Norihiro Ohtsubo

National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan

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Publications (24)51.86 Total impact

  • Ichiro Kasajima, Norihiro Ohtsubo, Katsutomo Sasaki
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    ABSTRACT: Purification of plant DNA involves lengthy ultracentrifugation using ethidium bromide. Here, ultracentrifugation method is improved by staining with GelRed. The resulting method is faster, safer and of higher sensitivity. Purified DNA quality was confirmed by treatment with restriction enzymes and isolation of gene promoters. New type of long adaptor with mismatch sequence was also developed for promoter isolation.
    Bioscience Biotechnology and Biochemistry 08/2014; 78(11):1-4. DOI:10.1080/09168451.2014.940831 · 1.21 Impact Factor
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    ABSTRACT: The class B genes DEFICIENS (DEF)/APETALA3 (AP3) and GLOBOSA (GLO)/PISTILLATA (PI), encoding MADS-box transcription factors, and their functions in petal and stamen development have been intensely studied in Arabidopsis and Antirrhinum. However, the functions of class B genes in other plants, including ornamental species exhibiting floral morphology different from these model plants, have not received nearly as much attention. Here, we examine the cooperative functions of TfDEF and TfGLO on floral organ development in the ornamental plant torenia (Torenia fournieri Lind.). Torenia plants co-overexpressing TfDEF and TfGLO showed a morphological alteration of sepals to petaloid organs. Phenotypically, these petaloid sepals were nearly identical to petals but had no stamens or yellow patches like those of wild-type petals. Furthermore, the inflorescence architecture in the co-overexpressing torenias showed a characteristic change in which, unlike the wild-types, their flowers developed without peduncles. Evaluation of the petaloid sepals showed that these attained a petal-like nature in terms of floral organ phenotype, cell shape, pigment composition, and the expression patterns of anthocyanin biosynthesis-related genes. In contrast, torenias in which TfDEF and TfGLO were co-suppressed exhibited sepaloid petals in the second whorl. The sepaloid petals also attained a sepal-like nature, in the same way as the petaloid sepals. The results clearly demonstrate that TfDEF and TfGLO play important cooperative roles in petal development in torenia. Furthermore, the unique transgenic phenotypes produced create a valuable new way through which characteristics of petal development and inflorescence architecture can be investigated in torenia.
    Plant Molecular Biology 08/2014; DOI:10.1007/s11103-014-0231-8 · 4.07 Impact Factor
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    ABSTRACT: Florescent proteins have been popularly used for studying genes and proteins of interest in various experiments at a cellular level, such as the analysis of intracellular localization and protein-protein interaction. However, the strength of fluorescence was insufficient for macro level observations of tissues or of the whole plant, and the fluorescent flowers that have been generated so far needed high-sensitive imaging equipment for the observation. Here we generated fluorescent Torenia flowers by the combined use of a high-performance fluorescent protein and the latest protein expression technologies, leading to the production of fluorescent proteins that can be easily and clearly observed. A coding sequence of a yellowish green fluorescent protein from the marine plankton Chiridius poppei (CpYGFP) was fused to the optimized sequences of the heat shock protein terminator and the 5′-untranslated region of the alcohol dehydrogenase gene of Arabidopsis to gain massive accumulation of the fluorescent protein. Strong fluorescence of CpYGFP was apparent in every part of the transgenic plant under the simple combination of a blue LED for excitation and an orange colored transparent acrylic filter for emission, while faint autofluorescence remained in the wild-type plants. By evaluating the combination of excitation wavelengths (excitation and emission filters) we were able to eliminate this undesired fluorescence. The fluorescent flowers could be used for ornamental purposes as well as for the analysis of fluorescent transgenic plants spatiotemporally in a nondestructive manner.
