Papers - KUNITAKE Hisato
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Uchida A., Sassa H., Takenaka S., Sakakibara Y., Suiko M., Kunitake H.
Plant OMICS 5 ( 4 ) 320 - 325 2012.7
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Plant OMICS
The differences between stylar protein of the Japanese pear (Pyrus pyrifolia (Burm.f.)) cultivars 'Kosui' (S 4 S 5 ) and 'Kikusui' (S 2 S 4 ) were compared by two-dimensional difference gel electrophoresis (2-D DIGE), and were labelled and visualized with different fluorescent dyes (IC3-OSu, IC 5 -OSu) on a single 2-D gel. The individual different expressed proteins spots were subjected to identification. The proteins were analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) to identify proteins related to gametophytic self-incompatibility (GSI). S 4 -RNase and thaumatin-like protein 1 were successfully detected as expected in the pistils of 'Kosui' and 'Kikusui'. S 5 -RNase was also detected in the pistils of 'Kosui'. However, we could not detect S 2 -RNase in 'Kikusui' in this study, possibly because the level of expression of S 2 -RNase might be minuscule, or the estimated isoelectric point (pI) of S 2 -RNase (pI:9.26) was more basic than S 4 -RNase (pI: 9.17) and S 5 -RNase (pI: 9.01). These results indicate that proteomic studies are effective tools for detection of the expected proteins and might be helpful for finding the unknown key proteins related to the mechanism of self-incompatibility (SI) in many other SI plants.
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Uchida A., Takenaka S., Takenaka S., Sakakibara Y., Kurogi S., Honsho C., Sassa H., Suiko M., Kunitake H.
Journal of the Japanese Society for Horticultural Science 81 ( 2 ) 150 - 158 2012.4
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of the Japanese Society for Horticultural Science
In Citrus, self-incompatibility (SI) regulation is gametophytic, and this phenomenon is an economically critical problem for some Citrus cultivars without high parthenocarpic ability. Few molecular biological studies of SI in Citrus have been performed, and the molecular mechanism of SI has not been clarified. To investigate the effect of different stages of style development on pollen tube behavior, flower buds of self-incompatible 'Hyuganatsu' (Citrus tamurana hort. ex Tanaka) were histologically assayed. When the flower buds were observed 168 hours after pollination, pollen tubes in the self-pollinated flower buds 1 and 3 days before anthesis (DBA) were arrested in the upper part of the styles, while those in flower buds self-pollinated 5 DBA reached the base of the styles. These results revealed that SI in 'Hyuganatsu' has not yet occurred in the flower buds 5 DBA, but generated in the flower buds 3 DBA. To search SI-related pistil proteins in Citrus, we profiled a number of protein expressions in 'Hyuganatsu' styles of 1, 3, and 5 DBA by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. One-hundred thirty-eight protein spots were significantly different in abundance among the three stages, and 17 up-regulated and 26 down-regulated protein spots could be identified. Among the 17 up-regulated proteins, nine up-regulated proteins exhibited the expression pattern of 1 DBA ≥ 3 DBA > 5 DBA, a pattern which reflected the transmission from SC to SI, evaluated by pollen tube growth. BLASTP homology search against the peptide sequences of these proteins was carried out and predicted the proteins related to the maintenance of cell shape, the signaling of pollen tube growth, the flavonol biosynthesis, the responses to various stresses, photosynthesis, and the methionine metabolic process. Among the nine proteins, some may be the SI-related pistil proteins; however, it was not possible to determine the SI-related key protein of the style in Citrus. © 2012.
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Comprehensive Analysis of Expressed Proteins in the Different Stages of the Style Development of Self-incompatible ‘Hyuganatsu’ (Citrus tamurana hort. Ex Tanaka) Reviewed
Uchida, A., S. Takenaka, Y. Sakakibara, S. Kurogi,, C. Honsho, H. Sassa, M. Suiko and H. Kunitake
J Japan Soc Hort Sci. 81 ( 2 ) 150 - 158 2012.4
Language：English Publishing type：Research paper (scientific journal)
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オリザリンおよびコルヒチン処理によるスノキ属植物における倍数体の作出. Reviewed
津田浩利・小島祥子・鉄村琢哉・小松春喜・國武久登.
園芸学研究 11 ( 2 ) 205 - 212 2012.4
Language：Japanese Publishing type：Research paper (scientific journal)
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ヒュウガナツ（Citrus tamurana hort. ex Tanaka）における成熟花粉の液体培養系の確立. Reviewed
内田飛香・安部秋晴・星野洋一郎・國武久登.
