Researcher Profile and Settings

Affiliation

• Field Science Center for Northern Biosphere

Job Title

• Specially Appointed Professor

Degree

• (BLANK)

J-Global ID

• 200901090873106422

Research Interests

• 作物生理学   Crop Physiology   

Research Areas

• Environmental science/Agricultural science / Landscape science
• Environmental science/Agricultural science / Environmental agriculture

Educational Organization

•  Bachelor's degree program　School of Agriculture
•  Master's degree program　Graduate School of Agriculture
•  Doctoral (PhD) degree program　Graduate School of Agriculture

Association Memberships

• 作物学会   植物生理学会   植物学会   

Research Activities

Published Papers

• Metagenomic Analyses of Viruses in the Orchid Mycorrhizal Interaction Using Improved Assemble Tools

  Kota Kambara, Hanako Shimura, Kaien Fujino, Chikara Masuta

  Methods in Molecular Biology 67 - 81 1064-3745 2023/12/08
• Construction of a de novo assembly pipeline using multiple transcriptome data sets from Cypripedium macranthos (Orchidaceae)

  Kota Kambara, Kaien Fujino, Hanako Shimura

  PLOS ONE 2023/06/06 [Refereed]
  
• Biological Activity of Anti-bolting Compound, alpha-(7Z,10Z,13Z)-hexadeca-7,10,13-trienoic Acid Monoglyceride to Reduce the Endogenous Amount of Gibberellins

  Tsuyoshi Ogihara, Shunpei Shikama, Akihisa Ishii, Syotaro Hirota, Junichi Kashiwagi, Kaien Fujino, Yuki Mitsui, Takafumi Shimizu, Mitsunori Seo, Naoki Kitaoka, Yasunori Koda, Hideyuki Matsuura

  JOURNAL OF PLANT GROWTH REGULATION 0721-7595 2023/04 [Refereed]
   

  A monoacylglycerol, alpha-(7Z,10Z,13Z)-hexadeca-7,10,13-trienoic acid monoglyceride, extracted from leaves of Japanese radish (Raphanus sativus) has been reported as an anti-bolting compound (ABC); however, the mechanism how ABC inhibits the plant bolting has been remained to be elucidated. In this paper, it was found that exogenous applications of ABC led Arabidopsis thaliana seedlings decrease transcriptional levels of AtKO and increase those of AtGA2ox to reduce endogenous levels of gibberellins (GAs) to retard A. thaliana growth, whose physiological phenomena were counteracted by exogenous applications of ent-kaurenoic acid and GA(3), respectively. Furthermore, alpha-oleanolic acid monoglyceride having both activities of supressing KO and enhancing GA2ox inductions retarded A. thaliana growth post germination, although alpha-palmitic acid monoglyceride having only the effect of supressing KO induction was not enough to suppress the growth. These experimental data supported the idea that dual function was essential for ABC to show biological activity.
• Functional characterization and vacuolar localization of fructan exohydrolase derived from onion (Allium cepa)

  Satoshi Oku, Keiji Ueno, Yukiko Sawazaki, Tomoo Maeda, Yutaka Jitsuyama, Takashi Suzuki, Shuichi Onodera, Kaien Fujino, Hanako Shimura

  Journal of Experimental Botany 73 (14) 4908 - 4922 0022-0957 2022/08/11 [Refereed]
   

  Abstract Fructans such as inulin and levan accumulate in certain taxonomic groups of plants and are a reserve carbohydrate alternative to starch. Onion (Allium cepa L.) is a typical plant species that accumulates fructans, and it synthesizes inulin-type and inulin neoseries-type fructans in the bulb. Although genes for fructan biosynthesis in onion have been identified so far, no genes for fructan degradation had been found. In this study, phylogenetic analysis predicted that we isolated a putative vacuolar invertase gene (AcpVI1), but our functional analyses demonstrated that it encoded a fructan 1-exohydrolase (1-FEH) instead. Assessments of recombinant proteins and purified native protein showed that the protein had 1-FEH activity, hydrolyzing the β-(2,1)-fructosyl linkage in inulin-type fructans. Interestingly, AcpVI1 had an amino acid sequence close to those of vacuolar invertases and fructosyltransferases, unlike all other FEHs previously found in plants. We showed that AcpVI1 was localized in the vacuole, as are onion fructosyltransferases Ac1-SST and Ac6G-FFT. These results indicate that fructan-synthesizing and -degrading enzymes are both localized in the vacuole. In contrast to previously reported FEHs, our data suggest that onion 1-FEH evolved from a vacuolar invertase and not from a cell wall invertase. This demonstrates that classic phylogenetic analysis on its own is insufficient to discriminate between invertases and FEHs, highlighting the importance of functional markers in the nearby active site residues.
• The plant nuclear lamina proteins NMCP1 and NMCP2 form a filamentous network with lateral filament associations.

  Kiyoshi Masuda, Riku Hikida, Kaien Fujino

  Journal of experimental botany 72 (18) 6190 - 6204 2021/09/30 [Refereed]
   

  Plant genomes lack genes encoding intermediate filament proteins, including lamins; however, functional lamin analogues are presumed to exist in plants. Plant-specific coiled-coil proteins, that is, nuclear matrix constituent proteins (NMCPs), are the most likely candidates as the structural elements of the nuclear lamina because they exhibit a lamin-like domain arrangement. They are exclusively localized at the nuclear periphery and have functions that are analogous to those of lamins. However, their assembly into filamentous polymers has not yet been confirmed. In this study, we examined the higher-order structure of NMCP1 and NMCP2 in Apium graveolens cells by using stimulated emission depletion microscopy combined with immunofluorescence cell labelling. Our analyses revealed that NMCP1 and NMCP2 form intricate filamentous networks, which include thick segments consisting of filament bundles, forming a dense filamentous layer extending across the nuclear periphery. Furthermore, the outermost chromatin distribution was found to be in the nucleoplasm-facing region of the nuclear lamina. Recombinant Daucus carota NMCP1 with a His-tag produced in Escherichia coli refolded into dimers and self-assembled into filaments and filament bundles. These results suggest that NMCP1 and NMCP2 organize into the nuclear lamina by forming a filamentous network with filament bundles that localize at the nuclear periphery.
• Revision of the relationship between anther morphology and pollen sterility by cold stress at the booting stage in rice.

  Koichi Yamamori, Kei Ogasawara, Seiya Ishiguro, Yohei Koide, Itsuro Takamure, Kaien Fujino, Yutaka Sato, Yuji Kishima

  Annals of botany 128 (5) 559 - 575 2021/09/07 [Refereed]
   

  BACKGROUND AND AIMS: Cold stress in rice (Oryza sativa) plants at the reproductive stage prevents normal anther development and causes pollen sterility. Tapetum hypertrophy in anthers has been associated with pollen sterility in response to cold at the booting stage. Here, we re-examined whether the relationships between anther abnormality and pollen sterility caused by cold stress at the booting stage in rice can be explained by a monovalent factor such as tapetum hypertrophy. METHODS: After exposing plants to a 4-d cold treatment at the booting stage, we collected and processed anthers for transverse sectioning immediately and at the flowering stage. We anatomically evaluated the effect of cold treatment on anther internal morphologies, pollen fertilities and pollen numbers in the 13 cultivars with various cold sensitivities. KEY RESULTS: We observed four types of morphological anther abnormalities at each stage. Pollen sterility was positively correlated with the frequency of undeveloped locules, but not with tapetum hypertrophy as commonly believed. In cold-sensitive cultivars grown at low temperatures, pollen sterility was more frequent than anther morphological abnormalities, and some lines showed remarkably high pollen sterility without any anther morphological alterations. Most morphological anomalies occurred only in specific areas within large and small locules. Anther length tended to shorten in response to cold treatment and was positively correlated with pollen number. One cultivar showed a considerably reduced pollen number, but fertile pollen grains under cold stress. We propose three possible relationships to explain anther structure and pollen sterility and reduction due to cold stress. CONCLUSIONS: The pollen sterility caused by cold stress at the booting stage was correlated with the frequency of entire locule-related abnormalities, which might represent a phenotypic consequence, but not a direct cause of pollen abortion. Multivalent factors might underlie the complicated relationships between anther abnormality and pollen sterility in rice.
• NDR/LATS-family protein kinase genes are indispensable for embryogenesis in Arabidopsis.

  Hyuk Sung Yoon, Kaien Fujino, Shenkui Liu, Tetsuo Takano, Daisuke Tsugama

  FEBS open bio 11 (9) 2600 - 2606 2021/09 [Refereed]
   

  NDR/LATS-family protein kinases are conserved among eukaryotes. These protein kinases in yeast and animals phosphorylate specific targets and regulate the cell cycle. Arabidopsis thaliana has eight NDR/LATS-family protein kinase genes (NDR1-8), of which NDR2, NDR4, and NDR5 are involved in regulating pollen development. However, the functions of the other NDR/LATS-family protein kinase genes in plants are unclear. Here, we show that three putative phosphorylation sites of an Arabidopsis basic leucine zipper transcription factor, VIP1, correspond to NDR/LATS-family protein kinase phosphorylation motifs and that two of these three sites are phosphorylated by NDR2, NDR3, or NDR8 in vitro. Expression of NDR1-8 was detected in various tissues. An NDR4 NDR6 NDR7 NDR8 quadruple mutation caused embryonic lethality These results suggest that different NDR/LATS-family protein kinases in plants have distinct physiological roles.
• The B″-family subunits of protein phosphatase 2A are necessary for in-vitro dephosphorylation of the Arabidopsis mechanosensory transcription factor VIP1.

  Hyuk Sung Yoon, Kaien Fujino, Shenkui Liu, Tetsuo Takano, Daisuke Tsugama

  Biochemical and biophysical research communications 534 353 - 358 2021/01/01 [Refereed]
   

  Protein phosphatase 2A (PP2A) B″-family subunits have Ca2+-binding EF-hand motifs and can bind PP2A substrates. Arabidopsis thaliana PP2A B″-family subunits are encoded by six genes, and bind a transcription factor, VIP1. VIP1 is dephosphorylated and nuclear-localized by hypo-osmotic stress. However, whether PP2A B″-family subunits mediate the VIP1 dephosphorylation is unclear. Here, we show by yeast two-hybrid and in vitro pull down assays that Arabidopsis PP2A B″-family subunits bind Arabidopsis PP2A A (scaffold) subunits. We also show that VIP1 dephosphorylation in vitro can be induced by a PP2A B″-family subunit.
• A GDSL-type esterase/lipase gene, GELP77, is necessary for pollen dissociation and fertility in Arabidopsis.

  Daisuke Tsugama, Kaien Fujino, Shenkui Liu, Tetsuo Takano

  Biochemical and biophysical research communications 526 (4) 1036 - 1041 2020/04/15 [Refereed][Not invited]
   

  Pollen wall characteristics are dramatically changed during pollen maturation. Many genes have been identified as regulators of such changes in pollen wall characteristics, but mechanisms of such changes have not been completely understood. Here, a GDSL-type esterase/lipase gene, GELP77, is shown to regulate such changes in Arabidopsis thaliana. GELP77-deficient (gelp77) plants exhibited male sterility, and this phenotype was suppressed by introduction of a GELP77 genomic fragment. Mature pollen grains of wild-type Arabidopsis plants have an organized reticulate surface structure and are dissociated from each other. In contrast, pollen grains of gelp77 lacked such a structure and were shrunken and stuck to each other. Nuclei were not detectable in gelp77 microspores at a putative uninucleate stage, suggesting that GELP77 is required as early as this stage. In plants that have the GELP77 promoter-GELP77-GFP transgene, the GELP77-GFP fusion protein was detected in microspores, tapetal cells and middle layer cells in anthers at post-meiotic stages, whereas not anthers at pre-meiotic stages. Analysis of amino acid sequences suggests that GELP77 is phylogenetically distant from the other 104 GDSL-type esterase/lipase genes in Arabidopsis and that GELP77 orthologs are present in various plant species. Together, these results indicate that GELP77 regulates pollen wall characteristics in Arabidopsis.
• A putative AGAMOUS ortholog is a candidate for the gene determining ease of dehulling in Tartary buckwheat (Fagopyrum tataricum).

