研究者情報

マスター

アカウント（マスター）

• 氏名

  荒川　圭太(アラカワ　ケイタ), アラカワ　ケイタ

所属（マスター）

• 農学研究院　基盤研究部門　森林科学分野

所属（マスター）

• 農学研究院　基盤研究部門　森林科学分野

独自項目

syllabus

• 2021, 林産学特論Ⅱ, Advanced Forest Products Science II, 修士課程, 農学院, 木材、材質、木質材料、木質構造、木部細胞、細胞壁、樹幹、環境応答
• 2021, 森林資源科学特論, Advanced Forest Resources Science, 修士課程, 農学院, 森林資源、樹木/林木、木材、バイオリファイナリ?、きのこ、生理活性物質
• 2021, 森林資源科学特論演習, Advanced Seminar on Forest Resources Science, 修士課程, 農学院, 森林資源、樹木/林木、木材、バイオリファイナリ?、きのこ、生理活性物質
• 2021, 林産学特論演習Ⅱ, Advanced Seminar on Forest Products II, 修士課程, 農学院, 木材、材質、木質材料、木質構造、木部細胞、細胞壁、環境応答、樹幹
• 2021, 樹木生理学, Tree Physiology, 学士課程, 農学部, 栄養成長と生殖成長、樹木細胞の基本構造、遺伝子発現調節、代謝、物質輸送と貯蔵、成長と分化、環境応答
• 2021, 樹木生物学実験, Laboratory Work on Wood Biology, 学士課程, 農学部, 樹木・構造解析・顕微鏡観察・環境応答・生理学実験
• 2021, 林産学実習, Practical Field Work on Conversion and Engineering of Forest Products, 学士課程, 農学部, 木炭、活性炭、木酢液、きのこ、製材、樹幹解析、繊維傾斜、等級区分
• 2021, 木材理学実験, Laboratory Work on Physics and Identification of Wood, 学士課程, 農学部, 材鑑識別，木材，力学的性質，試験方法
• 2021, 英語演習, English Seminar, 学士課程, 全学教育, 森林生態系の管理・保全、樹木の構造と性質、木質バイオマスの利活用
• 2021, 一般教育演習(ﾌﾚｯｼｭﾏﾝｾﾐﾅｰ), Freshman Seminar, 学士課程, 全学教育, 組織構造、環境応答、強度、木質バイオマス、樹木の化学成分、オゾン・酸性雨、保全管理、森林管理、生物多様性、生態系サービス、国土保全、森林政策

researchmap

プロフィール情報

学位

• 博士（農学）（名古屋大学）

プロフィール情報

• 姓

  荒川, アラカワ

• 名

  圭太, ケイタ

• ID各種

  200901069220147094

対象リソース

http://woodyplantbiology.wixsite.com/woodyplantbiology
http://www.agr.hokudai.ac.jp/rfoa/env/env3-4.html

業績リスト

研究キーワード

• 環境適応   寒冷環境   樹木生理学   

研究分野

• 環境・農学 / ランドスケープ科学
• 環境・農学 / 環境農学
• ライフサイエンス / 木質科学

論文

• 植物粗抽出液の抗氷核活性(過冷却促進活性)による凍結傷害軽減の試み

  荒川圭太, 工藤尚美, 鈴木伸吾, 鈴木伸吾, 高橋淳, 後藤高秋, 中澤佑哉, 福士幸治, 藤川清三

  低温生物工学会誌 69 1 2023年
• A possibility of influence factors on winter physical damage of grapevines in a snowy vineyard

  Y. Jitsuyama, Y. Kita, K. Arakawa, T. Suzuki

  VITIS 61 4 125 - 132 2022年 

  Hokkaido in northern Japan is a typical subpolar region and wine production using traditional cultivars can be done recently. However, there is an issue that the grapevines have short life spans in snowy central Hokkaido. This study investigates the effect of snow it-self and its interaction with vineyard slope and grape-vine stiffness on physical stress. In comparison of the flatness of the vineyard, the distortion of the basal trunk in the slope zone was greater than in the flat zones in the snowy mid-season. In comparison between cultivars, the distortion of the basal trunk in the flexible 'Kerner' was greater than in rigid 'Gewurztraminer', even in the flat zones. The magnitude of the distortion coincides with the mortality of grapevines. Although the chang-es in distortion showed a transition in just two winter durations, these results suggested a possibility that the snow cover might be linked to one of the physical dam-ages of grapevines in vineyards in heavy snowy regions.
• Freezing resistance and behavior of winter buds and canes of wine grapes cultivated in northern Japan

  Reiko Horiuchi, Keita Arakawa, Jun Kasuga, Takashi Suzuki, Yutaka Jitsuyama

  Cryobiology 101 44 - 51 2021年08月 [査読有り]
  
• Cryo-plate法を用いた交雑ポプラ培養体の超低温保存技術の開発

  田中大介, 佐久間義範, 安井雅範, 川村浩平, 荒川圭太

  低温生物工学会誌 67 1 47 - 51 2021年04月 [査読有り][通常論文]
  