園芸学研究 11 ( 2 ) 173 - 179 2012.4
Language：Japanese Publishing type：Research paper (scientific journal)
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我が国自生のスノキ属植物とブルーベリー栽培品種における植物組織培養と試験管外発根を利用したクローン増殖. Reviewed
山内（佐藤）真希子・津田浩利・荒木啓輔・内田飛香・安田喜一・鉄村琢哉・小松春喜・國武久登.
園芸学研究 11 ( 1 ) 13 - 19 2012.1
Language：Japanese Publishing type：Research paper (scientific journal)
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Unreduced 2n pollen production in 'Nishiuchi Konatsu' Hyuganatsu as inferred by pollen characteristics and progeny ploidy level. Reviewed
Honsho, C., E.Yamamura, K. Tsuruta, Y. Yoshimaru, K. Yasuda, A. Uchida, H. Kunitake and T. Tetsumura.
J Japan Soc Hort Sci. 81 ( 1 ) 19 - 26 2012.1
Language：English Publishing type：Research paper (scientific journal)
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Honsho C., Yamamura E., Tsuruta K., Yoshimaru Y., Yasuda K., Uchida A., Kunitake H., Tetsumura T.
Journal of the Japanese Society for Horticultural Science 81 ( 1 ) 19 - 26 2012.1
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of the Japanese Society for Horticultural Science
Hyuganatsu is a self-incompatible Citrus species with no parthenocarpic traits. Pollinizer requirements and seedy fruit production are therefore a limiting factor for Hyuganatsu growers. 'Nishiuchi Konatsu', a bud mutation of Hyuganatsu, can set fruit by self-pollination and aborts most seeds. To obtain a better understanding of the reproductive mechanisms of 'Nishiuchi Konatsu', its pollen was characterized by pollen observation and viability testing in vitro in addition to pollen tube observation of the pistil following various pollination combinations. Furthermore, the ploidy level of 'Nishiuchi Konatsu' and the progenies obtained after controlled pollination were analyzed using a flow cytometer. As a result, 'Nishiuchi Konatsu' pollen was shown to be functional in vitro, as indicated by both the pollen germination test and FDA staining. A curious feature of 'Nishiuchi Konatsu' pollen is that a giant pollen of about 42-44 μm in diameter, which is about 1.3-fold the average diameter of original Hyuganatsu pollen (33.4 μm), was partially produced. Observation of in vivo pollen germination and pollen tube elongation revealed that Hyuganatsu (or 'Nishiuchi Konatsu') × 'Nishiuchi Konatsu' showed imperfect incompatibility; that is, some pollen tubes were able to enter the style while others could not. Flow cytometry analysis indicated that 'Nishiuchi Konatsu' is diploid while all of its self-pollinated progenies, which were rarely obtained as normal seeds, were tetraploid. Progenies from other combinations were diploid. These results strongly suggest that unreduced 2n pollen production occurred in 'Nishiuchi Konatsu'. In addition to self-compatibilty and seed abortion of 'Nishiuchi Konatsu' self-pollination, it has been reported tha t the proportion of normal to aborted seeds differs according to the cultivar of the seed parent crossed with 'Nishiuchi Konatsu' pollen (Honsho et al., 2009). Taking into account the unreduced pollen production as well as the effect of polyploidization on self-incompatibility and embryo development, we finally discussed why such a phenomenon was induced by pollination with 'Nishiuchi Konatsu' pollen and why 'Nishiuchi Konatsu' became self-compatible. © 2012.
