Mar. 2010, 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.

Dynamics of high pathogenicity avian influenza virus infection with multiple introductions in a crow flock in an urban park in Hokkaido, Japan.
Norikazu Isoda, Takahiro Hiono, Yik Lim Hew, Fumihito Takaya, Bao Linh Nguyen, Daiki Kobayashi, Kaien Fujino, Yoshihiro Sakoda
Comparative immunology, microbiology and infectious diseases, 121, 102367, 102367, Aug. 2025, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Since 2021, high pathogenicity avian influenza viruses (HPAIVs) of the H5N1 clade 2.3.4.4b has been circulating globally, not only in domestic poultry but also in wild birds, both migratory and resident species. In March to May 2022, March to April 2023, and January to April 2024, crow die-offs were reported in an urban garden in Hokkaido, Japan, raising suspicions of HPAIV infection. Since August 2022, all dead carcasses were investigated for HPAIV detection and isolation. Phylogenetic analysis of the H5 hemagglutinin gene revealed that all detected HPAIVs belonged to clade 2.3.4.4b, a dominant lineage in Hokkaido since early 2022. Two distinct subgroups were identified: G2d (in 2022-2024) and G2a (in 2024). A maximum clade credibility tree, based on concatenated nucleotide sequences of the isolates, suggested that multiple distinct types of HPAIVs were introduced into the garden in rotation during the winters of 2022-2023 and 2023-2024. Infectious HPAIVs were isolated not only from the lungs and brains but also from the rectal contents of the dead crows, with no apparent difference in viral titers between the two subgroups. The case reproduction numbers of HPAIV infection in the crow flock ranged from 0.52 and 1.57 in the spring of 2022 and from 0.55 to 1.78 in the spring of 2023, suggesting that the contiguous HPAIV infections in the crows were due to multiple introductions into the flock. Crow can play a key role of potential spread to other animals, poultry and wildlife in urban areas or humans in rural areas.

Anther transcriptomes in cold-tolerant rice cultivars tend to show insensitive responses
Koichi Yamamori, Seiya Ishiguro, Kei Ogasawara, Kayyis Muayadah Lubba, Kaien Fujino, Kazumitsu Onishi, Yutaka Sato, Yuji Kishima
Plant Stress, 100700, 100700, Elsevier BV, Jan. 2025, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
Scientific journal

VIP1 and its close homologs confer mechanical stress tolerance in Arabidopsis leaves.
Hyuk Sung Yoon, Kaien Fujino, Shenkui Liu, Tetsuo Takano, Daisuke Tsugama
Plant physiology and biochemistry : PPB, 215, 109021, 109021, 06 Aug. 2024, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, VIP1, an Arabidopsis thaliana basic leucine zipper transcription factor, and its close homologs are imported from the cytoplasm to the nucleus when cells are exposed to mechanical stress. They bind to AGCTG (G/T) and regulate mechanical stress responses in roots. However, their role in leaves is unclear. To clarify this, mutant lines (QM1 and QM2) that lack the functions of VIP1 and its close homologs (bZIP29, bZIP30 and PosF21) were generated. Brushing more severely damaged QM1 and QM2 leaves than wild-type leaves. Genes regulating stress responses and cell wall properties were downregulated in brushed QM2 leaves and upregulated in brushed VIP1-GFP-overexpressing (VIP1-GFPox) leaves compared to wild-type leaves in a transcriptome analysis. The VIP1-binding sequence AGCTG (G/T) was enriched in the promoters of genes downregulated in brushed QM2 leaves compared to wild-type leaves and in those upregulated in brushed VIP1-GFPox leaves. Calmodulin-binding transcription activators (CAMTAs) are known regulators of mechanical stress responses, and the CAMTA-binding sequence CGCGT was enriched in the promoters of genes upregulated in the brushed QM2 leaves and in those downregulated in the brushed VIP1-GFPox leaves. These findings suggest that VIP1 and its homologs upregulate genes via AGCTG (G/T) and influence CAMTA-dependent gene expression to enhance mechanical stress tolerance in leaves.