  Yuka Fukuie, Hana Shimoyama, Toshikazu Morishita, Daisuke Tsugama, Kaien Fujino

  Planta 251 (4) 85 - 85 2020/03/20 [Refereed][Not invited]
   

  MAIN CONCLUSION: Tartary buckwheat rice-type cultivars, which allow easy dehulling, lacked periclinal cell divisions that proceed underneath the epidermis in the proximity of ovary midribs in non-rice-type cultivars. The easy dehulling in these cultivars was associated with a G→A substitution in an AGAMOUS ortholog. Ease of dehulling in Tartary buckwheat (Fagopyrum tataricum) can affect the quality of its products. Tartary buckwheat cultivars that allow easy dehulling are called rice-type cultivars. The rice and non-rice hull types are determined by a single gene, but this gene is unclear. Here, we show that cells underneath the epidermis in the proximity of ovary midribs undergo periclinal cell divisions in non-rice-type cultivars but do not in a rice-type cultivar. The cells that arose from the periclinal cell divisions later underwent lignification, which should increase mechanical strength of hulls. In RNA sequencing, a partial mRNA of an AGAMOUS ortholog in Tartary buckwheat (FtAG) was found to be absent in the rice-type cultivar. Cloning of this gene revealed that this is a 42-bp deletion due to a G→A substitution at a splice acceptor site in the FtAG genomic region. In F2 progeny derived from a cross between non-rice-type and rice-type cultivars, all the rice-type plants exhibited the homozygous A/A allele at this site, whereas all the Tartary-type plants exhibited either the homozygous G/G allele or the heterozygous A/G allele. These results suggest that FtAG is a candidate for the gene that determines ease of dehulling in Tartary buckwheat. The DNA marker that we developed to distinguish the FtAG alleles can be useful in breeding Tartary buckwheat cultivars.
• Data of whole genome sequencing of five garden asparagus (Asparagus officinalis) individuals with the MinION nanopore sequencer.

  Daisuke Tsugama, Kaien Fujino

  Data in brief 28 104838 - 104838 2020/02 [Refereed][Not invited]
   

  Garden asparagus (Asparagus officinalis) is a perennial, dioecious crop. Genomic DNA samples were prepared from five A. officinalis individuals that differ in sex and phenotypes, and sequenced with the MinION nanopore sequencer. The obtained data were 1.5-5 Gb/sample, and the average read length was larger than 1.4 kb for all the samples. The resulting reads were mapped to the existing A. officinalis genome sequence. The existing A. officinalis transcript sequences were mapped to the MinION-derived reads. On the basis of these mapping results, flanking sequences of five partial gene fragments that previously had not been mapped to any region of the existing genome were determined by genomic PCR followed by Sanger sequencing. These sequences enabled to estimate the genomic positions of those five partial gene fragments. The MinION-derived data and the flanking sequences of the five gene fragments were deposited in the NCBI (National Center for Biotechnology Information) SRA (Sequence Read Archive) database and the NCBI Nucleotide database, respectively.
• VIP1, a bZIP protein, interacts with the catalytic subunit of protein phosphatase 2A in Arabidopsis thaliana.

  Hyuk Sung Yoon, Kaien Fujino, Shenkui Liu, Tetsuo Takano, Daisuke Tsugama

  Plant signaling & behavior 15 (2) 1706026 - 1706026 2020 [Refereed][Not invited]
   

  VirE2-INTERACTING PROTEIN1 (VIP1) is a basic leucine zipper protein in Arabidopsis thaliana. VIP1 changes its subcellular localization from the cytoplasm to the nucleus when cells are exposed to mechanical or hypo-osmotic stress. The nuclear localization of VIP1 is inhibited either by inhibitors of calcium signaling or by inhibitors of protein phosphatases 1, 2A and 4 (PP1, PP2A and PP4, respectively). VIP1 binds to the PP2A B"-family subunits, which have calcium-binding EF-hand motifs and which act as the regulatory, substrate-recruiting B subunit of PP2A. The VIP1 de-phosphorylation can therefore be mediated by PP2A. However, details of the PP2A-mediated de-phosphorylation of VIP1 are unclear. Here, with yeast two-hybrid assays and in-vitro pull-down assays, we show that VIP1 does not interact with the scaffolding A subunit of PP2A, but that VIP1 does interact with the catalytic C subunits. Our data raise the possibility that not only the B"-family B subunit of PP2A but also its C subunit contributes to the PP2A-mediated de-phosphorylation of VIP1.
• Protein phosphatase 2A regulates the nuclear accumulation of the Arabidopsis bZIP protein VIP1 under hypo-osmotic stress.

  Daisuke Tsugama, Hyuk Sung Yoon, Kaien Fujino, Shenkui Liu, Tetsuo Takano

  Journal of experimental botany 70 (21) 6101 - 6112 2019/11/18 [Refereed][Not invited]
   

  VIP1 is a bZIP transcription factor in Arabidopsis thaliana. When cells are exposed to mechanical stress, VIP1 transiently accumulates in the nucleus, where it regulates the expression of its target genes and suppresses mechanical stress-induced root waving. The nuclear-cytoplasmic shuttling of VIP1 is regulated by phosphorylation and calcium-dependent signaling, but specific regulators of these processes remain to be identified. Here, inhibitors of protein phosphatase 2A (PP2A) are shown to inhibit both the mechanical stress-induced dephosphorylation and nuclear accumulation of VIP1. The PP2A B subunit, which recruits substrates of PP2A holoenzyme, is classified into B, B', B'', and B''' families. Using bimolecular fluorescence complementation, in vitro pull-down, and yeast two-hybrid assays, we show that VIP1 interacts with at least two of the six members of the Arabidopsis PP2A B''-family subunit, which have calcium-binding EF-hand motifs. VIP1AAA, a constitutively nuclear-localized VIP1 variant with substitutions in putative phosphorylation sites of VIP1, suppressed the root waving induced by VIP1-SRDX (a repression domain-fused variant of VIP1). These results support the idea that VIP1 is dephosphorylated by PP2A and that the dephosphorylation suppresses the root waving. The phosphorylation sites of VIP1 and its homologs were narrowed down by in vitro phosphorylation, yeast two-hybrid, and protein subcellular localization assays.
• Development of a DNA marker for variety discrimination specific to 'Manten-Kirari' based on an NGS-RNA sequence in Tartary buckwheat (Fagopyrum tataricum).

  Kenjiro Katsu, Tatsuro Suzuki, Kaien Fujino, Toshikazu Morishita, Takahiro Noda

  Food chemistry 295 51 - 57 2019/10/15 [Refereed]
   

  To discriminate the trace-rutinosidase variety of Tartary buckwheat 'Manten-Kirari', we developed DNA markers based on RNA polymorphism. Specifically, we mapped 17.76 GB RNA sequences, obtained using HiSeq2000, to create 11,358 large contigs constructed de novo from 'Manten-Kirari' RNA derived from GS-FLX+ titanium. From these, we developed eight DNA markers corresponding to single- to four-nucleotide polymorphisms between 'Manten-Kirari' and 'Hokkai T8', which is representative of normal rutinosidase content varieties in Japan. Using these markers, 'Manten-Kirari' was discriminated from 'Hokkai T8' by eight markers, from major Tartary buckwheat varieties by three markers, and from common buckwheats by two markers. We also performed direct PCR from flour and dried noodle made with 'Manten-Kirari' and 'Hokkai T8'. Based on the results, the DNA markers developed are promising for discriminating 'Manten-Kirari'. This is the first study to develop a DNA marker to discriminate varieties in the Polygonaceae family including buckwheat species.
• Death of female flower microsporocytes progresses independently of meiosis-like process and can be accelerated by specific transcripts in Asparagus officinalis

  Ide M, Masuda K, Tsugama D, Fujino K

  Scientific Reports 9 (1) 2703 - 2703 2019/02 [Refereed][Not invited]
   

  Asparagus officinalis (garden asparagus) is a dioecious perennial crop, and the dioecy (i.e., sex) of A. officinalis can affect its productivity. In A. officinalis, flower anthers in female plants fail to accumulate callose around microsporocytes, fail to complete meiosis, and degenerate due to cell death. Although 13 genes have been implicated in the anther development of male and female flowers, it is unclear how these genes regulate the cell death in female flower anthers. The aim of this study was to narrow down factors involved in this process. TUNEL staining and Feulgen staining of female flower microsporocytes suggest that female microsporocytes enter a previously undetected meiosis-like process, and that the cell death occurs independently of this meiosis-like process, excluding the possibility that the cell death is caused by the cessation of meiosis. RNA sequencing with individual floral organs (tepals, pistils and stamens) revealed that several genes possibly regulating the cell death, such as metacaspase genes and a Bax inhibitor-1 gene, are differentially regulated between female and male flower anthers, and that genes involved in callose accumulation are up-regulated only in male flower anthers. These genes are likely involved in regulating the cell death in female flower anthers in A. officinalis.
• CRISPR/Cas9-Mediated Editing of Genes Encoding rgs-CaM-like Proteins in Transgenic Potato Plants.

  Zhila Osmani, Shinnosuke Jin, Masafumi Mikami, Masaki Endo, Hiroki Atarashi, Kaien Fujino, Tetsuya Yamada, Kenji S Nakahara

  Methods in molecular biology (Clifton, N.J.) 2028 153 - 165 2019 [Refereed]
   

  A tobacco calmodulin-like protein, rgs-CaM, has been shown to interact with viruses in a variety of ways; it contributes to geminivirus infections but is also involved in primed immunity to the cucumber mosaic virus. Sequence similarity searches revealed several calmodulin-like proteins similar to rgs-CaM (rCML) in Arabidopsis and other Solanaceae plants, including potato (Solanum tuberosum). To analyze the functions of each rCML, mutations were introduced into potato rCMLs using the CRISPR/Cas9 system. Here, we describe our protocol of the CRISPR/Cas9-mediated targeted mutagenesis in stably transformed potato plants.
• Calcium signalling regulates the functions of the bZIP protein VIP1 in touch responses in Arabidopsis thaliana.

  Daisuke Tsugama, Shenkui Liu, Kaien Fujino, Tetsuo Takano

  Annals of botany 122 (7) 1219 - 1229 2018/12/31 [Refereed]
   

  Background and Aims: VIP1 is a bZIP transcription factor in Arabidopsis thaliana. VIP1 and its close homologues transiently accumulate in the nucleus when cells are exposed to hypo-osmotic and/or mechanical stress. Touch-induced root bending is enhanced in transgenic plants overexpressing a repression domain-fused form of VIP1 (VIP1-SRDXox), suggesting that VIP1, possibly with its close homologues, suppresses touch-induced root bending. The aim of this study was to identify regulators of these functions of VIP1 in mechanical stress responses. Methods: Co-immunoprecipitation analysis using VIP1-GFP fusion protein expressed in Arabidopsis plants identified calmodulins as VIP1-GFP interactors. In vitro crosslink analysis was performed using a hexahistidine-tagged calmodulin and glutathione S-transferase-fused forms of VIP1 and its close homologues. Plants expressing GFP-fused forms of VIP1 and its close homologues (bZIP59 and bZIP29) were submerged in hypotonic solutions containing divalent cation chelators, EDTA and EGTA, and a potential calmodulin inhibitor, chlorpromazine, to examine their effects on the nuclear-cytoplasmic shuttling of those proteins. VIP1-SRDXox plants were grown on medium containing 40 mm CaCl2, 40 mm MgCl2 or 80 mm NaCl. MCA1 and MCA2 are mechanosensitive calcium channels, and the hypo-osmotic stress-dependent nuclear-cytoplasmic shuttling of VIP1-GFP in the mca1 mca2 double knockout mutant background was examined. Key Results: In vitro crosslink products were detected in the presence of CaCl2, but not in its absence. EDTA, EGTA and chlorpromazine all inhibited both the nuclear import and the nuclear export of VIP1-GFP, bZIP59-GFP and bZIP29-GFP. Either 40 mm CaCl2or 80 mm NaCl enhanced the VIP-SRDX-dependent root bending. The nuclear-cytoplasmic shuttling of VIP1 was observed even in the mca1 mca2 mutant. Conclusions: VIP1 and its close homologues can interact with calmodulins. Their nuclear-cytoplasmic shuttling requires neither MCA1 nor MCA2, but does require calcium signalling. Salt stress affects the VIP1-dependent regulation of root bending.
• B-family subunits of protein phosphatase 2A are necessary for pollen development but not for female gametophyte development in Arabidopsis

  Tsugama D, Liu S, Fujino K, Takano T

  Biochemical and Biophysical Research Communications 505 (1) 176 - 180 2018/10 [Refereed][Not invited]
   

  Protein phosphatase 2A (PP2A) is a heterotrimeric protein complex conserved among eukaryotes. The B subunit of PP2A determines the substrate specificity of the PP2A holoenzyme, and is classified into the B, B', B″ and B‴ families. Arabidopsis thaliana has two isoforms of the B-family subunit (ATBA and ATBB). A double knockout of their genes is lethal, but which developmental process is primarily impaired by the double knockout is unclear. Identifying such a process helps understand PP2A-mediated signaling more deeply. Here, genetic characterization of new knockout mutants for these genes shows that they are necessary for pollen development but not for female gametophyte development. Compared to wild-type pollen grains, the mutant pollen grains exhibited lower enzyme activities, germinated less frequently on stigmas, and exhibited the aberrant numbers of sperm cell nuclei, suggesting that ATBA and ATBB play pleiotropic roles in pollen development. The amino acids stabilizing the interaction between the human PP2A A and B-family subunits are conserved in an Arabidopsis A subunit (AtPP2AA2), ATBA and ATBB. His-tagged AtPP2AA2 co-immunoprecipitated with either Myc-tagged ATBA or Myc-tagged ATBB in vitro, confirming their interactions. Proteins that regulate pollen development and that undergo dephosphorylation are likely primary targets of ATBA and ATBB.
• Possible inhibition of Arabidopsis VIP1-mediated mechanosensory signaling by streptomycin.