• カツラ樹皮由来の氷核活性物質に関する研究

  古賀 泰雅, 鉄穴口 晃, 鈴木 伸吾, 重冨 顕吾, 荒川 圭太

  低温生物工学会誌 67 2 141 - 145 低温生物工学会 2021年 [査読有り]
  
• Cryo-scanning electron microscopy reveals that supercooling of overwintering buds of freezing-resistant interspecific hybrid grape ‘Yamasachi’ is accompanied by partial dehydration

  Jun Kasuga, Yusuke Tsumura, Daisuke Kondoh, Yukata Jitsuyama, Reiko Horiuchi, Keita Arakawa

  Journal of Plant Physiology 253 153248 - 153248 2020年08月 [査読有り][通常論文]
  
• Supercooling-promoting (Anti-ice nucleation) substances

  Seizo Fujikawa, Chikako Kuwabara, Jun Kasuga, Keita Arakawa

  Advances in Experimental Medicine and Biology 1081 289 - 320 2018年 [査読有り][通常論文]
   

  © Springer Nature Singapore Pte Ltd. 2018. Studies on supercooling-promoting substances (SCPSs) are reviewed introducing name of chemicals, experimental conditions and the supercooling capability (SCC) in all, so far recognized, reported SCPSs and results of our original study are presented in order to totally show the functional properties of SCPSs which are known in the present state. Many kinds of substances have been identified as SCPSs that promote supercooling of aqueous solutions in a non-colligative manner by reducing the ice nucleation capability (INC) of ice nucleators (INs). The SCC as revealed by reduction of freezing temperature (°C) by SCPSs differs greatly depending on the INs. While no single SCPS that affects homogeneous ice nucleation to reduce ice nucleation point has been found, many SCPSs have been found to reduce freezing temperatures by heterogeneous ice nucleation with a large fluctuation of SCC depending on the kind of heterogeneous IN. Not only SCPSs increase the degree of SCC (°C), but also some SCPSs have additional SCC to stabilize a supercooling state for a long term to stabilize supercooling against strong mechanical disturbance and to reduce sublimation of ice crystals. The mechanisms underlying the diverse functions of SCPSs remain to be determined in future studies.
• Mechanism of overwintering in trees

  Keita Arakawa, Jun Kasuga, Naoki Takata

  Advances in Experimental Medicine and Biology 1081 129 - 147 2018年 [査読有り][通常論文]
   

  © Springer Nature Singapore Pte Ltd. 2018. Boreal trees possess very high freezing resistance, which is induced by short-day length and low temperatures, in order to survive severe subzero temperatures in winter. During autumn, cooperation of photoreceptors and circadian clock system perceiving photoperiod shortening results in growth cessation, dormancy development, and first induction of freezing resistance. The freezing resistance is further enhanced by subsequent low temperature during seasonal cold acclimation with concomitant changes in various morphological and physiological features including accumulation of sugars and late embryogenesis abundant proteins. The mechanism of adaptation to freezing temperatures differs depending on the type of tissue in boreal trees. For example, bark, cambium, and leaf cells tolerate freezing-induced dehydration by extracellular freezing, whereas xylem parenchyma cells avoid intracellular freezing by deep supercooling. In addition, dormant buds in some trees respond by extraorgan freezing. Boreal trees have evolved overwintering mechanisms such as dormancy and high freezing resistance in order to survive freezing temperatures in winter.
• Screening of plant resources with anti-ice nucleation activity for frost damage prevention.

  Shingo Suzuki, Satoshi Fukuda, Yukiharu Fukushi, Keita Arakawa

  Bioscience, biotechnology, and biochemistry 81 11 2090 - 2097 2017年11月 [査読有り][通常論文]
   

  Previous studies have shown that some polyphenols have anti-ice nucleation activity (anti-INA) against ice-nucleating bacteria that contribute to frost damage. In the present study, leaf disk freezing assay, a test of in vitro application to plant leaves, was performed for the screening of anti-INA, which inhibits the ice nucleation activity of an ice-nucleating bacterium Erwinia ananas in water droplets on the leaf surfaces. The application of polyphenols with anti-INA, kaempferol 7-O-β-glucoside and (-)-epigallocatechin gallate, to the leaf disk freezing assay by cooling at -4--6 °C for 3 h, revealed that both the compounds showed anti-INAs against E. ananas in water droplets on the leaf surfaces. Further, this assay also revealed that the extracts of five plant leaves showed high anti-INA against E. ananas in water droplets on leaf surfaces, indicating that they are the candidate resources to protect crops from frost damage.
• 寒冷地樹木の耐寒性と過冷却促進物質について (特集 極限環境生物のストレス応答とその産業応用)

  荒川 圭太

  冷凍 = Refrigeration 92 1078 547 - 553 日本冷凍空調学会 2017年08月 [査読無し][通常論文]
  