    Plant Biotechnology 01/2014; 31(4):309-318. DOI:10.5511/plantbiotechnology.14.0907a · 1.06 Impact Factor
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    ABSTRACT: We present a set of DNA extraction/purification/quantification procedures for preparation of large-amount and pure DNA samples from mature leaves of cyclamen and tree species, from which high quality and quantity of DNA would not be extractable with any other protocols. Cyclamen is a popular flowering plant throughout the temperate world. Only minute amounts or non-detectable levels of cyclamen DNA can be extracted from its leaves by standard methods such as the CTAB method. We have developed a new DNA extraction buffer which we call the ‘PVPP buffer’. This buffer can extract ∼20 μg of DNA from 1 g fresh weight of mature cyclamen leaves. Following extraction, DNA was successfully purified by CsCl ultracentrifugation, but purification was not successful with CTAB precipitation. Qualitative and quantitative analyses of the DNA extract were performed by gel electrophoresis, not by the popular UV absorbance-based analysis, which seems to overestimate DNA quality and quantity. This fact suggests the possibility that quality and quantity of DNA extracts were not necessarily enough in the former reports. Pure DNA in large amounts was also extractable from mature leaves of all species tested (camellia, chrysanthemum, orchid, pine, rose and tea), which include ‘recalcitrant’ ones, using the PVPP buffer and CsCl purification. Extraction using PVPP buffer does not require special equipment and CsCl purification requires access to an ultracentrifuge.
    Scientia Horticulturae 12/2013; 164:65–72. DOI:10.1016/j.scienta.2013.09.011 · 1.50 Impact Factor
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    ABSTRACT: Cyclamen persicum (cyclamen) is a commercially valuable, winter-blooming perennial plant. We cloned two cyclamen orthologues of AGAMOUS (AG), CpAG1 and CpAG2, which are mainly expressed in the stamen and carpel, respectively. Cyclamen flowers have 5 petals, but expression of a chimeric repressor of CpAG1 (CpAG1-SRDX) caused stamens to convert into petals, resulting in a flower with 10 petals. By contrast, CpAG2-SRDX only caused incomplete formation of stamens and carpels. Expression in Arabidopsis thaliana showed similar effects on flower organ specification. Simultaneous expression of CpAG1-SRDX and CpAG2-SRDX in cyclamen induced rose-like, multi-petal flowers, a potentially valuable trait in commercial ornamental varieties. Expression of CpAG2-SRDX in a cyclamen mutant lacking expression of CpAG1 more effectively produced multi-petal flowers. Here, we controlled the number of petals in cyclamen by simple genetic engineering with a chimeric repressor. This strategy may be applicable useful for other ornamental plants with two distinct AG orthologues.
    Scientific Reports 09/2013; 3:2641. DOI:10.1038/srep02641 · 5.58 Impact Factor
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    ABSTRACT: The waxy plant cuticle protects cells from dehydration, repels pathogen attack, and prevents organ fusion during development. The transcription factor WAX INDUCER1/SHINE1 (WIN1/SHN1) regulates the biosynthesis of waxy substances in Arabidopsis thaliana. Here, we show that the MIXTA-like MYB transcription factors MYB106 and MYB16, which regulate epidermal cell morphology, also regulate cuticle development coordinately with WIN1/SHN1 in Arabidopsis and Torenia fournieri. Expression of a MYB106 chimeric repressor fusion (35S:MYB106-SRDX) and knockout/down of MYB106 and MYB16 induced cuticle deficiencies characterized by organ adhesion and reduction of epicuticular wax crystals and cutin nanoridges. A similar organ fusion phenotype was produced by expression of a WIN1/SHN1 chimeric repressor. Conversely, the dominant active form of MYB106 (35S:MYB106-VP16) induced ectopic production of cutin nanoridges and increased expression of WIN1/SHN1 and wax biosynthetic genes. Microarray experiments revealed that MYB106 and WIN1/SHN1 regulate similar sets of genes, predominantly those involved in wax and cutin biosynthesis. Furthermore, WIN1/SHN1 expression was induced by MYB106-VP16 and repressed by MYB106-SRDX. These results indicate that the regulatory cascade of MIXTA-like proteins and WIN1/SHN1 coordinately regulate cutin biosynthesis and wax accumulation. This study reveals an additional key aspect of MIXTA-like protein function and suggests a unique relationship between cuticle development and epidermal cell differentiation.