  Daisuke Tsugama, Shenkui Liu, Kaien Fujino, Tetsuo Takano

  Plant signaling & behavior 13 (10) e1521236  2018 [Refereed]
   

  VIP1 (VIRE2-INTERACTING PROTEIN 1) and its close homologues are Arabidopsis thaliana bZIP proteins regulating stress responses and root tropisms. They are present in the cytoplasm under steady conditions, but transiently accumulate in the nucleus when cells are exposed to mechanical stress such as hypo-osmotic stress and touch. This pattern of changes in subcellular localization is unique to VIP1 and its close homologues, and can be useful to further characterize mechanical stress signaling in plants. A recent study showed that calcium signaling regulates this pattern of subcellular localization. Here, we show that a possible calcium channel inhibitor, streptomycin, also inhibits the nuclear accumulation of VIP1. Candidates for the specific regulators of the mechanosensitive calcium signaling are further discussed.
• Determination of the Absolute Configuration of a Monoglyceride Antibolting Compound and Isolation of Related Compounds from Radish Leaves (Raphanus sativus)

  Tsuyoshi Ogihara, Naruki Amano, Yuki Mitsui, Kaien Fujino, Hiroyuki Ohta, Kosaku Takahashi, Hideyuki Matsuura

  JOURNAL OF NATURAL PRODUCTS 80 (4) 872 - 878 0163-3864 2017/04 [Refereed][Not invited]
   

  A monoglyceride (1) has been reported to possess an antibolting effect in radish (Raphanus sativus), but its absolute configuration at the C-2 position was not determined earlier. In this work, the absolute configuration of 1 was determined to be (2S), and it was also accompanied by one new (2) and two known monoglycerides (3 and 4). The chemical structure of 2 was determined as beta-(7'Z,10'Z,13'Z)-hexadecatrienoic acid monoglyceride monoglyceride (beta-16:3 monoglyceride). Qualitative and quantitative analytical methods for compounds 1-4 were developed, using two deuterium-labeled compounds (8 and 9) as internal standards. The results revealed a broader range of distribution of 1-4 in several annual winter crops. It was also found that these isolated compounds have an inhibitory effect on the root elongation of Arabidopsis thaliana seedlings at concentrations of 25 and 50 /AM in the medium. However, the inhibitory effect of 1 was not dependent on coronatin-insensitive 1 (COD) protein, which may suggest the involvement of an unidentified signaling system other than jasmonic acid signaling.
• Detainment of Tam3 Transposase at Plasma Membrane by Its BED-Zinc Finger Domain

  Hua Zhou, Megumi Hirata, Ryo Osawa, Kaien Fujino, Yuji Kishima

  PLANT PHYSIOLOGY 173 (2) 1492 - 1501 0032-0889 2017/02 [Refereed][Not invited]
   

  Transposable elements (TEs) are considered to be parasites of host genomes because they act as powerful mutagens. If not kept in check, they can cause gene disruption, genome rearrangement, and genomic takeover. Hence, activities of TEs are under the rigid control of hosts. To date, all identified TE regulations have been epigenetic dependent, with the exception of the DNA transposon Tam3. Blocking nuclear translocation of Tam3 transposase (TPase) is consistent with the suppression of Tam3 in Antirrhinum majus. In this article, we discovered that epigenetic-independent regulation of Tam3 is mediated by the BED-zinc finger (Znf-BED) domain of Tam3 TPase. The host targets the N terminus of the Znf-BED domain, which contains two highly conserved aromatic amino acids, to detain Tam3 TPase at the plasma membrane and to silence Tam3. Zinc finger proteins perform broader functions in transcriptional regulation through their DNA binding ability. Our data revealed that the posttranslational epigenetic-independent silencing against TEs was a result of the protein binding ability of the Znf-BED domain.
• A putative MYB35 ortholog is a candidate for the sex-determining genes in Asparagus officinalis

  Daisuke Tsugama, Kohei Matsuyama, Mayui Ide, Masato Hayashi, Kaien Fujino, Kiyoshi Masuda

  SCIENTIFIC REPORTS 7 41497  2045-2322 2017/02 [Refereed][Not invited]
   

  Asparagus officinalis (garden asparagus) is a dioecious perennial crop. For agricultural production of A. officinalis, male plants have advantages over female plants. The dioecism of A. officinalis is determined by the single dominant masculinizing M locus, which is involved in tapetal cell development in stamens, but thus far no specific M locus genes have been identified. We re-analyzed previously published RNA-Seq data for the A. officinalis transcriptome, cloned some genes, and discovered that a putative ortholog of MYB35, which is indispensable for tapetal cell development in Arabidopsis thaliana, is absent in the genome of female plants in A. officinalis. In a reverse transcription-PCR analysis, this gene (AoMYB35) exhibited strong expression in stamens in male flowers at an early developmental stage. In an in situ hybridization analysis, AoMYB35 mRNA was detected in tapetal cells in young male flowers. GFP-fused AoMYB35 was detected in the nucleus when expressed in onion epidermal cells. These results suggest that AoMYB35 is a male-specific gene encoding a putative transcription factor that acts in tapetal cells at an early stage of flower development in A. officinalis. Together, the results support the idea that AoMYB35 is a candidate for one of the M locus genes in A. officinalis.
• Identification of candidates for interacting partners of the tail domain of DcNMCP1, a major component of the Daucus carota nuclear lamina-like structure

  Ryota Mochizuki, Daisuke Tsugama, Michihiro Yamazaki, Kaien Fujino, Kiyoshi Masuda

  NUCLEUS 8 (3) 312 - 322 1949-1034 2017 [Refereed][Not invited]
   

  NMCP/CRWN (NUCLEAR MATRIX CONSTITUENT PROTEIN/CROWDED NUCLEI) is a major component of a protein fibrous meshwork (lamina-like structure) on the plant inner nuclear membrane. NMCP/CRWN contributes to regulating nuclear shape and nuclear functions. An NMCP/CRWN protein in Daucus carota (DcNMCP1) is localized to the nuclear periphery in interphase cells, and surrounds chromosomes in cells in metaphase and anaphase. The N-terminal region and the C-terminal region of DcNMCP1 are both necessary for localizing DcNMCP1 to the nuclear periphery. Here candidate interacting partners of the amino acid position 975-1053 of DcNMCP1 (T975-1053), which is present in the C-terminal region and contains a conserved sequence that plays a role in localizing DcNMCP1 to the nuclear periphery, are screened for. Arabidopsis thaliana nuclear proteins were subjected to far-Western blotting with GST-fused T975-1053 as a probe, and signals were detected at the positions corresponding to approximate to 70, approximate to 40, and approximate to 18kDa. These approximate to 70, approximate to 40, and approximate to 18kDa nuclear proteins were identified by mass spectrometry, and subjected to a yeast 2-hybrid (Y2H) analysis with T975-1053 as bait. In this analysis, the approximate to 40kDa protein ARP7, which is a nuclear actin-related protein possibly involved in regulating chromatin structures, was confirmed to interact with T975-1053. Independently of the far-Western blotting, a Y2H screen was performed using T975-1053 as bait. Targeted Y2H assays confirmed that 3 proteins identified in the screen, MYB3, SINAT1, and BIM1, interact with T975-1053. These proteins might have roles in NMCP/CRWN protein-mediated biologic processes.
• A Single-Nucleotide Polymorphism in an Endo-1,4-beta-Glucanase Gene Controls Seed Coat Permeability in Soybean

  Seong-Jin Jang, Masako Sato, Kei Sato, Yutaka Jitsuyama, Kaien Fujino, Haruhide Mori, Ryoji Takahashi, Eduardo R. Benitez, Baohui Liu, Tetsuya Yamada, Jun Abe

  PLOS ONE 10 (6) e0128527  1932-6203 2015/06 [Refereed][Not invited]
   

  Physical dormancy, a structural feature of the seed coat known as hard seededness, is an important characteristic for adaptation of plants against unstable and unpredictable environments. To dissect the molecular basis of qHS1, a quantitative trait locus for hard seededness in soybean (Glycine max (L) Merr.), we developed a near-isogenic line (NIL) of a permeable (soft-seeded) cultivar, Tachinagaha, containing a hard-seed allele from wild soybean (G. soja) introduced by successive backcrossings. The hard-seed allele made the seed coat of Tachinagaha more rigid by increasing the amount of beta-1,4-glucans in the outer layer of palisade cells of the seed coat on the dorsal side of seeds, known to be a point of entrance of water. Fine-mapping and subsequent expression and sequencing analyses revealed that qHS1 encodes an endo-1,4-beta-glucanase. A single-nucleotide polymorphism (SNP) introduced an amino acid substitution in a substrate-binding cleft of the enzyme, possibly reducing or eliminating its affinity for substrates in permeable cultivars. Introduction of the genomic region of qHS1 from the impermeable (hard-seeded) NIL into the permeable cultivar Kariyutaka resulted in accumulation of beta-1,4-glucan in the outer layer of palisade cells and production of hard seeds. The SNP allele found in the NIL was further associated with the occurrence of hard seeds in soybean cultivars of various origins. The findings of this and previous studies may indicate that qHS1 is involved in the accumulation of beta-1,4-glucan derivatives such as xyloglucan and/or beta-(1,3)(1,4)-glucan that reinforce the impermeability of seed coats in soybean.
• Isolation and sequence analysis of Old Stabiliser that suppresses activity of transposon Tam3 in Antirrhinum

  Hirata M, Ebinuma I, Fujino K, Kishima Y

  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan 日本作物学会 (55) 15 - 16 2014/12
• Molecular basis of a shattering resistance boosting global dissemination of soybean

  Hideyuki Funatsuki, Masaya Suzuki, Aya Hirose, Hiroki Inaba, Tetsuya Yamada, Makita Hajika, Kunihiko Komatsu, Takeshi Katayama, Takashi Sayama, Masao Ishimoto, Kaien Fujino

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 111 (50) 17797 - 17802 0027-8424 2014/12 [Refereed][Not invited]
   

  Pod dehiscence (shattering) is essential for the propagation of wild plant species bearing seeds in pods but is a major cause of yield loss in legume and crucifer crops. Although natural genetic variation in pod dehiscence has been, and will be, useful for plant breeding, little is known about the molecular genetic basis of shattering resistance in crops. Therefore, we performed map-based cloning to unveil a major quantitative trait locus (QTL) controlling pod dehiscence in soybean. Fine mapping and complementation testing revealed that the QTL encodes a dirigent-like protein, designated as Pdh1. The gene for the shattering-resistant genotype, pdh1, was defective, having a premature stop codon. The functional gene, Pdh1, was highly expressed in the lignin-rich inner sclerenchyma of pod walls, especially at the stage of initiation in lignin deposition. Comparisons of near-isogenic lines indicated that Pdh1 promotes pod dehiscence by increasing the torsion of dried pod walls, which serves as a driving force for pod dehiscence under low humidity. A survey of soybean germplasm revealed that pdh1 was frequently detected in landraces from semiarid regions and has been extensively used for breeding in North America, the world's leading soybean producer. These findings point to a new mechanism for pod dehiscence involving the dirigent protein family and suggest that pdh1 has played a crucial role in the global expansion of soybean cultivation. Furthermore, the orthologs of pdh1, or genes with the same role, will possibly be useful for crop improvement.
• Isolation of a major genetic interaction associated with an extreme phenotype using assorted F2 populations in rice

  Yuya Ota, Seiya Ishiguro, Eiko Aoyama, Ryosuke Aiba, Reika Iwashiro, Takanari Tanabata, Itsuro Takamure, Kaien Fujino, Yuji Kishima

  MOLECULAR BREEDING 33 (4) 997 - 1003 1380-3743 2014/04 [Refereed][Not invited]
   