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本勝 千歳, 山村 恵梨, 鶴田 今日子, 芳丸 由佳子, 安田 喜一, 内田 飛香, 國武 久登, 鉄村 琢哉
Journal of the Japanese Society for Horticultural Science 81 ( 1 ) 19 - 26 2012
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：一般社団法人 園芸学会
ヒュウガナツは自家不和合性で，かつ単為結果性を持たない．そのため，受粉樹が必要であることと，果実中に多数の種子が含まれることが，生産者にとって問題であった．このヒュウガナツの枝変わり品種である'西内小夏'は自家受粉で結実し，また種子のほとんどがしいな化する性質を持つ．このメカニズムについての知見を得るために，本研究では花粉の観察，花粉稔性の調査，また花柱内における花粉管伸長の調査を行い，さらに'西内小夏'自身および'西内小夏'を交雑に用いて得られた後代の倍数性を，フローサイトメトリーによって分析した．その結果，まず'西内小夏'花粉は十分な稔性を持っていることが明らかとなった．そして興味深い結果として，'西内小夏'花粉には一般的なヒュウガナツの平均花粉径（33.4 μm）の約 1.3 倍の大きさである巨大花粉が一部生産されていることが確認された．次に花柱内の花粉管伸長を観察したところ，ヒュウガナツあるいは'西内小夏'の雌ずいに'西内小夏'花粉を受粉したとき，多くの花粉が柱頭と花柱の境界周辺で花粉管伸長が阻害されていた一方で，一部の花粉が花柱基部まで伸長していた．そしてフローサイトメトリーの結果，'西内小夏'自体は二倍体であったが，'西内小夏'を自家受粉してまれに得られた正常種子由来の実生は全て四倍体であった．これらの結果は，'西内小夏'が非還元花粉を形成していることを強く示唆している．これまでに'西内小夏'の自家和合化および種子のしいな化に加えて，'西内小夏'の花粉をいくつかの単胚性カンキツに受粉すると，種子親に応じてしいなの形成程度が異なることが報告されている（Honsho ら，2009）．これらの現象について，非還元花粉形成と倍数化が自家不和合性に及ぼす影響を考慮して，考察を行った．<br>
DOI： 10.2503/jjshs1.81.19
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産学官連携によるブルーベリー葉茶の開発(大学発!美味しいバイオ)
國武 久登, 甲斐 孝憲
生物工学会誌 : seibutsu-kogaku kaishi 90 ( 8 ) 520 - 522 2012
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：日本生物工学会
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自家不和合性の'ヒュウガナツ'（<i>Citrus tamurana</i> hort. ex Tanaka）花柱の異なる発達ステージにおける発現タンパク質の網羅的解析
内田 飛香, 竹中 聡, 榊原 陽一, 黒木 重文, 本勝 千歳, 佐々 英徳, 水光 正仁, 國武 久登
Journal of the Japanese Society for Horticultural Science 81 ( 2 ) 150 - 158 2012
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：一般社団法人 園芸学会
これまでにカンキツにおける自家不和合性の分子生物学的研究が行われてきたが，そのメカニズムについては未だ明らかになっていない．花柱の成育ステージの違いが引き起こす花粉管の挙動の経時的変化を調査するため，自家不和合性品種'ヒュウガナツ'の花蕾の組織学的調査を行った．自家受粉 168 時間後において，開花 1 日前と 3 日前に受粉した花蕾の花粉管は花柱上部に停滞していた．一方，開花 5 日前に受粉した花蕾では花粉管が花柱基部まで到達していた．これらの結果，'ヒュウガナツ'の開花 5 日前の花蕾においては自家不和合性反応は生じていないが，開花 3 日前の花蕾においては自家不和合性反応が生じていることが明らかとなった．そこで，カンキツにおける自家不和合性に関連する雌ずいタンパク質を探索するため，'ヒュウガナツ'の開花 1，3，5 日前の花蕾における発現タンパク質のプロファイリングを二次元電気泳動法と MALDI-TOF/MS 法を用いて行った．3 つの異なるステージにおいて，138 個のタンパク質の発現量に有意な差が観察され，そのうち 17 個の発現量が上昇しているタンパク質と，26 個の発現量が減少しているタンパク質が同定された．17 個の発現量が上昇していたタンパク質のうち，9 個のタンパク質が示す開花 1 日前 ≧ 開花 3 日前 ＞ 開花 5 日前の発現パターンが，花粉管の挙動に基づく自家和合性から自家不和合性への移行を反映していると思われた．BLASTP によるペプチド配列の相同性探索により，これらは細胞形成の維持や花粉管伸長におけるシグナル，フラボノイド生合成系，ストレス応答，光合成系やメチオニン代謝経路に関するタンパク質であると推定された．これら 9 個のタンパク質のうち，いずれかが自家不和合性と関連を持つ花柱タンパク質である可能性があると考えられた．しかしながら，これらの中からカンキツにおける自家不和合性反応の鍵となるタンパク質を明らかにすることはできなかった．<br>
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Nepal A., Sugimoto Y., Nishiwaki A., Kunitake H., Anugroho F., Kirimura M., Muranaka C.
European Journal of Scientific Research 52 ( 1 ) 123 - 131 2011.4
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：European Journal of Scientific Research
Microbial additive compost on microbial growth, activity and N mineralization of the soil amendments were examined, and their relationships were analyzed. Composts were prepared from commercial cattle manure with/without additive containing thermophilic bacteria. Soils unamended and amended compost with/without additive were incubated at 25oC for 125 days. The microbial additive increased microbial populations and activity during the first 13 days of incubation and accelerated N mineralization, which was enhanced throughout the rest of the incubation. Therefore, N mineralization can be increased by the increased microbial growth and activity caused by the microbial additive. © EuroJournals Publishing, Inc. 2011.
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Effects of microbial additive on composting process and on Swiss chard growth and nutrient uptake
Nepal A., Sugimoto Y., Nishiwaki A., Kunitake H., Anugroho F., Kirimura M., Muranaka C.