Characterizing the role of PP2A B'' family subunits in mechanical stress response and plant development through calcium and ABA signaling in Arabidopsis thaliana.
Hyuk Sung Yoon, Kaien Fujino, Shenkui Liu, Tetsuo Takano, Daisuke Tsugama
PloS one, 19, 11, e0313590, 2024, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Protein phosphatase 2AB'' (PP2A B'') family subunits have calcium-binding EF-hand motifs, facilitating interaction with PP2A substrates. In Arabidopsis thaliana, the PP2A B'' family subunits consist of six members, AtB''α-ε and FASS. These subunits can interact with a basic leucine zipper transcription factor, VIP1, and its close homologs. Mechanical stress triggers PP2A-mediated dephosphorylation of VIP1 and its close homologs, leading to nuclear localization and gene upregulation to alleviate touch-induced root bending and leaf damage. However, the physiological roles of PP2A B'' family subunits in the mechanical stress response in Arabidopsis remain unclear. This study aims to characterize such roles. A quadruple knockout mutant with T-DNA insertions in AtB''α, AtB''β, AtB''γ, and AtB''δ was generated. atb''αβγδ mutants exhibited no significant damage upon brushing or touch-induced root bending compared to the wild type. Transcriptome analysis showed a significant decrease in the expression of CYP707A3, a gene potentially targeted by VIP1 that regulates abscisic acid (ABA) catabolism, in the atb''αβγδ mutant compared to wild type leaves. However, other genes, including XTH23, EXLA1, and CYP707A1, also VIP1 targets, exhibited similar induction in both brushed atb''αβγδ mutants and wild type leaves. We observed an enrichment of the CAMTA motif, CGCG(C/T) in the promoters of genes showing downregulated expression levels in brushed atb''αβγδ leaves compared to brushed wild type leaves. These findings suggest that PP2A B'' family subunits exhibit functional redundancy in the VIP1-dependent pathway but influence CAMTA-dependent gene expression under mechanical stress. Under calcium-deficient and ABA-supplemented conditions, growth of atb''αβγδ seedlings was retarded when compared to wild type and single knockout mutants, atb''γ and atb''δ, indicating a crucial role in plant development by modulating calcium or ABA signaling.

Transcriptomic Analyses Reveal the Role of Cytokinin and the Nodal Stem in Microtuber Sprouting in Potato (Solanum tuberosum L.).
Xia Zhang, Kaien Fujino, Hanako Shimura
International journal of molecular sciences, 24, 24, 15 Dec. 2023, [Peer-reviewed], [Corresponding author], [International Magazine]
English, Scientific journal, In potatoes, tuber secondary growth, especially sprouting, deforms the tubers and severely lowers their commercial value. Tuber sprouting is induced by signal substances, such as gibberellin (GA), which are transported to the tuber from the plant body. The molecular mechanism underlying GA-induced sprouting remains ambiguous. Here, we tried to recreate tuber secondary growth using in vitro stemmed microtubers (MTs) (with the nodal stem attached) and MT halves (with the nodal stem entirely removed). Our experiments showed that GA alone could initiate the sprouting of stemmed microtubers; however, GA failed to initiate MT halves unless 6-benzyladenine, a synthetic cytokinin CK, was co-applied. Here, we analyzed the transcriptional profiles of sprouting buds using these in vitro MTs. RNA-seq analysis revealed a downregulation of cytokinin-activated signaling but an upregulation of the "Zeatin biosynthesis" pathway, as shown by increased expression of CYP735A, CISZOG, and UGT85A1 in sprouting buds; additionally, the upregulation of genes, such as IAA15, IAA22, and SAUR50, associated with auxin-activated signaling and one abscisic acid (ABA) negative regulator, PLY4, plays a vital role during sprouting growth. Our findings indicate that the role of the nodal stem is synonymous with CK in sprouting growth, suggesting that CK signaling and homeostasis are critical to supporting GA-induced sprouting. To effectively control tuber sprouting, more effort is required to be devoted to these critical genes.