  Detection of quantitative trait loci (QTLs) is dependent on the materials used in the analysis, as different combinations of parental materials may lead to different outcomes in QTLs for the same trait. On the other hand, an extreme phenotype associated with a given trait implies the potential involvement of a particular allele in various allelic interactions. A genetic factor associated with such an extreme phenotype may frequently be identified from various genetic populations consisting of different parental combinations. In this study, we attempted to uncover the genetic factor associated with extremely early heading date in rice, using various F2 populations. Heading date in rice has been characterized by at least 19 QTLs, from which 12 genes have been identified. A58, a rice strain with an extremely early heading date, is adapted to Hokkaido, the northernmost limit of rice cultivation. Six F2 populations derived from crosses of A58 with six other strains displayed a range of heading dates. Genotyping using 19 QTL markers indicated that the A58 allele of the Ghd7 locus was present in most F2 individuals exhibiting extremely early heading dates. This analysis also demonstrated that when the wild-type Ehd1 allele was present, the Ghd7 allele from A58 accelerated floral induction. The results of this study demonstrate that assorted F2 populations are valuable materials for comprehensive genotyping to explore major genetic factors for extreme phenotypes, and that this methodology is broadly applicable to other unknown traits.
• Low Temperature-Responsive Changes in the Anther Transcriptome's Repeat Sequences Are Indicative of Stress Sensitivity and Pollen Sterility in Rice Strains

  Seiya Ishiguro, Kei Ogasawara, Kaien Fujino, Yutaka Sato, Yuji Kishima

  PLANT PHYSIOLOGY 164 (2) 671 - 682 0032-0889 2014/02 [Refereed][Not invited]
   

  Genome-wide transcriptome analyses using microarray probes containing genes and repeat sequences have been performed to examine responses to low temperatures in rice (Oryza sativa). We focused particularly on the rice anther at the booting stage, because a low temperature at this stage can result in pollen abortion. The five rice strains examined in this study showed different pollen fertilities due to a low-temperature treatment during the booting stage. The microarray analyses demonstrated that the low-temperature stress caused genome-wide changes in the transcriptional activities not only of genes but also of repeat sequences in the rice anther. The degree of the temperature-responsive changes varied among the five rice strains. Interestingly, the low-temperature-sensitive strains revealed more changes in the transcriptome when compared with the tolerant strains. The expression patterns of the repeat sequences, including miniature inverted-repeat transposable elements, transposons, and retrotransposons, were correlated with the pollen fertilities of the five strains, with the highest correlation coefficient being 0.979. Even in the low-temperature-sensitive strains, the transcriptomes displayed distinct expression patterns. The elements responding to the low temperatures were evenly distributed throughout the genome, and the major cis-motifs involved in temperature-responsive changes were undetectable from the upstream sequences in the corresponding repeats. The genome-wide responses of transcription to the temperature shift may be associated with chromatin dynamics, which facilitates environmental plasticity. A genome-wide analysis using repeat sequences suggested that stress tolerance could be conferred by insensitivity to the stimuli.
• Localization of Daucus carota NMCP1 to the nuclear periphery: the role of the N-terminal region and an NLS-linked sequence motif, RYNLRR, in the tail domain

  Yuta Kimura, Kaien Fujino, Kana Ogawa, Kiyoshi Masuda

  FRONTIERS IN PLANT SCIENCE 5 62  1664-462X 2014/02 [Refereed][Not invited]
   

  Recent ultrastructural studies revealed that a structure similar to the vertebrate nuclear lamina exists in the nuclei of higher plants. However, plant genomes lack genes for lamins and intermediate-type filament proteins, and this suggests that plant-specific nuclear coiled-coil proteins make up the lamina-like structure in plants. NMCP1 is a protein, first identified in Daucus carota cells, that localizes exclusively to the nuclear periphery in interphase cells. It has a tripartite structure comprised of head, rod, and tail domains, and includes putative nuclear localization signal (NLS) motifs. We identified the functional NLS of DcNMCP1 (carrot NMCP1) and determined the protein regions required for localizing to the nuclear periphery using EGFP-fused constructs transiently expressed in Apium graveolens epidermal cells. Transcription was driven under a CaMV35S promoter, and the genes were introduced into the epidermal cells by a DNA-coated microprojectile delivery system. Of the NLS motifs, KRRRK and RRHK in the tail domain were highly functional for nuclear localization. Addition of the N-terminal 141 amino acids from DcNMCP1 shifted the localization of a region including these NLSs from the entire nucleus to the nuclear periphery. Using this same construct, the replacement of amino acids in RRHK or its preceding sequence, YNL, with alanine residues abolished localization to the nuclear periphery, while replacement of KRRRK did not affect localization. The sequence R/Q/HYNLRR/H, including YNL and the first part of the sequence of RRHK, is evolutionarily conserved in a subclass of NMCP1 sequences from many plant species. These results show that NMCP1 localizes to the nuclear periphery by a combined action of a sequence composed of R/Q/HYNLRR/H, NLS, and the N-terminal region including the head and a portion of the rod domain, suggesting that more than one binding site is implicated in localization of NMCP1.
• Mapping and use of QTLs controlling pod dehiscence in soybean

  Hideyuki Funatsuki, Makita Hajika, Tetsuya Yamada, Masaya Suzuki, Seiji Hagihara, Yoshinori Tanaka, Shohei Fujita, Masao Ishimoto, Kaien Fujino

  BREEDING SCIENCE 61 (5) 554 - 558 1344-7610 2011/01 [Refereed][Not invited]
   

  While the cultivated soybean, Glycine max (L.) Merr., is more recalcitrant to pod dehiscence (shattering-resistant) than wild soybean, Glycine sofa Sieb. & Zucc., there is also significant genetic variation in shattering resistance among cultivated soybean cultivars. To reveal the genetic basis and develop DNA markers for pod dehiscence, several research groups have conducted quantitative trait locus (QTL) analysis using segregated populations derived from crosses between G. max accessions or between a G. max and G. soja accession. In the populations of G. max, a major QTL was repeatedly identified near SSR marker Sat_366 on linkage group J (chromosome 16). Minor QTLs were also detected in several studies, although less commonality was found for the magnitudes of effect and location. In G. max x G. soja populations, only QTLs with a relatively small effect were detected. The major QTL found in G. max was further fine-mapped, leading to the development of specific markers for the shattering resistance allele at this locus. The markers were used in a breeding program, resulting in the production of near-isogenic lines with shattering resistance and genetic backgrounds of Japanese elite cultivars. The markers and lines developed will hopefully contribute to the rapid production of a variety of shattering-resistant soybean cultivars.
• Temperature controls nuclear import of Tam3 transposase in Antirrhinum

  Kaien Fujino, Shin-nosuke Hashida, Takashi Ogawa, Tomoko Natsume, Takako Uchiyama, Tetsuo Mikami, Yuji Kishima

  PLANT JOURNAL 65 (1) 146 - 155 0960-7412 2011/01 [Refereed][Not invited]
   

  P>It has been proposed that environmental stimuli can activate transposable elements (TEs), whereas few substantial mechanisms have been shown so far. The class-II element Tam3 from Antirrhinum majus exhibits a unique property of low-temperature-dependent transposition (LTDT). LTDT has proved invaluable in developing the gene isolation technologies that have underpinned much of modern plant developmental biology. Here, we reveal that LTDT involves differential subcellular localization of the Tam3 transposase (TPase) in cells grown at low (15 degrees C) and high (25 degrees C) temperatures. The mechanism is associated with the nuclear import of Tam3 TPase in Antirrhinum cells. At high temperature, the nuclear import of Tam3 TPase is severely restricted in Antirrhinum cells, whereas at low temperature, the nuclear localization of Tam3 TPase is observed in about 20% of the cells. However, in tobacco BY-2 and Allium cepa (onion) cells, Tam3 TPase is transported into most nuclei. In addition to three nuclear localization signals (NLSs), the Tam3 TPase is equipped with a nuclear localization inhibitory domain (NLID), which functions to abolish nuclear import of the TPase at high temperature in Antirrhinum. NLID in Tam3 TPase is considered to interact with Antirrhinum-specific factor(s). The host-specific regulation of the nuclear localization of transposase represents a new repertoire controlling class-II TEs.
• Fine mapping and development of DNA markers for the qPDH1 locus associated with pod dehiscence in soybean

  Masaya Suzuki, Kaien Fujino, Yumi Nakamoto, Masao Ishimoto, Hideyuki Funatsuki

  MOLECULAR BREEDING 25 (3) 407 - 418 1380-3743 2010/03 [Refereed][Not invited]
   

  Pod dehiscence (shattering) is a major cause of yield loss in mechanical harvesting of soybeans. To develop useful selection markers, we conducted a high-resolution mapping of a major quantitative trait locus (QTL) controlling pod dehiscence, designated as qPDH1. The progeny of a residual heterozygous line, which was a recombinant inbred line segregating only for the genomic region around qPDH1, was screened for flanking markers to obtain various recombinants in the vicinity of the QTL. Analysis of the relationship between degree of pod dehiscence and graphical genotype of these lines confined the location of qPDH1 to a 134-kb region on chromosome 16 (formerly linkage group J), where ten putative genes were predicted to be present. None of these genes showed significant sequence homology with the Arabidopsis genes that have previously been reported to be associated with pod dehiscence, suggesting the presence of a novel gene and mechanism underlying pod dehiscence in soybean. Sequencing analysis of the parental shattering-resistant and -susceptible cultivars for the candidate genes revealed a high-frequency nucleotide polymorphism in this genomic region between the cultivars. Three markers were developed using insertion/deletion variations in the region. Polymorphism at these marker loci was basically conserved between diverse shattering-resistant and -susceptible cultivars/lines, suggesting the versatility and usefulness of these markers for marker-assisted selection.
• A major QTL, qPDH1, is commonly involved in shattering resistance of soybean cultivars

  Tetsuya Yamada, Hideyuki Funatsuki, Seiji Hagihara, Shohei Fujita, Yoshinori Tanaka, Hiroyuki Tsuji, Masao Ishimoto, Kaien Fujino, Makita Hajika

  BREEDING SCIENCE 59 (4) 435 - 440 1344-7610 2009/12 [Refereed][Not invited]
   

  A major quantitative trait locus (QTL) controlling pod dehiscence (shattering) in soybean, designated qPDH1, has previously been identified using progeny of shattering-resistant cultivars derived from a Thai cultivar, SJ2. The QTL was located near a simple sequence repeat marker, Sat_366, on linkage group J. To determine whether shattering-resistance genes originating from different resources are located at qPDH1 in general, we conducted genetic analysis using DNA markers for several populations. In an F-2 Population derived from a cross between a shattering-susceptible cultivar, Toyomusume, and a shattering-resistant cultivar, Harosoy, a major QTL for pod dehiscence was identified in the region near qPDH1, which was confirmed in the progeny of F-4:5 populations. A major QTL was identified near qPDH1 also in F-2 populations derived from crosses including Wasekogane and Kariyutaka as shattering-resistant parents. The heterozygous genotypes at the QTL showed high degrees of pod dehiscence, suggesting that shattering resistance behaves as a nearly recessive trait. In F-2 Populations derived from crosses between shattering-resistant cultivars, heterozygous genotypes at the Sat_366 locus were shattering-resistant. These results suggest that shattering-resistant cultivars harbor recessive shattering-resistance allele(s) at qPDH1 regardless of their origin and that molecular markers near qPDH1 could be used for marker-assisted selection for shattering resistance in soybean.
• Stable Transcription Activities Dependent on an Orientation of Tam3 Transposon Insertions into Antirrhinum and Yeast Promoters Occur Only within Chromatin

  Takako Uchiyama, Kaien Fujino, Takashi Ogawa, Akihito Wakatsuki, Yuji Kishima, Tetsuo Mikami, Yoshio Sano

  PLANT PHYSIOLOGY 151 (3) 1557 - 1569 0032-0889 2009/11 [Refereed][Not invited]
   