European Journal of Scientific Research 52 ( 1 ) 132 - 141 2011.4
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：European Journal of Scientific Research
We investigated the effect of a microbial additive on composting and on growth of Swiss chard (Beta vulgaris var. cicla). Composts were prepared from commercial cattle manure with/without a microbial additive containing thermophilic bacteria. Compost with microbial additive (AC) had greater temperature rise and microbial activity (measured by ATP levels) during first-phase composting than compost without additive (NAC), possibly because of increased thermophilic bacterial activity. Application of AC in Swiss chard cultivation gave higher SPAD values, photosynthetic activity, leaf yield, and nutrient contents than with NAC. The microbial additive including thermophilic bacteria produced good-quality compost that characterized by lower moisture and C:N ratio, and good growth of Swiss chard. © EuroJournals Publishing, Inc. 2011.
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Abnormality of gamete formation in a pummelo [Citrus maxima (Burm.) Merr.] haploid
Yahata M., Yahata M., Yahata M., Kunitake H., Yasuda K., Hirai T., Yabuya T., Yamashita K., Komatsu H.
Journal of the Japanese Society for Horticultural Science 80 ( 1 ) 14 - 18 2011.2
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of the Japanese Society for Horticultural Science
We investigated female and male gamete formation in a pummelo [Citrus maxima (Burm.) Merr.] haploid. During female gamete formation, no embryo-sac mother cell (EMC) was formed in ovules, and consequently no normal embryo sac was observed at anthesis, which was confirmed as the cause of female sterility in the haploid. During male gamete formation, on the other hand, meiosis in the pollen mother cells (PMCs) of the haploid occurred twice, but abnormalities were observed in most dividing cells. In some dividing cells, univalents did not distribute to the opposite poles at anaphase I; instead, they remained near the equatorial plate. The nine univalents then performed mitosis, which distributed equally forward opposite poles. Furthermore, at the tetrad stage, the tetrad comprised 99.3% of the 'Banpeiyu' pummelo, whereas sporads from monads to hexads with a relatively high frequency of dyads (24.7%) were produced in the haploid. These results suggest that fertile pollen grains observed in the haploid pummelo were produced by abnormalities in the first meiotic division such as first division restitution (FDR). JSHS © 2011.
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Relationship between Weight of Seeds and the Ploidy Level of the Seedlings Obtained from the Cross Between ‘Banpeiyu’ Pummelo and Somatic Hybrid ‘Citrus parental line No.4’.（共著） Reviewed
Yahata, M., T. Sekimoto, K. Yasuda, H. Komatsu and H. Kunitake
Bulletin of the Faculty of Agriculture, University of Miyazaki 57 63 - 70 2011.2
Language：English Publishing type：Research paper (bulletin of university, research institution)
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Analytical study on the relationships between microbial growth and N mineralization influenced by microbial additive compost. "jointly worked" Reviewed
Arati P. N., Y. Sugimoto, A. Nishiwaki, H. Kunitake, F. Anugroho, M. Kirimura and C. Muranaka
European Journal of Scientific Research 52 ( 1 ) 123 - 131 2011.2
Language：English Publishing type：Research paper (scientific journal)
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Effects of microbial additive on composting process and on Swiss chard growth and nutrient uptake. "jointly worked" Reviewed
Arati P. N., Y. Sugimoto, A. Nishiwaki, H. Kunitake, F. Anugroho, M. Kirimura and C. Muranaka
European Journal of Scientific Research 52 ( 1 ) 132 - 141 2011.2
Language：English Publishing type：Research paper (scientific journal)
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Morphological characteristics of fruit in a haploid pummelo."jointly worked" Reviewed
Yahata, M., H. Kunitake, K. Yasuda, T. Yabuya, K. Yamashita, H. Komatsu
宮崎大学農学部研究報告 57 57 - 61 2011.2
Language：English Publishing type：Research paper (bulletin of university, research institution)
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Abnormality of gamete formation in a pummelo [Citrus maxima (Burm.) Merr.] haploid. "jointly worked" Reviewed
Yahata M., H. Kunitake, K. Yasuda, T. Hirai, T. Yabuya, K. Yamashita and H. Komatsu.
Journal of Japanese Society for Horticultural Science 80 ( 1 ) 14 - 18 2011.1
Language：English Publishing type：Research paper (scientific journal)
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ニンポウキンカン珠心胚へのコルヒチン処理によって得た倍数体の特性とそれらの三倍体育種への利用（共著） Reviewed
糠谷綱希・太田知宏・安田喜一・八幡昌紀・國武久登・小松春喜・新居直祐・向井啓雄・原田久・高木敏彦
園芸学研究 10 ( 1 ) 1 - 8 2011.1
Language：Japanese Publishing type：Research paper (scientific journal)