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, Springer US, 08 Dec. 2023
In book

Construction of a de novo assembly pipeline using multiple transcriptome data sets from Cypripedium macranthos (Orchidaceae)
Kota Kambara, Kaien Fujino, Hanako Shimura
PLOS ONE, 06 Jun. 2023, [Peer-reviewed]
Scientific journal

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, Apr. 2023, [Peer-reviewed]
English, Scientific journal

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, Oxford University Press (OUP), 11 Aug. 2022, [Peer-reviewed]
Scientific journal, 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, 30 Sep. 2021, [Peer-reviewed], [Corresponding author], [International Magazine]
English, Scientific journal, 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, 07 Sep. 2021, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Sep. 2021, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, 01 Jan. 2021, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, 15 Apr. 2020, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, 20 Mar. 2020, [Peer-reviewed], [Corresponding author], [International Magazine]
English, Scientific journal, 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, Feb. 2020, [Peer-reviewed], [Last author], [International Magazine]
English, Scientific journal, 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, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, 18 Nov. 2019, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, 15 Oct. 2019, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Feb. 2019, [Peer-reviewed], [Last author], [International Magazine]
English, Scientific journal, 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, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, 31 Dec. 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Oct. 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Apr. 2017, [Peer-reviewed]
English, Scientific journal

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, Feb. 2017, [Peer-reviewed]
English, Scientific journal

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, Feb. 2017, [Peer-reviewed]
English, Scientific journal

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, 2017, [Peer-reviewed]
English, Scientific journal

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, Jun. 2015, [Peer-reviewed]
English, Scientific journal

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, 55, 15, 16, The Crop Science Society of Japan, Dec. 2014
Japanese

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, Dec. 2014, [Peer-reviewed]
English, Scientific journal

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, Apr. 2014, [Peer-reviewed]
English, Scientific journal

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, Feb. 2014, [Peer-reviewed]
English, Scientific journal

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, Feb. 2014, [Peer-reviewed]
English, Scientific journal

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, Jan. 2011, [Peer-reviewed]
English

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, Jan. 2011, [Peer-reviewed]
English, Scientific journal

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, Mar. 2010, [Peer-reviewed]
English, Scientific journal

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, Dec. 2009, [Peer-reviewed]
English, Scientific journal

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, Nov. 2009, [Peer-reviewed]
English, Scientific journal

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, Apr. 2009, [Peer-reviewed]
English, Scientific journal

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, 58, 1, 63, 69, Japanese Society of Breeding, 18 Apr. 2008, [Peer-reviewed], [Last author]
English

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, 2008, [Peer-reviewed]
English, Scientific journal

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, 01 Apr. 2006, [Peer-reviewed]

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, 222, 0, 238, 238, CROP SCIENCE SOCIETY OF JAPAN, 2006, [Peer-reviewed]

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, 46, 83, 84, The Crop Science Society of Japan, Dec. 2005
Japanese

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, May 2004
English, Scientific journal

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, 43, 113, 114, The Crop Science Society of Japan, Nov. 2002
Japanese

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, 37, 102, 103, The Crop Science Society of Japan, Dec. 1996
Japanese

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, Jul. 1995, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal

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, Mar. 1995, [Peer-reviewed]
English, Scientific journal

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, Nov. 1994, [Peer-reviewed]
English, Scientific journal

EXPANSION OF POTATO CELLS IN RESPONSE TO JASMONIC ACID
K TAKAHASHI, K FUJINO, Y KIKUTA, Y KODA
PLANT SCIENCE, 100, 1, 3, 8, 1994, [Peer-reviewed]
English, Scientific journal

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, Dec. 1993, [Peer-reviewed]
English, Scientific journal

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, Feb. 1993, [Peer-reviewed]
English

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, 32, 48, 49, The Crop Science Society of Japan, Dec. 1991
Japanese

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, 2, 101, 101, Japanese Society for Plant Cell and Molecular Biology, 1985
Japanese

Genome sequencing of two strains of mycorrhizal fungi from Cypripedium macranthos var. rebunense
神原昂太, 藤野介延, 志村華子, 日本菌学会大会講演要旨集, 68th, 2024

Virus detection in Cypripedium macranthum var. rebunense and its mycorrhizal fungi using multiple de novo assembly tools
神原昂太, 志村華子, 藤野介延, 増田税, 日本ウイルス学会学術集会プログラム・予稿集(Web), 70th, 2023

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

Transcriptome analysis using RNA-seq of watermelon parthenocarpy induced by pollen from different genera in the Cucurbitaceae
濱田寛也, 杉山慶太, 藤野介延, 志村華子, 園芸学研究 別冊, 21, 2, 2022

Analysis of factors that are related to seed germination in orchid
神原昂太, 志村華子, 藤野介延, 園芸学研究 別冊, 21, 1, 2022