    The Plant Cell 05/2013; 25(5). DOI:10.1105/tpc.113.110783 · 9.58 Impact Factor
  • Journal- Japanese Society for Horticultural Science 01/2013; 82(1):39-50. DOI:10.2503/jjshs1.82.39 · 0.82 Impact Factor
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    ABSTRACT: We identified a Torenia fournieri Lind. mutant (no. 252) that exhibited a sepaloid phenotype in which the second whorls were changed to sepal-like organs. This mutant had no stamens, and the floral organs consisted of sepals and carpels. Although the expression of a torenia class B MADS-box gene, GLOBOSA (TfGLO), was abolished in the 252 mutant, no mutation of TfGLO was found. Among torenia homologs such as APETALA1 (AP1), LEAFY (LFY), and UNUSUAL FLORAL ORGANS (UFO), which regulate expression of class B genes in Arabidopsis, only accumulation of the TfUFO transcript was diminished in the 252 mutant. Furthermore, a missense mutation was found in the coding region of the mutant TfUFO. Intact TfUFO complemented the mutant phenotype whereas mutated TfUFO did not; in addition, the transgenic phenotype of TfUFO-knockdown torenias coincided with the mutant phenotype. Yeast two-hybrid analysis revealed that the mutated TfUFO lost its ability to interact with TfLFY protein. In situ hybridization analysis indicated that the transcripts of TfUFO and TfLFY were partially accumulated in the same region. These results clearly demonstrate that the defect in TfUFO caused the sepaloid phenotype in the 252 mutant due to the loss of interaction with TfLFY.
    The Plant Journal 05/2012; 71(6):1002-14. DOI:10.1111/j.1365-313X.2012.05047.x · 6.82 Impact Factor
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    ABSTRACT: miR156/157 is a small RNA molecule that is highly conserved among various plant species. Overexpression of miR156/157 has been reported to induce bushy architecture and delayed phase transition in several plant species. To investigate the effect of miR157 overexpression in a horticultural plant, and to explore the applicability of miRNA to molecular breeding, we introduced Arabidopsis MIR157b (AtMIR157b) into torenia (Torenia fournieri). The resulting 35S:AtMIR157b plants showed a high degree of branching along with small leaves, which resembled miR156/157-overexpressing plants of other species. We also isolated torenia SBP-box genes with target miR156/157 sequences and confirmed that their expression was selectively downregulated in 35S:AtMIR157b plants. The reduced accumulation of mRNA was probably due to sequence specificity. Moreover, expression of torenia homologs of the SBP-box protein-regulated genes TfLFY and TfMIR172 was also reduced by AtmiR157 overexpression. These findings suggest that the molecular mechanisms of miR156/157 regulation are conserved between Arabidopsis and torenia. The bushy architecture and small leaves of 35S:AtMIR157b torenia plants could be applied in molecular breeding of various horticultural plants as well as for increasing biomass and crop production.
    Planta 05/2012; 236(4):1027-35. DOI:10.1007/s00425-012-1649-3 · 3.38 Impact Factor
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    ABSTRACT: While heavy-ion beam irradiation is becoming popular technology for mutation breeding in Japan, the combination with genetic manipulation makes it more convenient to create greater variation in plant phenotypes. We have succeeded in producing over 200 varieties of transgenic torenia (Torenia fournieri Lind.) from over 2,400 regenerated plants by this procedure in only 2 years. Mutant phenotypes were observed mainly in flowers and showed wide variation in colour and shape. Higher mutation rates in the transgenics compared to those in wild type indicate the synergistic effect of genetic manipulation and heavy-ion beam irradiation, which might be advantageous to create greater variation in floral traits.