  Transposon insertions occasionally occur in the promoter regions of plant genes, many of which are still capable of being transcribed. However, it remains unclear how transcription of such promoters is able to occur. Insertion of the Tam3 transposon into various genes of Antirrhinum majus can confer leaky phenotypes without its excision. These genes, named Tam3-permissible alleles, often contain Tam3 in their promoter regions. Two alleles at different anthocyanin biosynthesis loci, nivea(recurrens::Tam3) (niv(rec)) and pallida(recurrens::Tam3) (pal(rec)), both contain Tam3 at a similar position immediately upstream of the promoter TATA-box; however, these insertions had different phenotypic consequences. Under conditions where the inserted Tam3 is immobilized, the niv(rec) line produces pale red petals, whereas the pal(rec) line produces no pigment. These pigmentation patterns are correlated with the level of transcripts from the niv(rec) or pal(rec) alleles, and these transcriptional activities are independent of DNA methylation in their promoter regions. In niv(rec), Tam3 is inserted in an orientation that results in the 3' end of Tam3 adjacent to the 5' region of the gene coding sequence. In contrast, the pal(rec) allele contains a Tam3 insertion in the opposite orientation. Four of five different nonrelated genes that are also Tam3-permissible alleles and contain Tam3 within the promoter region share the same Tam3 orientation as niv(rec). The different transcriptional activities dependent on Tam3 orientation in the Antirrhinum promoters were consistent with expression of luciferase reporter constructs introduced into yeast chromosomes but not with transient expression of these constructs in Antirrhinum cells. These results suggest that for Tam3 to sustain stable transcriptional activity in various promoters it must be embedded in chromatin.
• A Major Soybean QTL, qPDH1, Controls Pod Dehiscence without Marked Morphological Change

  Masaya Suzuki, Kaien Fujino, Hideyuki Funatsuki

  PLANT PRODUCTION SCIENCE 12 (2) 217 - 223 1343-943X 2009/04 [Refereed][Not invited]
   

  Pod dehiscence (shattering) is a major source of yield loss in the mechanically harvested soybean. We examined near-isogenic lines (NILs) for a major quantitative trait locus (QTL,) controlling pod dehiscence, designated a; gPDH1, to reveal the mechanism underlying the effect of this QTL on shattering resistance. The degree of shattering resistance differed among the NILs; as pod dehiscence percentage after 3 hr heat treatment was under 50% and over 90% for the genotypes resistant to shattering and those susceptible to shattering, respectively. Oil the other hand, there were no significant differences in the length, width and thickness of pods among the NILs. Anatomical analysis of the dorsal sutures of pods, at which pod dehiscence was found to continence most frequently, revealed no marked differences between the NILs. These results suggest drat qPDH1 controls pod dehiscence without markedly changing the morphology of the pods.
• Confirmation of the location and the effects of a major QTL controlling pod dehiscence, qPDH1, in soybean

  Hideyuki Funatsuki, Makita Hajika, Seiji Hagihara, Tetsuya Yamada, Yoshinori Tanaka, Hiroyuki Tsuji, Masao Ishimoto, Kaien Fujino

  Breeding Science Japanese Society of Breeding 58 (1) 63 - 69 1344-7610 2008/04/18 [Refereed][Not invited]
   

  Pod dehiscence (shattering) is a major source of yield loss in mechanical harvest of soybean. To develop a marker-assisted selection system for a major quantitative trait locus (QTL) controlling pod dehiscence, designated as qPDH1, we confirmed the usefulness of flanking markers and the effect of qPDH1 under different genetic backgrounds. The progeny of a residual heterozygous line for the genomic region around qPDH1 was screened for four flanking markers to obtain various recombinants in the vicinity of the QTL. The analysis of the relationship between the pod dehiscence degree and the graphical genotype of these lines confirmed the presence of qPDH1 in the region between the SSR markers, Sat_366 and Sat_093, on linkage group J. At these marker loci, the alleles from a Thai cultivar, SJ2, the donor of the shattering resistance, were inherited by most of the shattering-resistant, SJ2-derived cultivars and were distinct from those of the shatteringsusceptible cultivars tested. The effect of the allele from SJ2 at qPDH1 was confirmed by association tests under four genetic backgrounds derived from crosses with three susceptible cultivars in the northern to southwestern regions of Japan and a susceptible accession of Indonesian origin at three locations. These results suggest that the allele from SJ2 at qPDH1 and the linked markers could be widely used for the improvement of the shattering resistance in soybean.
• Multiple regulatory mechanisms influence the activity of the transposon, Tam3, of Antirrhinum

  Takako Uchiyama, Yumiko Saito, Hiroyuki Kuwabara, Kaien Fujino, Yuji Kishima, Cathie Martin, Yoshio Sano

  NEW PHYTOLOGIST 179 (2) 343 - 355 0028-646X 2008 [Refereed][Not invited]
   

  In Antirrhinum, several unique regulations of the transposon, Tam3, have been described. Tam3 activity in Antirrhinum is strictly controlled by the growing temperature of plants (low-temperature-dependent transposition: LTDT), by chromosomal position of Tam3 copy and by two specific repressor genes Stabiliser (St) and New Stabiliser (NSt). Here, the effects of the St and NSt loci on Tam3 transposition are compared. In cotyledons and hypocotyls, Tam3 is active even at high growing temperatures, indicating that LTDT does not operate when these organs are developing. This developmental regulation of Tam3 activity is differentially influenced by the St and NSt loci: St permits Tam3 transposition in cotyledons and hypocotyls, whereas NSt suppresses it in these organs. The effects of these host genes on Tam3 activity at the molecular level were examined. It was found that neither of these genes inhibits the transcription of the Tam3 transposase gene nor its translation, and that the Tam3 transposase has the potential to catalyze transposition in the St and NSt lines. The differences between the effects of St and NSt imply that they regulate Tam3 activity independently. Our molecular data indicate that their influence on Tam3 transposition seems to be nonepigenetic; possible mechanisms for their activity are discussed.
• Simple sequence repeat markers linked to a major QTL controlling pod shattering in soybean

  H. Funatsuki, M. Ishimoto, H. Tsuji, K. Kawaguchi, K. Kawaguchi, M. Hajika, K. Fujino

  Plant Breeding 125 (2) 195 - 197 0179-9541 2006/04/01 [Refereed][Not invited]
   

  Shattering of soybean pods prior to harvest leads to a reduction in yield. In order to identify simple sequence repeat (SSR) markers linked to quantitative trait loci (QTLs) conditioning pod shattering, QTL analysis was conducted using an recombinant inbred line (RIL) population segregating for this trait. The degrees of pod-shattering resistance were evaluated by heat treatment applied to pods harvested from plants in the field and in a growth chamber. Composite interval mapping identified one major QTL between SSR markers Sat_093 and Sat_366 on linkage group J for both environments. The position and the effect of this QTL were confirmed in an F2 population derived from a cross between the pod shattering-susceptible parental cultivar and a pod shattering-resistant RIL. The SSR markers linked to the major QTL will be useful for marker-assisted selection in soybean-breeding programmes. © 2006 Blackwell Verlag.
• QTL analysis of pod shattering in soybean

  Funatsuki Hideyuki, Ishimoto Masao, Fujino Kaien, Hajika Makita, Yamada Tetsuya, Tsuji Hiroyuki, Tanaka Yoshinori, Kimura Yoshiaki, Higihara Seiji, Yuzawa Masaaki, Shinozaki Atsushi

  Abstracts of Meeting of the CSSJ 日本作物学会 222 (0) 238 - 238 2006 [Refereed][Not invited]
  
• Expression of gibberellin related genes on potato tuberization

  Nakaichigo Y, Koda Y, Fujino K

  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan 日本作物学会 (46) 83 - 84 2005/12
• Molecular characterization of a 10-kDa buckwheat molecule reactive to allergic patients' IgE

  R Matsumoto, K Fujino, Y Nagata, S Hashiguchi, Y Ito, Y Aihara, Y Takahashi, K Maeda, K Sugimura

  ALLERGY 59 (5) 533 - 538 0105-4538 2004/05 [Not refereed][Not invited]
   

  Background: Using the sera from buckwheat (BW)-allergic patients, several putative causative molecules were reported. However, few molecules were determined on the molecular structure. We demonstrated in 2000 that the major allergen with 24 kDa (BW24KD) is a legumin-like storage protein. Objective: The aim of this study was to isolate and characterize further a major allergen with 10 kDa by molecular cloning. Methods and results: Buckwheat allergens were identified by immunoblotting analysis using sera from 14 allergic and two nonallergic individuals. We identified a protein with 10 kDa (BW10KD) that reacted with immunoglobulin E (IgE) more strongly than with IgG and IgA in 57% of the allergic patients but not with IgE in nonallergic individuals. Analyses were performed by N-terminal amino acid sequencing and molecular cloning. Physiological significance was assessed by an immunoblotting experiment showing that the reactivity of an allergic patient's serum IgE to BW10KD was competitively inhibited by natural BW extracts. Conclusion: Molecular cloning experiments indicated that BW10KD as a BW allergen was a member of the 2S-albumin multigene family.
• Immunoblot-analysis using an antibodiy for an allergenic protein (BW24KD) to tartary buckwheat leaf proteins

  Maruyama-Funatsuki W., Nakatsuka K., Fujino K., Kato A., Funatsuki H.

  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan 日本作物学会 (43) 113 - 114 2002/11
• ABA-induced bud-formation in agrostis alba cv highland.

  Jamsran U., Fujino K., Kikuta Y., Nakashima H.

  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan 日本作物学会 (37) 102 - 103 1996/12
• REORIENTATION OF CORTICAL MICROTUBULES IN THE SUB-APICAL REGION DURING TUBERIZATION IN SINGLE-NODE STEM SEGMENTS OF POTATO IN CULTURE

  K FUJINO, Y KODA, Y KIKUTA

  PLANT AND CELL PHYSIOLOGY 36 (5) 891 - 895 0032-0781 1995/07 [Refereed][Not invited]
   

  The initial events in tuberization were examined in single-node stem segments of potato, in which the tuberization was easily regulated in culture. The addition of 8% sucrose to the culture medium caused the cessation of elongation of lateral shoots and the swelling of the sub-apical region of each shoot. Swelling was first induced by lateral cell expansion, which was followed by periclinal cell division. The divided cells then expanded laterally. The alteration in the direction of growth was accompanied by the reorientation of arrays of cortical microtubules (MTs), which was monitored by immunofluorescence microscopy. Cells in the sub-apical region of elongating shoots had prominent transverse arrays of MTs. The MTs in swelling cells were oriented longitudinally with respect to the axis of the shoot. Finally, the arrays of MTs became completely disorganized. By contrast, the elongation of lateral shoots continued in GA(3)-treated segments and the cells in the sub-apical region of such shoots retained conspicuous transverse arrays of MTs during culture, even in the presence of a high concentration (8%) of sucrose.
• PLANTLET REGENERATION AND NOVEL PROTEIN-SYNTHESIS IN A LONG-TERM CULTURED CALLUS OF RICE IN RESPONSE TO ABSCISIC-ACID

  ZJ XU, K FUJINO, C FURUYA, Y KIKUTA

  JAPANESE JOURNAL OF CROP SCIENCE 64 (1) 109 - 114 0011-1848 1995/03 [Refereed][Not invited]
   

  Some physiological changes induced by abscisic acid (ABA) were investigated in a long-term cultured callus of rice, Oryza sativa, L. cultivar YUHKARA. The results were as follows: (1) The retardation of callus growth was evident, while dry matter content was apparently increased with the addition of abscisic acid (ABA). (2) Soluble protein content was increased both in fresh weight and dry matter bases. (3) The novel proteins (14, 18.5, 25, 45 kDa) were induced by ABA and disappeared after plantlet regeneration, while proteins of the same molecular weight existed in intact mature seed embryos and disappeared when the seeds were germinating. (4) Moreover, the electrophoretic patterns of total soluble proteins from callus being cultured with ABA, and from callus with regenerating plantlets, were very similar to those of total soluble proteins from mature seed embryos and germinating seed embryos in SDS polyacrylamide gel plates. (5) The practice that the callus precultured with addition of ABA, especially 10 mgL(-1) ABA, followed by transferring to a regeneration medium gave us a high frequency of plantlet regeneration observed in the long-term culture of callus.
• DETECTION OF IMMUNOLOGICALLY RELATED KUNITZ AND BOWMAN-BIRK PROTEINASE-INHIBITORS EXPRESSED DURING POTATO-TUBER DEVELOPMENT

  C MITSUMORI, K YAMAGISHI, K FUJINO, Y KIKUTA

  PLANT MOLECULAR BIOLOGY 26 (3) 961 - 969 0167-4412 1994/11 [Refereed][Not invited]
   

  Antiserum against a potato Kunitz-type proteinase inhibitor (PKPI) expressed in Escherichia coli was produced. In immunoblotting assays of proteins from potato tubers cultured in vitro, three proteins reacted to the antiserum, two of 20 kDa and one of 10 kDa. Their N-termini were sequenced. While the 20 kDa proteins showed 59 and 90% identity to PKPI, the 10 kDa one had 65% identity to soybean C-II proteinase inhibitor. Characterization of the temporal expression of these proteins showed that both could be detected from 10 days after induction of tuberization (DAI) in vitro, but the times when maximum amounts of PKPI and 10 kDa protein could be detected were different, corresponding to 22 and 32 DAI, respectively. The amounts of these proteins decreased in the following stages, and no positive reaction of the antiserum with mature tuber proteins could be found. The 20 kDa proteins were also detected in early stages of development of potato tubers grown in the held, indicating that these proteins are expressed during normal tuber development, and differ from the PKPIs reported previously.
• EXPANSION OF POTATO CELLS IN RESPONSE TO JASMONIC ACID