Responses to phytohormones in Potato StCRCT-overexpressing line
松下大輝, 永山大貴, 志村華子, 森田隆太郎, 深山浩, 藤野介延, 日本作物学会講演会要旨集, 254th, 2022

Time-course analysis of parthenocarpy-related genes and examination of gene transfer methods in watermelon
長岡恒平, 杉山慶太, 鈴鹿明広, 藤野介延, 志村華子, 園芸学研究 別冊, 20, 1, 2021

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
Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan, Japanese

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
Japanese

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, Dec. 2016
English, Summary international conference

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, 23 Oct. 2015
Japanese

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, 21 Mar. 2015
Japanese

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, 09 Mar. 2015
Japanese

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
Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan, Japanese

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, 09 Sep. 2012
Japanese

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, 29 Mar. 2012
Japanese

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, 04 Dec. 2009
北海道農業研究センター, Japanese

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, 29 Sep. 2009
Japanese

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, 30 Jun. 2009
Japanese

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, 27 Mar. 2009
Japanese

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, 24 Sep. 2008
The Crop Science Society of Japan, Japanese

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, 27 Mar. 2008
The Crop Science Society of Japan, Japanese

Analysis of useful genes in buckwheat seed
藤野 介延, 年報, 2005, 109, 114, 2005
飯島記念食品科学振興財団, Japanese

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
Hokkaido Branch, the Japanese Society of Breeding and Hokkaido Branch, the Crop Science Society of Japan, Japanese

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, 20 Oct. 2004
The Crop Science Society of Japan, Japanese

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, 20 Oct. 2004
The Crop Science Society of Japan, Japanese

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, 20 Oct. 2004
The Crop Science Society of Japan, Japanese

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, 20 Oct. 2004
The Crop Science Society of Japan, Japanese

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, 25 Aug. 2002
The Crop Science Society of Japan, Japanese

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
一般社団法人 日本アレルギー学会, Japanese

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, 27 Sep. 2001
The Crop Science Society of Japan, Japanese

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, 27 Sep. 2001
The Crop Science Society of Japan, Japanese

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
一般社団法人 日本アレルギー学会, Japanese

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, 04 Oct. 2000
The Crop Science Society of Japan, Japanese

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, 01 Oct. 1999
The Crop Science Society of Japan, Japanese

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
Hokkaido University, English

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, 08 Oct. 1998
The Crop Science Society of Japan, Japanese

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, 08 Oct. 1998
The Crop Science Society of Japan, Japanese

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
一般社団法人 日本アレルギー学会, Japanese

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, 02 Apr. 1997
The Crop Science Society of Japan, Japanese

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, 02 Apr. 1997
The Crop Science Society of Japan, Japanese

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, Mar. 1996
English

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, Mar. 1995
English

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, 13 Oct. 1994
植物化学調節学会, Japanese

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
Hokkaido University, English

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, 18 Oct. 1993
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, The Crop Science Society of Japan, Japanese

89 Trangent gene expression of potato protoplasts
Fukase Takako, Fujino Kaien, Kikuta Yoshio, Japanese Journal of Crop Science, 62, 2, 177, 178, 18 Oct. 1993
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., The Crop Science Society of Japan, Japanese

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, 30 Oct. 1991
日本作物学会, Japanese

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, 10 Oct. 1990
日本作物学会, Japanese

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
Hokkaido University, English

Viability, DNA Synthesis and Cell Wall Regeneration on Potato Protoplasts　　　　　　　　　　　　　　　
Biotechnology in Agriculture and Forestry, 1987