    Methods in molecular biology (Clifton, N.J.) 01/2012; 847:275-89. DOI:10.1007/978-1-61779-558-9_24 · 1.29 Impact Factor
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    ABSTRACT: To establish an efficient way to create novel floral traits in horticultural flowers, we have introduced many chimeric repressors of Arabidopsis transcription factors into torenia. Among them, we found a transgenic torenia exhibiting unopened flower buds and glossy dark green leaves with curled margins as a consequence of overexpression of Arabidopsis MYB24 with a transcriptional repression domain (MYB24-SRDX). Petals inside the flower buds exhibited a distinct coloration pattern. To bring out this favorable petal trait without inducing the unfavorable phenotypes due to the constitutive expression of chimeric repressors by the cauliflower mosaic virus 35S (35S) promoter, we tested the ability of a floral organs-pecific Arabidopsis APETALA1 (AP1) promoter, which was found to be active in both petals and flower buds of torenia. As expected, AP1 pro:MYB24-SRDX transgenic torenias resulted in the opening of flowers and a normal leaf phenotype. Furthermore, these AP1 pro:MYB24-SRDX torenias exhibited wavy petals with a characteristic configuration. This is a good example of the utilization of a floral organ-specific promoter for creating distinct flower phenotypes without causing unfavorable morphological and physiological changes in other organs.
    Plant Biotechnology 03/2011; 28(2):181-188. DOI:10.5511/plantbiotechnology.11.0124b · 1.06 Impact Factor
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    ABSTRACT: Chimeric repressor gene-silencing technology is a useful tool for changing morphology of ornamental plants. It has previously been demonstrated that the chimeric repressor TCP3SRDX, which consists of Arabidopsis TCP3 and an ERF-associated amphiphilic repression motif repression domain, perturbs the marginal morphology of Arabidopsis leaves and flowers. To obtain new rose cultivars that have ornamental values, we attempted to alter the morphology of Rosaxhybrida cv. Lavande with TCP3SRDX. The TCP3SRDX transgenic rose plants showed interesting phenotypes: the number of leaflets and the size of leaf teeth increased, the petals were wavy, and the sepals were compound-leafy. We succeeded in altering rose morphology using Arabidopsis TCP3 without the sequence information of a TCP3 homologue in the target plant species.
    Plant Biotechnology 03/2011; 28(2):149-152. DOI:10.5511/plantbiotechnology.10.1214a · 1.06 Impact Factor
  • Plant Biotechnology 03/2011; 28(2):123-130. · 1.06 Impact Factor
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    ABSTRACT: While torenia (Torenia fournieri Lind.) is a useful model flower for molecular biological studies of floral architecture, the maintenance of plant materials and resulting transgenic plants requires vegetative propagation due to its heterozygous nature. Reduction of labor and costs for maintaining thousands of in vitro torenia cultures is therefore a critical issue. We found that substituting trehalose for sucrose drastically extended the culture period to 70 days, which is more than twice as long as for the common, sucrose-based medium, without reduction in plant viability. Comparative measurement of the plant mass indicated that the increased survival benefit of the trehalose-based medium might be on account of improvement in the rhizosphere environment through reduction of root density in the culture, rather than by reduced plant growth. No harmful effects arising from the trehalose-based medium were observed in 1,800 laboratory lines during the bimonthly subculture for over 12 months, except for a wilting on the first transfer to the trehalose-based medium. In conjunction with the use of the commercial food additive, Okome-ni-TREHA (R) rather than reagent-grade trehalose, we have succeeded in reducing the costs and labor associated with the culture medium to less than one third of those for the sucrose-based system.