  K TAKAHASHI, K FUJINO, Y KIKUTA, Y KODA

  PLANT SCIENCE 100 (1) 3 - 8 0168-9452 1994 [Refereed][Not invited]
   

  Since jasmonic acid (JA) has strong potato tuber-inducing activity and tuberization of potato plants is initiated mainly by expansion of cells, it is highly likely that JA is capable of inducing expansion of potato cells. When disks cut from potato tubers were cultured on medium that contained JA, the disks began to swell markedly after 1 day in culture. Within 5 days in culture, the fresh weight of the disks doubled in the presence of JA at 3 x 10(-5) M. Light microscopy revealed that the swelling was due to the expansion and not the division of cells. JA exhibited this expansion-inducing activity at concentrations above 10(-5) M. Air-borne methyl jasmonate (JA-Me) also exhibited this activity. The cells that expanded in response to JA in the medium or to airborne JA-Me were localized on the lower side of each disk. The localization seemed to be a result of the greater availability of water. Sucrose in the culture medium was not necessary for the expansion of cells. The expansion-inducing activity appeared to be specific to JA and related compounds, since various plant hormones and a precursor of ethylene had no appreciable effects on the size of cells. Abscisic acid at concentrations above 10(-5) M and benzyl adenine at concentrations above 10(-4) M markedly inhibited the JA-induced expansion of cells. The expansion-inducing activities of JA and JA-Me seem to be associated with their tuber-inducing activities.
• REGENERATION VIA SOMATIC EMBRYOGENESIS AND/OR ORGANOGENESIS IN CALLUS FROM MATURE SEEDS OF ECHINOCHLOA-ORYZICOLA VASING

  ZJ XU, K FUJINO, Y KIKUTA

  JAPANESE JOURNAL OF CROP SCIENCE 62 (4) 614 - 620 0011-1848 1993/12 [Refereed][Not invited]
   

  Callus was initiated From mature seeds without the rinds of barnyard grass, Echinochloa oryzicola Vasing, on a modified Murashige and Skoog (MS) medium containing 2.0 similar to 20.0 mgl(-1) 2, 4-D. The rate of callus formation was greatly improved with the addition of 4 mgl(-1) tryptophan. Upon transfer to MS medium containing 6.0 mgl(-1) of 2, 4-D, the calli were sustained in subcultures. The plantlets were regenerated under light on MS medium involving various hormone combinations of 2, 4-D and cytokinins. In the regenerated plantlets 55% were normal plants and 45% abnormal plants including albinos. The processes of plantlet regeneration were observed after the plantlets were embedded in paraffin. The ratio of embryogenesis to organogenesis was found to be 25:75 and this ratio could not be effectively controlled by media using varying concentration of 2, 4-D, BA, kinetin, and zeatin.
• JASMONIC ACID-INDUCIBLE GENE-EXPRESSION OF A KUNITZ-TYPE PROTEINASE-INHIBITOR IN POTATO-TUBER DISKS

  K YAMAGISHI, C MITSUMORI, K TAKAHASHI, K FUJINO, Y KODA, Y KIKUTA

  PLANT MOLECULAR BIOLOGY 21 (3) 539 - 541 0167-4412 1993/02 [Refereed][Not invited]
   

  Messenger RNAs of a potato (Solanum tuberosum L.) Kunitz-type proteinase inhibitor(s) (PKPI) were present in potato disks excised from tubers stored for 14 months (old tubers) or 2 months (young tubers) after harvest, and disappeared during the aseptic culture. The PKPI mRNA accumulation was found to be induced in potato disks from the old tubers by the addition of jasmonic acid (JA) [3-oxo-2-(2'-cis-pentenyl)-cyclopentane-1-acetic acid].
• Effect of ABA on plantlet regeneration from rice-seed callus culture

  Xu Z., Fujino K., Kikuta Y.

  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan 日本作物学会 (32) 48 - 49 1991/12
• 馬鈴薯のプロトプラスト培養および再生個体について

  藤野 介延, 喜久田 嘉郎, 増田 清, 岡澤 養三

  植物組織培養 Japanese Society for Plant Cell and Molecular Biology 2 101 - 101 0289-5773 1985

Books etc

• Viability, DNA Synthesis and Cell Wall Regeneration on Potato Protoplasts

  Biotechnology in Agriculture and Forestry 1987

MISC

• Expression analysis of genes related to sugar metabolism during onion development

  清水こはる, 上野敬司, 小野寺秀一, 藤野介延, 志村華子  園芸学研究 別冊  22-  (1)  2023
• The screening of genes that show insensitivity to low tempeeature during rice booting stage

  山森晃一, 石黒聖也, 小笠原慧, 小出陽平, 藤野介延, 佐藤裕, 貴島祐治  育種学研究  22-  2020
• ダイズの柱頭上花粉発芽に関わるFTオルソログの機能

  平田万季, 針谷康平, 竹島亮馬, 松浦英幸, 藤野介延, XU M., 山田哲也, 阿部純  育種学研究  21-  2019
• Speciation of interceller localization mechanism of transposase from Antirrhinum mujus

  Ohigashi T, Zhou H, Yuasa I, Mitsui S, Koide Y, Tsugama D, Fujino K, Kishima Y  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan  59-  (0)  2  -3  2018  [Not refereed][Not invited]
  
• Comparative analyses of anther structure and genome-wide expression at booting stage under low-temperature stress among rice varieties

  山森晃一, 菊池駿太, 石黒聖也, 小笠原慧, 小出陽平, 藤野介延, 佐藤裕, 貴島祐治  日本育種学会・日本作物学会北海道談話会会報(Web)  58-  8‐9(J‐STAGE)  2017  [Not refereed][Not invited]
  
• Epigenetic-independent regulation of Tam3 in Antirrhinum majus detains transposase to the plasma membrane by targeting its BED-zinc finger domain

  Zhou Hua, Megumi Hirata, Ryo Osawa, Kaien Fujino, Yuji Kishima  GENES & GENETIC SYSTEMS  91-  (6)  359  -359  2016/12  [Not refereed][Not invited]
  
• ディリジェント様タンパク質の新たな機能の提案:ダイズの莢のねじれへの関わりについて

  片山健至, 船附秀行, 鈴木雅也, 廣瀬亜矢, 稲葉大貴, 山田哲也, 羽鹿牧太, 小松邦彦, 佐山貴司, 石本政男, 藤野介延  リグニン討論会講演集  60th-  10  -13  2015/10/23  [Not refereed][Not invited]
  
• ダイズの脱粒性を左右する裂莢性QTLのマップベースクローニング

  船附秀行, 鈴木雅也, 廣瀬亜矢, 稲葉大貴, 山田哲也, 羽鹿牧太, 小松邦彦, 片山健至, 佐山貴司, 石本政男, 藤野介延  育種学研究  17-  74  2015/03/21  [Not refereed][Not invited]
  
• ダイズ耐裂莢性遺伝子はディリジェント様タンパク質が関与する新たな裂莢機構を示す

  藤野介延, 鈴木雅也, 廣瀬亜矢, 稲葉大貴, 山田哲也, 羽鹿牧太, 小松邦彦, 片山健至, 佐山貴司, 石本政男, 船附秀行  日本植物生理学会年会要旨集  56th-  261  2015/03/09  [Not refereed][Not invited]
  
• Salt tolerance conferred by a plant endogenous peptide elicitor

  Doman K, Watanabe T, Fujino K, Yamaguchi Y  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan  54-  (0)  23  -24  2013  [Not refereed][Not invited]
  
• An endogenous factor that inhibits bolting in spinach plants, and analysis of bolting-related genes

  ABE Erika, FUJINO Kaien, YAMAGUCHI Yube, KODA Yasunori  日本作物學會紀事  81-  314  -315  2012/09/09
• Function of plant hormone for secondary growth of potato tuber

  TANAKA Hideyuki, KODA Yasunori, FUJINO Kaien  日本作物學會紀事  81-  158  -159  2012/03/29
• ダイズの難裂莢性DNAマーカー

  船附秀行, 羽鹿牧太, 山田哲也, 萩原誠司, 田中義則, 藤田正平, 石本政男, 辻博之, 藤野介延  新しい研究成果 北海道地域  2008-  144  -146  2009/12/04  [Not refereed][Not invited]
  
• Morphological analysis in pod-shattering using NILs for pod-shattering resistance QTL in soybean

  FUJINO Kaien, SATO Arisa, SUZUKI Masaya, FUNATSUKI Hideyuki  日本作物學會紀事  78-  134  -135  2009/09/29
• ダイズの難裂莢性DNAマーカー

  船附秀行, 羽鹿牧太, 山田哲也, 萩原誠司, 田中義則, 藤田正平, 石本政男, 辻博之, 藤野介延  研究成果情報 北海道農業  2008-  230  -231  2009/06/30  [Not refereed][Not invited]
  
• High-resolution mapping of a gene related to shattering resistance in soybean

  SUZUKI Masaya, FUJINO Kaien, ISHIMOTO Masao, FUNATSUKI Hideyuki  日本作物學會紀事  78-  344  -345  2009/03/27
• 106 Intracellular localization of antioxidant protein (peroxiredoxin) in rice

  Fujino Kaien, Tao Yan, Ogawa Kana, Koda Yasunori  Japanese Journal of Crop Science  77-  (2)  212  -213  2008/09/24
• Morphological analysis of pods using RHLs for pod-shattering resistance QTL in soybean

  FUJINO Kaien, TANAKA Yuka, SUZUKI Masaya, FUNATSUKI Hideyuki  Japanese Journal of Crop Science  77-  216  -217  2008/03/27
• Analysis of useful genes in buckwheat seed

  藤野 介延  年報  2005-  109  -114  2005
• Genomic location of a pod-shattering resistance gene from soybean cultivar 'Hayahikari'

  Funatsuki H, Ishimoto M, Tsuji H, Kawaguchi K, Hajika M, Fujino K  Report of the Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan  46-  (0)  43  -44  2005
• 16 Characterization of peroxiredoxin in rice suspension cells

  Tao Yan, Ogawa Kana, Koda Yasunori, Fujino Kaien  Japanese Journal of Crop Science  73-  (2)  32  -33  2004/10/20
• 137 Characterization of a gene expressed around dormant bud of potato microtuber

  Ogawa Kana, Koda Yasunori, Fujino Kaien  Japanese Journal of Crop Science  73-  (2)  274  -275  2004/10/20
• 138 Analysis of gene expressed in phloem tissues of potato

  Sato Mana, Manabe Hiroyuki, Ogawa Kana, Koda Yasunori, Fujino Kaien  Japanese Journal of Crop Science  73-  (2)  276  -277  2004/10/20
• 139 Molecular cloning of rutinase of Fagopyrum tataricum

  Harada Shotaro, Iida Akihito, Koda Yasunori, Fujino Kaien  Japanese Journal of Crop Science  73-  (2)  278  -279  2004/10/20
• Analysis of a novel allergen expressed during seed development of buckwheat : Fagopyrum esculentum Moench

  Fujino K., Maeda M., Ogawa K., Koda Y., Sugimura K., Funatsuki M.  Japanese Journal of Crop Science  71-  (2)  212  -213  2002/08/25
• 471 Immunoblotting法によるそばアレルゲン分子,BW8KDの解析

  松元 亮, 橋口 周平, 相原 雄幸, 藤野 介延, 伊東 祐二, 杉村 和久  アレルギー  51-  (9)  1024  -1024  2002  [Not refereed][Not invited]
  
• Characterization of transgenic potato plants expressed GUS gene at lateral buds.

  Ogawa Kana, Fujino Kaien, Miura Asuka, Koda Yasunori, Kikuta Yoshio  Japanese Journal of Crop Science  70-  (2)  347  -348  2001/09/27
• Characterization of antioxidant protein(Peroxiredoxin)in rice seeds.

