maize nucleosomes and demethylation by environmental stress.

When maize seedlings were exposed to cold stress, a genome-wide demethylation occurred in root tissues. Screening of genomic DNA identified one particular fragment that was demethylated during chilling. This 1.8-kb fragment, designated ZmMI1, contained part of the coding region of a putative protein and part of a retrotransposon-like sequence. ZmMI1 was transcribed only under cold stress. Direct methylation mapping revealed that hypomethylated regions spanning 150 bases alternated with hypermethylated regions spanning 50 bases. Analysis of nuclear DNA digested with micrococcal nuclease indicated that these regions corresponded to nucleosome cores and linkers, respectively. Cold stress induced severe demethylation in core regions but left linker regions relatively intact. Thus, methylation and demethylation were periodic in nucleosomes. The following biological significance is conceivable. First, because DNA methylation in nucleosomes induces alteration of gene expression by changing chromatin structures, vast demethylation may serve as a common switch for many genes that are simultaneously controlled upon environmental cues. Second, because artificial demethylation induces heritable changes in plant phenotype (Sano, H., Kamada, I., Youssefian, S., Katsumi, M., and Wabilko, H. (1990) Mol. Gen. Genet. 220, 441-447), altered DNA methylation may result in epigenetic inheritance, in which gene expression is modified without changing the nucleotide sequence.

Acclimatization↗
PubMed2002

A chloroplast-resident DNA methyltransferase is responsible for hypermethylation of chloroplast genes in Chlamydomonas maternal gametes.

Chloroplast DNA of the green alga Chlamydomonas reinhardtii is maternally inherited. Methylation mapping directly revealed that, before mating, chloroplast DNA of maternal (mating type plus; mt(+)) gametes is heavily methylated whereas that of paternal (mating type minus; mt(-)) gametes is not. Indirect immunofluorescence analyses with anti-5-methylcytosine mAbs visually showed methylation to occur exclusively in chloroplast DNA of mt(+) gametes, and not in mt(-) gametes or nuclear DNA of either mt. To clarify the relationship between methylation and maternal inheritance of chloroplast DNA, we have isolated and characterized a cDNA encoding a DNA methyltransferase. The deduced protein, CrMET1, consists of 1,344 aa and contains a conserved catalytic domain at the C terminal and a nonconserved N-terminal region. The predicted N-terminal region has an arginine-rich domain, suggesting CrMET1 is transferred to chloroplasts. This finding could be directly shown by green fluorescent protein epifluorescence microscopy analyses. CrMET1 transcripts were found to be absent in both mt(+) and mt(-) vegetative cells. Upon gametogenesis, however, transcript levels clearly increased in mt(+) but not mt(-) cells. These experiments suggest that the CrMET1 protein is located in chloroplasts and that it specifically methylates cytosine residues of chloroplast DNA in mt(+) gametes. This conclusion was further strengthened by the observation that, during gametogenesis, CrMET1 is expressed in a mt(-) mutant, mat-1, whose chloroplast DNA is heavily methylated in gametes and paternally inherited. The results provide evidence that cytosine methylation plays a critical role in maternal inheritance of chloroplast genes in C. reinhardtii.

Animals↗
PubMed2002

Functional characterization of a heavy metal binding protein CdI19 from Arabidopsis.

Heavy metals are potentially highly toxic for organisms. Plants possess the ability to minimize damage but the underlying molecular mechanisms have yet to be detailed. Screening Cd-responsive genes in Arabidopsis, we previously identified a gene encoding a putative metal binding protein CdI19, which, upon introduction into yeast cells, conferred marked toleration of Cd exposure. Here we describe that bacterially expressed CdI19 directly interacts with Cd at its CXXC motif, as revealed by circular dichroism analysis, and that it is exclusively localized at plasma membranes, as revealed by heterologous expression of fusion product with a green fluorescent protein in BY2 cells. Northern blot analyses indicated that CdI19 transcripts were induced not only by Cd, but also by dicationic forms of Hg, Fe and Cu. Histochemical assays using transgenic Arabidopsis expressing the CdI19 promoter::GUS showed CdI19 to be expressed in petiole, hypocotyl, peduncle and vascular bundles in root tissues. Overexpression of the CdI19 cDNA conferred Cd tolerance in transgenic Arabidopsis. These results suggest that CdI19 plays an important role in the maintenance of heavy metal homeostasis and/or in detoxification by endowing plasma membranes with the capacity to serve as an initial barrier against inflow of free heavy metal ions into cells.

Adaptation, Physiological↗