    Plant Biotechnology 01/2011; 28(2):263-266. DOI:10.5511/plantbiotechnology.11.0124c · 1.06 Impact Factor
  • Norihiro Ohtsubo
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    ABSTRACT: There is a significant difference between cereal crops and floricultural crops in the concept of genetic modification. This is due to the difference in requirements for these agricultural products; wide variation of flower color, shape and fragrance for floricultural plants in contrast to productivity improvements or fine-tuning of physiological traits for crop plants. Bringing GM flowers to the market against their short product life involves the following factors: (1) a high-efficiency production and screening system of elite lines, (2) reliable methods to minimize biodiversity impact and evaluation of their efficacy, and (3) social receptivity based on intensive education and information sharing. We have developed an efficient system by merging the genetic information resources of the Arabidopsis genome and a transcription factor-based gene silencing system called CRES-T through the Flower CRES-T Project. In this project, we have demonstrated the applicability and general versatility of CRES-T in various plant species through experiments on eight different flower species with over 100 transcription factors. In addition to providing phenotypic information via the original database, we are attempting to improve public knowledge, for example, through the production of resin-embedded specimens of GM-flowers and reviving lost garden varieties of morning glory and using them in educational programs, to gain public acceptance of GM plants. Multi-petal cyclamens with complete sterility, which have been produced by suppressing a pair of floral-organ identity genes, will be released in the near future as the third GM commodity following Suntory's blue carnations and rose. This cyclamen represents the best example of a GM product with a low diversity impact ever produced.
    Plant Biotechnology 01/2011; 28(2):113-121. DOI:10.5511/plantbiotechnology.11.0120a · 1.06 Impact Factor
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    ABSTRACT: Molecular breeding with genetic modification enables the production of novel floral traits in floricultural plants that could not be obtained by traditional breeding. To facilitate novel flower production, we collectively introduced 2 sets of 42 and 50 chimeric repressors of Arabidopsis transcription factors into Agrobacterium and then used these to co-transform torenia (Torenia fournieri). We generated 750 transgenic torenias, and identification of the transgenes revealed that more than 80% of the transgenic torenias had a single transgene. A total of 264 plants showed phenotypic modification, and 91.2% displayed modified flower colors and/or shapes, such as altered color patterns, curled petal margins, and wavy petals. These results indicated that the collective transformation system can be applied to molecular breeding of flowers. Detailed analysis of the phenotypes revealed that PETAL LOSS could control blotch sizes and that modification of cell shape could change the texture of petals. We found that the chimeric repressors of functionally unknown transcription factors also induced novel floral traits, and therefore, the transgenic torenias provide an understanding of the functions of transcription factors that could not be revealed by previous studies in Arabidopsis.
    Plant Biotechnology 01/2011; 28(2):189-199. DOI:10.5511/plantbiotechnology.10.1216a · 1.06 Impact Factor
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    ABSTRACT: Chimeric REpressor gene-Silencing Technology (CRES-T) is a powerful gene-silencing tool to analyze the function of Arabidopsis transcription factors. To investigate whether CRES-T is also applicable to horticultural plants inadequate for genetic engineering because of their limited molecular biological characterization and polyploidy, we applied CRES-T to torenia and the hexaploid chrysanthemum and produced their transgenic plants expressing the chimeric repressor derived from the Arabidopsis TEOSINTE BRANCHED1, CYCLOIDEA, and PCF family transcription factor 3 (TCP3) fused with a plant-specific transcriptional repression domain named SRDX, consisting of 12 amino acids originated from the EAR-motif (TCP3-SRDX). Transgenic torenia and chrysanthemum expressing TCP3-SRDX exhibited fringed leaves and short pistils, while those expressing TCP3 fused with either the mutated repression domain (TCP3-mSRDX) or the overexpressor of TCP3 (TCP3-ox) did not exhibit phenotypic changes. In addition to fringed leaves, TCP3-SRDX transgenic torenia plants exhibited petals with fringed margins, distinctive color patterns, and reduced anthocyanin accumulation. In TCP3-SRDX transgenic chrysanthemum plants, floral organ development was suppressed as compared with the wild type. These results indicate that the Arabidopsis-derived TCP3-SRDX induced morphological changes in transgenic torenia and chrysanthemum although the observed phenotypes partially differ from each other. CRES-T may function in various plant species including polyploid species and modify their biological characteristics.