  Tao Yan, Fujino Kaien, Xu Zheng-Jun, Koda Yasunori, Kikuta Yoshio  Japanese Journal of Crop Science  70-  (2)  345  -346  2001/09/27
• 449 そばアレルギー患者血清を用いたアレルゲン分子の同定

  松元 亮, 橋口 周平, 藤野 介延, 相原 雄幸, 伊東 祐二, 杉村 和久  アレルギー  50-  (9)  1011  -1011  2001  [Not refereed][Not invited]
  
• Somatic embryogenesis induced by abscisic acid from Carrot(Daucus carota L.)embryos and seedlings

  Nishiwaki M., Fujino K., Koda Y., Masuda K., Kikuta Y.  Japanese Journal of Crop Science  69-  (2)  236  -237  2000/10/04
• Production of antiserum against a mejor allergen in buckwheat seed

  Fujino Kaien, Funatsuki Wakako, Funatsuki Hideyuki, Aoki Tomoyuki, Kikuta Yoshio  Japanese Journal of Crop Science  68-  (2)  210  -211  1999/10/01
• Dormant bud formation in temperate grass species (Poaceae) cultured in vitro system

  Jamsran, U, Fujino, K, Kikuta, Y, Nakashima H  Journal of the Faculty of Agriculture Hokkaido University  69-  (3)  151  -169  1999  [Not refereed][Not invited]
  
• Analysis of gene action revealed by gene trap tagging of regulatory elements in potato

  三浦 明日香, 中谷 浩樹, 下野 嘉子, 藤野 介延, 喜久田 嘉郎  Japanese Journal of Crop Science  67-  (2)  178  -179  1998/10/08
• Analysis of an allergen expressed during seed development of buckwheat(Fagopyrum esculentum Moench)

  青木 智之, 藤野 介延, 丸山 雅子, 船附 秀行, 喜久田 嘉郎  Japanese Journal of Crop Science  67-  (2)  180  -181  1998/10/08
• 275 8KDaそばアレルゲンのN末アミノ酸配列の決定

  長田 由紀, 伊東 祐二, 前田 恵治, 高橋 由利子, 藤野 介延, 杉村 和久  アレルギー  47-  (9)  1013  -1013  1998  [Not refereed][Not invited]
  
• 122 Analysis of cDNAs corresponding to genes differentially expressed during seed development of buckwheat (Fagopyrum esculentum Moench)

  Fujino Kaien, Funatsuki Hideyuki, Inada Mizue, Shimono Yoshiko, Kikuta Yoshio  Japanese Journal of Crop Science  66-  (1)  244  -245  1997/04/02
• 144 ABA induced dormant bud formation in grasses

  Undarmaa Jamsran, Fujino Kaien, Kikuta Yoshio, Nakashima Hiroshi  Japanese Journal of Crop Science  66-  (1)  288  -289  1997/04/02
• ANALYSIS OF ABSCISIC ACID RESPONSIVE PROTEIN(24.5KD)OF RICE(olyza sativa L. )

  TANAKA Kaori, FUJINO Kaien, ASANO Tetsumoto, FURUYA Chihiro, XU Zheng-jun, KIKUTA Yoshio  Plant and cell physiology  37-  141  -141  1996/03
• JASMONIC ACID INDUCES TRANSVERSEORIENTATION OF CORTICAL MICROTUBULES IN CELLS OF POTATO TUBER

  TAKAHASHI Kiyoshi, FUJINO Kaien, KIKUTA Yoshio, KODA Yosunori  Plant and cell physiology  36-  S131  1995/03
• C3-84 ジャスモン酸による馬鈴薯塊茎細胞の肥大におけるブラシノライドの促進作用

  高橋 淳, 藤野 介延, 喜久田 嘉郎, 幸田 泰則  植物化学調節学会研究発表記録集  (29)  155  -156  1994/10/13
• Preferential Synthesis of mitochondrial DNA during the initial stage of tissue Growth in Potato explant cultures.

  Masuda, K, Kikuta, Y, Fujino, K, Okazawa, Y  Journal of the Faculty of Agriculture Hokkaido University  66-  (1)  13  -25  1994  [Not refereed][Not invited]
  
• 88 Effect of abscisic acid on rice cultured cells

  Furuya Chihiro, Xu Zhengjun, Fujino Kaien, Kikuta Yoshio  Japanese Journal of Crop Science  62-  (2)  175  -176  1993/10/18  

  イネの培養細胞の蛋白質に及ぼすアブシジン酸の影響について
• 89 Trangent gene expression of potato protoplasts

  Fukase Takako, Fujino Kaien, Kikuta Yoshio  Japanese Journal of Crop Science  62-  (2)  177  -178  1993/10/18  

  バレイショプロトプラストのエレクトロポレーション法による遺伝子導入の最適条件について検討した.
• 142 タイヌビエ種子カルスの不定胚形成

  徐 正君, 藤野 介延, 喜久田 嘉郎  日本作物學會紀事  60-  (2)  283  -284  1991/10/30
• 93 馬鈴薯茎断片培養系の塊茎形成過程における細胞壁構成成分の変化

  藤野 介延, 喜久田 嘉郎, 入宇田 尚樹, 幸田 泰則, 岡澤 養三  日本作物學會紀事  59-  (2)  185  -186  1990/10/10
• Protoplast Culture of Potato : an Improved Procedure for Isolating Viable Protoplasts

  Kikuta, Y, Fujino, K, Saito, W, Masuda, K, Okazawa Y  Journal of the Faculty of Agriculture Hokkaido University  62-  (4)  614  -620  1986  [Not refereed][Not invited]
  

Awards & Honors

• 2010/03 日本育種学会 日本育種学会論文賞
   A major QTL, qPDH1, is commonly involved in shattering resistance of soybean cultivars.

Research Grants & Projects

• 植物の防御機構等に関与する不溶性プロアントシアニジン合成機構の生理的解析

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2021/04 -2025/03 

  Author : 鈴木 達郎, 藤野 介延, 澤井 祐典
• ジャガイモ塊茎における分裂組織の成長制御メカニズムの解明

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2021/07 -2023/03 

  Author : 志村 華子, 藤野 介延

   

  二次肥大や二次成長などによって形が奇形になることはジャガイモ塊茎の品質を著しく低下させる。地上部で生じるとされる塊茎形成誘導シグナルが地下茎先端の分裂組織へ移動し、分裂組織の周辺組織が肥大して塊茎ができるといわれているが、このような劇的な形態変化の制御、塊茎の休眠や萌芽に関わる分子メカニズムはまだ不明な点が多い。本研究では塊茎発達や休眠の制御に関わる分子メカニズムを解明することを目的とし、マイクロチューバーを用いた培養系やジャガイモ塊茎に休眠不良を起こすウイロイドを用いた研究を進めている。今年度はジャガイモ培養物を用いてマイクロチューバーを誘導し、さらにマイクロチューバーの休眠を打破して二次成長や二次肥大を起こす条件の検討を行ったところ、マイクロチューバーで効率的に二次肥大を再現することができた。また、塊茎の休眠や萌芽に及ぼす遺伝子発現について網羅的に調べるために、RNAseq解析を行うことにした。材料の準備のため、ジベレリンやサイトカイニンの有無、マイクロチューバ基部組織の有無など条件の異なる培養物を大量に育成し、各試験区の萌芽直前の組織からRNA抽出を進めた。塊茎はデンプンが豊富であるため、一般的に純度の高いRNAを抽出するのが困難である。そこでRNA抽出方法の検討も進め、いくつかの条件検討の結果、品質の良いRNAを精製することができた。試験区の反復も加えて40サンプル程度を用意してRNAseqに供試し、得られたデータの解析を進めている。また、ウイロイド感染による奇形塊茎をマイクロチューバーで再現するために、ジャガイモ培養シュートへ無菌的にウイロイドを接種することを試みた。今後感染確認を行うとともに塊茎誘導条件で変化がみられるかを調査する。
• Physiological analysis of pericarp dehiscence mechanism involved in yield

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2017/04 -2020/03 

  Author : Fujino Kaien

   

  Tartary buckwheat cultivars that allow easy dehulling are called rice-type cultivars. The rice and non-rice hull types are determined by a single gene, but this gene is unclear. Here, I show that cells underneath the epidermis in the proximity of ovary midribs undergo periclinal cell divisions in non-rice-type cultivars but do not in a rice-type cultivar. The cells that arose from the periclinal cell divisions later underwent lignification, which should increase mechanical strength of hulls. In RNA sequencing, a partial mRNA of an AGAMOUS ortholog in Tartary buckwheat (FtAG) was found to be absent in the rice-type cultivar. By RNA sequencing and linkage analysis, we also show that FtAG is a candidate for the gene that promotes differentiation of the cells undergoing the periclinal cell divisions and that thereby determines the RT and NT phenotypes in Tartary buckwheat.
• Mechanism of enhanced salinity tolerance by an endogenouse peptide elicitor in Arabidopsis

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2014/04 -2017/03 

  Author : Yamaguchi Yube, DOMAN Kohei, FUJINO Kaien, MURAKAMI Yuhei, FUJII Kenta

   

  We investigated the mechanism of enhanced salinity tolerance in Arabidopsis thaliana overexpressing AtproPep1, a precursor protein of 23-amino acid peptide AtPep1. Amount of AtproPep1 protein in overexpression lines was increased in response to NaCl treatment. Expression of 2 matacaspase genes and SOS genes, which were predicted to process AtproPep1 and important for Na+ exclusion from the cells,respectively, were higher in the overexpression lines than in WT. Since Na+ content was kept lower in the overexpression lines under NaCl stress, we are speculating that promotion of AtPep1 generation by NaCl treatment in the AtproPep1 overexpression lines led to activation of Na+ exclusion from cells, which are directly related to enhanced salt tolerance.
• Possibility of active secretion of Arabidopsis enodgenous peptide elicitor

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2012/04 -2015/03 

  Author : YAMAGUCHI Yube, DOMAN Kohei, FUJINO Kaien

   

  Arabidopsis AtPep1, an endogenous peptide elicitor, is thought to be somehow released from infected cells and induce defense responses. To examine how Atpep1 is released from cells, we generated AtPep1 overexpression lines and tried to detect AtPep1 by antibody. AtPep1 overexpression lines showed shifted balance in plant hormones even under no stress growth conditions. Therefore it is possible that AtPep1 is release actively from living cells. We also grew AtPep1-overexpression lines under various stress conditions to see clear evidence for active release, and found that these lines were more tolerate to high salinity environment.
• Analysis of pod structure responsible for soybean shattering resistance

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2012/04 -2015/03 

  Author : FUJINO Kaien

   

  Pod dehiscence (shattering) causes a significant yield loss in legume crops. However, little is known about the genetic basis of the shattering resistance in those crops. Map-based cloning of a major quantitative trait locus controlling pod dehiscence in soybean and complementation test revealed that the gene identified, Pdh1, encoded a dirigent-like protein. In shattering-resistant cultivars, the gene was defective with a premature stop codon. Functional Pdh1 was specifically expressed in pod walls. Comparison of near-isogenic lines indicated that Pdh1 regulate torsion of dried pod walls, which functions as force to dehisce binding of pod walls. These findings provide useful information for plant breeding and point to a new biological role for the dirigent-like proteins.
• ANALYSIS OF ALLERGENS AND USEFUL GENES IN BUCKWHEAT SEED STORAGE PROTEINS

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2003 -2004 

  Author : FUJINO Kaien, MASUDA Kiyoshi

   

  Buckwheat (Fagopyrum esculentum M.) gene EL0847 that showed an increase in transcript during the early stages of seed development was isolated from immature seeds harvested 14 days after pollination by differential screening. The deduced amino acid sequence of EL0847 shows several repeats of alanine, glutamate and valine which suggests a structural role for the encoded protein, and reveals similarity to the deduced amino acid sequence of Hev b5, which is believed to be a major allergen from natural rubber latex. In an E.coli. expression system, the translation product of a cDNA encoding the EL0847 was not recognized by sera from allergic patients. Based on the N-terminal amino acid sequence of the rutinase which showed flavonol 3-glucosidase activity, a degenerate primer were synthesized and 3' RACE PCR amplification was carried out. The deduced amino acid sequence of rutinase gene shows similarity to the beta-glucosidase in plants. Antiserum was raised against the translation product of a cDNA encoding the rutinase in an E.coli. expression system and immunoblotting of total protein from tatary buckwheat seeds (Fagopyrum tataricum G.) revealed that two proteins reacted with the antiserum. The immunoblotting and genomic Southern analysis data indicated the rutinase gene were encoded by gene family. In an E.coli, and a S.pombe expression system, the translation product of a cDNA encoding the rutinase showed little flavonol 3-glucosidase activity.
• ANALYSIS OF ALLERGENS IN BUCKWHEAT SEED STORAGE PROTEINS

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2001 -2002 

  Author : FUJINO Kaien, MASUDA Kiyoshi

   