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
科学研究費助成事業
01 Apr. 2021 - 31 Mar. 2025
鈴木 達郎, 藤野 介延, 澤井 祐典
「１）iProA最終合成タンパク質・遺伝子の単離」については、昨年度に引き続き活性測定方法の開発を行った。基質としてカテキン、エピカテキンに加え、システイン-エピカテキンが重合反応の開始に重要との知見があるため、システインエ-ピカテキンを検討すこととした。システイン-エピカテキンは市販されていないことから、ソバ殻に含まれるiProAをシステイン存在下で加熱することで合成する方法の開発を試みた。具体的には温度、時間、各物質の存在割合等の検討を行った。検討結果、いくつかの条件において、反応後の液をLC/MSで分析したところ、システイン-エピカテキンに相当するm/zを有する化合物を見出すことができ、またイオントラップMSにて当該m/zを持つ物質は、コリジョンによりシステインおよびエピカテキンに相当するm/zに相当する物質から構成されていることが明らかとなった。以上をもってシステイン-エピカテキンを合成できたと判断した。ただし、合成量は現段階では極めて少ないため、より効率的な方法の検討を経て、合成したシステイン-エピカテキンを基質としたiProA合成系の開発を実施する。また、遺伝子発現の観点から原因遺伝子の推定を行った。RNAseqのデータ解析を実施しつつ、DeepFeature法による候補遺伝子の推定を実施した。DeepFeature法は、RNAseqデータを機械学習させることで特徴量とした原因遺伝子群（遺伝子名）を返り値として出力できる手法である。医学分野と異なりデータ量が限られる植物分野へ応用するため入力データ等を工夫することで適用できる可能性が見出された。現在RNAseqおよびDeepFeatur法の両方のデータを比較することで原因遺伝子を探索中である。
日本学術振興会, 基盤研究(B), 国立研究開発法人農業・食品産業技術総合研究機構, 23K21191

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
科学研究費助成事業
01 Apr. 2021 - 31 Mar. 2025
鈴木 達郎, 藤野 介延, 澤井 祐典
日本学術振興会, 基盤研究(B), 国立研究開発法人農業・食品産業技術総合研究機構, 21H02178

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
科学研究費助成事業
09 Jul. 2021 - 31 Mar. 2023
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。
日本学術振興会, 挑戦的研究(萌芽), 北海道大学, 21K19102

Physiological analysis of pericarp dehiscence mechanism involved in yield
Grants-in-Aid for Scientific Research
01 Apr. 2017 - 31 Mar. 2020
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 17H03754

Mechanism of enhanced salinity tolerance by an endogenouse peptide elicitor in Arabidopsis
Grants-in-Aid for Scientific Research
01 Apr. 2014 - 31 Mar. 2017
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Osaka Prefecture University, 26440153

Possibility of active secretion of Arabidopsis enodgenous peptide elicitor
Grants-in-Aid for Scientific Research
01 Apr. 2012 - 31 Mar. 2015
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists (B), 24770036

Analysis of pod structure responsible for soybean shattering resistance
Grants-in-Aid for Scientific Research
01 Apr. 2012 - 31 Mar. 2015
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido University, 24580017

ANALYSIS OF ALLERGENS AND USEFUL GENES IN BUCKWHEAT SEED STORAGE PROTEINS
Grants-in-Aid for Scientific Research
2003 - 2004
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), HOKKAIDO UNIVERSITY, 15580010

ANALYSIS OF ALLERGENS IN BUCKWHEAT SEED STORAGE PROTEINS
Grants-in-Aid for Scientific Research
2001 - 2002
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), HOKKAIDO UNIVERSITY, 13660012

GENE ACTION DURING REDIFFERENTIATION AND EMBRYOGENlC DEVELOPMENT IN CROPS CELLS CULTURED, IN VITRO
Grants-in-Aid for Scientific Research
1997 - 1999
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), HOKKAIDO UNIVERSITY, 09460007

禾本科植物の種子カルスに関する生理学的研究
科学研究費助成事業
1996 - 1996
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。
日本学術振興会, 奨励研究(A), 北海道大学, 08760009

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
科学研究費助成事業
1995 - 1995
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。
日本学術振興会, 奨励研究(A), 北海道大学, 07760009

GENE ACTION DURING CELLULAR DIFFERENTIATION AND RELATED ORGANOGENESIS IN CROPS CULTURED,IN VITRO
Grants-in-Aid for Scientific Research
1993 - 1995
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.
Japan Society for the Promotion of Science, Grant-in-Aid for General Scientific Research (B), HOKKAIDO UNIVERSITY, 05454042

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
科学研究費助成事業
1993 - 1993
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。
日本学術振興会, 奨励研究(A), 北海道大学, 05760010

Studies of developmental physiology of cell differenciation in crop tissues and cells cultured, in vitro.
Grants-in-Aid for Scientific Research
1989 - 1991
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.
Japan Society for the Promotion of Science, Grant-in-Aid for General Scientific Research (B), Hokkaido University, 01480040

バレイショの塊茎形成　　　　　　　　　　　　　　　
Competitive research funding

Tuber formation of potato　　　　　　　　　　　　　　　
Competitive research funding