    Plant Biotechnology 01/2011; 28(2):131-140. DOI:10.5511/plantbiotechnology.11.0121a · 1.06 Impact Factor
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    ABSTRACT: FioreDB (http://www.cres-t.org/fiore/public_db/) is a database of phenotypes induced by Chimeric REpressor gene-Silencing Technology (CRES-T) in ornamental and model plants. CRES-T induces a loss-of-function phenotype of a transcription factor (TF) gene by expression of a chimeric repressor produced by fusion of a TF to the strong transcriptional repression domain (SRDX). The earlier version of FioreDB provided phenotypic information induced by many kinds of chimeric repressors in various plants including torenia, chrysanthemum, cyclamen, gentian, morning glory, lisianthus and Arabidopsis. Phenotypic information, however, was not linked with gene information. We report here the development of the new FioreDB that provides more than 300 phenotypic information of various plants, linked to more than 100 TFs. FioreDB also provides information about classification of TFs, putative repression motifs found in TFs and other proteins, and incorporates publicly available gene information such as sequences and microarray data for all Arabidopsis genes. The new FioreDB described here, will be a valuable resource for basic research of TFs and for the manipulation of traits of agronomically important plants by CRES-T, especially from the point of view of horticulture.
    Plant Biotechnology 01/2011; 28(2):123-130. DOI:10.5511/plantbiotechnology.11.0106a · 1.06 Impact Factor
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    ABSTRACT: Homeotic class B genes GLOBOSA (GLO)/PISTILLATA (PI) and DEFICIENS (DEF)/APETALA3 (AP3) are involved in the development of petals and stamens in Arabidopsis. However, functions of these genes in the development of floral organs in torenia are less well known. Here, we demonstrate the unique floral phenotypes of transgenic torenia formed due to the modification of class B genes, TfGLO and TfDEF. TfGLO-overexpressing plants showed purple-stained sepals that accumulated anthocyanins in a manner similar to that of petals. TfGLO-suppressed plants showed serrated petals and TfDEF-suppressed plants showed partially decolorized petals. In TfGLO-overexpressing plants, cell shapes on the surfaces of sepals were altered to petal-like cell shapes. Furthermore, TfGLO- and TfDEF-suppressed plants partially had sepal-like cells on the surfaces of their petals. We isolated putative class B gene-regulated genes and examined their expression in transgenic plants. Three xyloglucan endo-1,4-beta-d-glucanase genes were up-regulated in TfGLO- and TfDEF-overexpressing plants and down-regulated in TfGLO- and TfDEF-suppressed plants. In addition, 10 anthocyanin biosynthesis-related genes, including anthocyanin synthase and chalcone isomerase, were up-regulated in TfGLO-overexpressing plants and down-regulated in TfGLO-suppressed plants. The expression patterns of these 10 genes in TfDEF transgenic plants were diverse and classified into several groups. HPLC analysis indicated that sepals of TfGLO-overexpressing plants accumulate the same type of anthocyanins and flavones as wild-type plants. The difference in phenotypes and expression patterns of the 10 anthocyanin biosynthesis-related genes between TfGLO and TfDEF transgenic plants indicated that TfGLO and TfDEF have partial functional divergence, while they basically work synergistically in torenia. Electronic supplementary material The online version of this article (doi:10.1007/s00438-010-0574-z) contains supplementary material, which is available to authorized users.
    MGG Molecular & General Genetics 11/2010; 284(5):399-414. DOI:10.1007/s00438-010-0574-z · 2.83 Impact Factor
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    Plant Biotechnology 03/2008; 25:45-53. DOI:10.5511/plantbiotechnology.25.45 · 1.06 Impact Factor