  Buckwheat (Fagopyrum esculenturn Moench) genes FA02 and FA18 were isolated from immature seeds harvested 14 days after polination, and were found to encode legumin-like proteins that are expressed during seed development. The deduced amino acid sequence of FA02 was identical to the N-terminal amino acid domain of BW24KD, which is believed to be a major buckwheat allergen. It was predicted that FA02 would be cleaved to generate two separate components, a 41.3 kDa alpha-subunit and a 21 kDa beta-subunit. It was deduced from molecular mass and N terminal amino acid that BW24KD was the beta-submit of FA02. Antiserum was raised against the deduced FA02 beta-subunit and immunoblotting of total protein from buckwheat seeds (Fagopyrum esculentum M. and Fagopyrum tartaricum M.) revealed that several groups of proteins reacted with the antiserum. Based on the N-terminal amino acid sequence of the 10 kDa buckwheat allergenic protein (BW10KD) which strongly reacted with IgE in 50% of allergic patients, a degenerate primer were synthesized and 3'RACE PCR amplification was carried out. Fe2SA1 cDNA clone, which was isolated from a cDNA library made of immature buckwheat seeds using as a probe a3' RACE PCR clone, was partially identical to 2S albumin seed storage protein. The deduced amino acid sequence of Fe2SA1 was partially identical to the 16 and 18 kDa molecule, which are believed to be major buckwheat allergens. In an E. coli. Expression system, the translation product of a cDNA encoding the Fe2SA1 was strongly recognized by sera from allergic patients.
• GENE ACTION DURING REDIFFERENTIATION AND EMBRYOGENlC DEVELOPMENT IN CROPS CELLS CULTURED, IN VITRO

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1997 -1999 

  Author : KIKUTA Yoshio, FUJINO Kaien, KODA Yasunori

   

  By using in vitro system of rice, bentgrass, carrot and potato, the accumulation of knowledge in redifferentiation and embryogenic callus cultures has been commenced. The physiological process of specifical gene activation was observed by the RT-PCR differential displays for somatic embryogenesis of rice, carrot and for dormant bud formation of bentgrass cultures. The role of ABA was found to be important for the induction of embryogenic callus formation in rice and carrot, and of dormant bud formation in bentgrass. On this line of research, the gene expression of peroxiredoxin (Per1) , dehydrin (DH-a18) and viviparous1 (VP1) occurred in cultured cells during the induction of ABA regime. The mRNAs of Per1, DHa18 and VP1 were induced by ABA in the long-term cultured callus of rice and dormant tissue of bentgrass cultures but disappeared after plentlet regeneration, or sprouting. While the transcripts existed in intact mature seed embryoes and disappeared when the seeds were germinating. Sustaining of the embryogenic callus cultures could be some extent achieved by the addition of ABA in medium. This hormone could be responsible for regenerating somatic embryoes and for normal embryogeny in rice plants. Eukaryotic translation initiation factor 5A(eIF-5A) is one of factors necessary for the initiation of eukaryotic cellular protein biosynthesis. Little is known in plant eIF-5A. We have isolated a cDNA encoding SteIF-5A. Five cDNA were cloned from potato showing differential expression, mainly developing tuber and fruit. This suggests that the expression is the highest in metabolic active cell. To protect cellular integrity against damage caused by reactive oxygen and free radicals, plants possess an array of antioxidant systems. Peroxiredoxins are one of enzymatic antioxidants, the functions of which are the most resposible for abiotic stresses upon redifferentiation of crop cells.
• 禾本科植物の種子カルスに関する生理学的研究

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1996 -1996 

  Author : 藤野 介延

   

  稲カルスにおいて形態形成を誘導する前にABA処理やストレスを与えることにより再分化率が向上する。そこでABA処理を行ったカルスよりタンパクを抽出しSDS-PAGEを行ったところ無処理区には存在しないタンパクがいくつかみられた。このうち24.5kDのタンパクを精製し抗体を作成した。このタンパクのアミノ酸シークエンス等を行なったところオオムギの種子の成熟過程に出現するタンパク質と高い相同性が見られた。このアミノ酸配列からプライマーを合成しRT-PCRを行い、増幅したDNA断片をプローブとしライブラリーよりcDNAをスクリーニングした。このcDNAのシークエンスを行い、塩基配列を決定し(D63917)RPer-1とした。RPer-1は酵母や人など生物に広く存在するTSA(Thiol-specific Antioxidant)又はペルオキシレドキシンと高い相同性を示した。RPer-1を大腸菌で発現させたところイネの24.5kDの抗体と反応し、またTSA活性を示した。RPer-1はイネの培養カルスにおいて、恒常的にわずかながら発現しているが、3mg/1 ABA処理に対し1時間以内に強く発現し、また乾燥処理においてはABAよりもはるかに強く発現した。 以上のように本研究では、ABAにより誘導される蛋白質のアミノ酸配列よりそれをコードする遺伝子の塩基配列を決定し、またその発現を研究した。今後はこの遺伝子が形態形成にどのように関与しているか検討し、これにより稲や大麦などの禾本科の組織培養における種子からのカルス誘導や効率の高い形態形成系の確率を図るものである。
• 禾本科植物のカルス培養における形態形成とそれに関する生理学的研究

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1995 -1995 

  Author : 藤野 介延

   

  イネカルスからの形態形成を誘導する際に、前処理としてABAやストレスを与えると再分化率が向上する。そこでABA処理を行ったイネカルス(Oryza sativa,L.cv.Yuhkara)よりタンパクを抽出しSDS-PAGEを行ったところABA無処理区には存在しないタンパクがいくつかみられた。このうち24.5kDのタンパクを精製し抗体を作成した。この抗体を使用しウェスタンブロッティングを行ったところABAだけではなく高濃度のNaClやマンニトールにも反応することが判明した。また、発芽後このタンパクは日を追って消失するが3日目の幼植物体を乾燥条件においたところこのタンパクの発現が確認された。このことはイネ品種ユ-カラだけではなく他のジャポニカ種やインディカ種、オオムギの幼植物体でも確認された。このタンパクのN末端アミノ酸シークエンスを行なったところオオムギの種子の成熟過程に出現するタンパク質と高い相同性が見られた。そこで登熟過程のイネの胚におけるこのタンパクの局在を免疫組織学的手法により調べたところ細胞質に存在しているものと考えられた。 以上の結果から、今後はこのタンパクの機能を確認するために塩基配列の決定を行い、また未熟胚や完熟胚における局在化を明らかにし種子の成熟過程とカルスからの形態形成過程での発現を比較検討する。またこのタンパクの特異性を知るために稲の他品種や他の禾本科での発現を検討し効率の高い形態形成系の確立を図る必要がある。
• GENE ACTION DURING CELLULAR DIFFERENTIATION AND RELATED ORGANOGENESIS IN CROPS CULTURED,IN VITRO

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1993 -1995 

  Author : KIKUTA Yoshio, FUJINO Kaien, KODA Yasunori

   

  By using in vitro tuberization system of potato, the accumulation of potato kunitz type proteinase inhibitor (PKPI) mRNA was specifically detected in tubers during the early stage of development, and thus expected to be a molecular marker of potato tuberization. Furthermore, the accumulation of PKPI-mRNA was also found when tissues were treated with cool to warm atmosphere by ascending temperature, and by addition of jasmonic acid. The novel proteins (14,18.5,24.5,45kDa) were induced by ABA in long-term cultured callus of rice and disappeared after plentlet regeneration, while proteins of the same molecular weight existed in intact mature seed embryos and disappeared when the seeds were germinating. Sustaining of embryogenic callus cultures could be some extent achieved by the addition of ABA in medium. This hormone could be responsible for regenerating somatic embryos and for normal embryogeny in rice plants. A protocpl of the trangent gene expression in electroporated protoplasts has been developed for the introduction of foreign DNA into cells from cultured potato, tobacco, carrot, and rice. The method yields high amounts of the reporter enzyme B-glucuronidase (GUS) when cognate genes are driven by the promotors of NOS and 35S,and by upstream sequences of PKPI from potato tuber and Actin-1 from rice actin genes. Comparisons with expression in potato, tobacco, carrot, and rice cells indicate that trangent assays can be used to investigate promotor acivation and enhancer function.
• 禾本科植物の種子カルス誘導とそれに関する生理学的研究

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1993 -1993 

  Author : 藤野 介延

   

  禾本科のうち稲では既にプロトプラストからの個体再生系が確立されている。そこで稲と大麦のプロトプラストにおけるエレクトロポレーションの条件を検討した。また大豆の振盪培養細胞ならびに馬鈴薯の葉肉プロトプラストとも比較した。振盪培養細胞からプロトプラストを調整しpBI221(35Sプロモーター+GUS)のエレクトロポレーションを行った。処理後3日目のプロトプラストのグロクローニダーゼの活性をMUGによる蛍光値により検討した。稲の場合、GUS活性は電気容量250muF時で電圧が900V/cmのときに最も高い活性を示した。また大麦の場合活性の最大値は120V/cmで稲・大豆の振盪培養細胞に比較し高い値を示した。供試するDNAの精製方法によっても影響を受けた。塩化セシウムによる精製とQIAGEN(QIAGEN社)により精製を比較した場合QIAGENによるほうが1.5倍近く活性が高くなった。しかしながら35Sプロモーターによる発現はトランジェントアッセイの場合においても双子葉植物と比較してかなり低かった。 大麦の誘導組織の違いによるカルスについて比較検討した。種子の芽生えの生長点からカルス誘導を行いそれらの振盪培養による維持・増殖を行なった。これらのカルスと未熟配胚より誘導したカルスのタンパク質の相違をSDS-PAGEにより検討した。これらのカルスは両者とも品種ディサより誘導した。培養期間は1年以上経過しており安定した細胞系統を使用した。幾つかのタンパク(主に90kD,40kD,33kD,19kD,17kD)において差が見られた。これが誘導組織による継代培養中の変異か不明であるが今後これらの系統を比較することで形態形成能の違いについて検討を加える必要がある。
• Studies of developmental physiology of cell differenciation in crop tissues and cells cultured, in vitro.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1989 -1991 

  Author : KIKUTA Yoshio, FUJINO Kaien, KODA Yasunori

   

  Studies on developmental physiology of cell differentiation in crop tissues and cells cultured, in vitro. Abstract By employing plant tissue and cell culture techniques, the process of growth and differentiation of plant cells can be controlled and modified. An investigation was undertaken with the hope that some clue might be found as to the relationship between the formation of tubers in potato, somatic embryos and of adventitious shootbuds in rice cells cultured, in vitro, and the effects of certain hormonal and enviromental conditions on the induction and expression of genes for the growth and differentiation of the crop cells. The material used was potato plant (Solanus tuberosum L. cv Irish Cobbler and cv May Queen) and rice plant (OryLa sativa L. cv Ishikari) and Barnyardgrass (Echinochloa oryzicola Vasing). In order to study the process of tuberization in potato, the major turber proteins, such as patatin, 22 k Da protein family, and proteinase inhibitors have been used as biochemical markers. We established a cDNA library from 'in vitro' tubers (cv Irish Cobbler), and a tuber specific cDNA clone, cPTI, was isolated by differential screening with the mRNA from leaves. The nucleotide sequence and the corresponding amino acid sequence were deduced. Transcripts of a potato Kunitz-type proteinase inhibitor (PKTI) gene were present in discs excised from tubers stored for 14 months as well as in those from 2 months after harvested, and the PKTI gene expression was induced in potato discs by the addition of jasmonic acid (JA) [3-oxo-2-(2'-cis-pentenyl)-cyclopentane-l-acetic acid], the most actively at 10 muM after 24 h treatment. While, the patatin transcripts were also present in discsfrom stored tubers but less than those of the PKPI transcripts. Callus was initiated from mature seed of Barnyardgrass on a modified Murashige and Skoog medium containing 2.0-20.0mg/1 2, 4-D, 30g/1 sucrose. The rate of callus initiation was increased with the addition of 4mg/1 Tryptophan. Calluses were subcultured in medium containing 6.0mg/1 2.4-D, 30g/1 sucrose. The plantlets were regenerated under the light on MS medium involving various hormonecombination (0.05- 0.5mg/1 2, 4-D ; 0.05mg/1 2, 4-D plus 3.0mg/1 BA ; 0.05mg/1 2, 4-D plus 5.0mg/1 kinetin or 0.05mg/1 2, 4-D plus 2.0mg/1 zeatin). In the regenerated plantlets, there were normal plant 55% and abnormal plant 45% (albino etc.). The processes of plantlet regeneration of rice were also observed through the supplementation of ABA and proline in medium with the relation this to the activity of nitrate reductase of cells. The proportion of embryogenesis and organgenesis could not be successfully controlled by medium ingredients using 2, 4-D, BA, kinetin and zeatin.
• バレイショの塊茎形成
• Tuber formation of potato

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