• Anti-Ice Nucleating Activity of Surfactants against Silver Iodide in Water-in-Oil Emulsions

  Takaaki Inada, Toshie Koyama, Hiroyuki Tomita, Takuya Fuse, Chikako Kuwabara, Keita Arakawa, Seizo Fujikawa

  JOURNAL OF PHYSICAL CHEMISTRY B 121 27 6580 - 6587 2017年07月 [査読有り][通常論文]
   

  Various water-soluble substances are known as anti-ice nucleating agents (anti-INAs), which inhibit heterogeneous ice nucleation initiated by ice nucleating agents (INAs). Among them, several surfactants are reportedly effective as anti-INAs especially against silver iodide (AgI), which is a typical inorganic INA that induces heterogeneous ice nucleation at relatively high temperatures. In this study, the anti-ice nucleating activities of seven surfactants were examined in emulsified surfactant solutions containing AgI particles. Among previously reported anti-INAs (e.g., antifreeze proteins (AFPs), polyphenol compounds and synthetic polymers), a cationic surfactant used in this study, hexadecyltrimethylammonium bromide (C16TAB), showed the highest anti-ice nucleating activity against AgI. Based on the unique concentration-dependent dispersibility of AgI particles in C16TAB solution, anti-ice nucleating activity of C16TAB must be caused by the adsorption of C16TAB molecules on AgI surfaces either as a monolayer or a bilayer depending on the C16TAB concentration.
• 過冷却促進（抗氷核）ポリフェノールによる 組換え氷核タンパク質の氷核活性阻害

  出原 信大, 桑原 慎子, 鈴木 伸吾, 小髙 優子, 藤川 清三, 荒川 圭太

  低温生物工学会誌 63 2 155 - 160 低温生物工学会 2017年 [査読有り][通常論文]
   

  It is known that some polyphenols with anti-ice nucleation activity decrease freezing temperature of thesolution containing ice nucleators, resulting in maintenance of supercooling state of the solution for a longperiods. In the previous study, recombinant proteins of Erwinia ananas ice nucleation protein, inaA withhistidine-tag (His-inaA) were expressed in Escherichia coli cells and ice nucleation activity was detected inthe cell suspension. In the present study, the purification and the characterization of His-inaA proteins fromtransformed E. coli cells were done to study the mechanism of anti-ice nucleation of these polyphenols insolutions containing inaA. When the extracts of E. coli cells expressing His-inaA were fractionated intosoluble, membrane and inclusion body fractions, ice nucleation activities were detected in all three fractions.Then, His-inaA was purified from the soluble fraction by affinity column chromatography and ice nucleationactivity of the purified His-inaA fraction was detected. Further, it is confirmed that anti-ice nucleationactivity of polyphenols was detected in solutions containing His-inaA.
• 植物抽出物の過冷却促進活性の比較

  鈴木 伸吾, 福田 怜, 福士 幸治, 荒川 圭太

  低温生物工学会誌 63 1 33 - 36 低温生物工学会 2017年 [査読有り][通常論文]
   

  Flavonol glycosides, which are a kind of flavonoid, are generally known as antioxidative substances andaccumulated in various plant species. Recent studies showed that some flavonol glycosides possess anti-icenucleation activities (or supercooling-facilitating activities), which results in the depression of freezingtemperatures of the solutions in the presence of ice nucleating substances such as Erwinia ananas and silveriodide. In this study, supercooling-facilitating activities in the crude extracts from leaves, which may includesupercooling-facilitating polyphenols, were compared among 65 plants (22 woody plants, 43 herbaceousplants) by droplet freezing assay using E. ananas as an ice nucleator. Relatively highsupercooling-facilitating activities (>2˚C) were detected in the crude extracts from 14 plants. Since it isexpected that the application of crude extracts with relatively high supercooling-facilitating activities mayprotect plant tissues from frost damages by ice-nucleating bacteria (Pseudomonas, Erwinia, etc.) on thesurface of the tissues at relatively high subzero temperatures (about -2 ~ -3˚C), further characterization wereexamined using crude extract of Sasa senanensis with relatively high supercooling-facilitating activity. Whenpartial purification was examined, the HPLC recovered fraction which may include flavone glycosides,which are also a kind of flavonoid, showed supercooing-facilitating activity.
• ガラス化法による交雑ポプラの超低温保存

  川村 浩平, 嘉見 大助, 鈴木 伸吾, 田中 大介, 藤川 清三, 荒川 圭太

  低温生物工学会誌 62 2 123 - 126 低温生物工学会 2016年 [査読有り][通常論文]
   

  The objective of this study is to establish a cryopreservation protocol for shoot apices of hybrid aspen (Populus tremula Å~ P. alba). Cryopreservation was carried out using a vitrification method on shoot apices excised from in vitro cultures. Excised shoot apices were precultured at 23oC for 2 days on hormone-free 1/2 MS medium with 0.4 M sucrose, then treated with liquid 1/2 MS medium supplemented with 0.4 M sucrose and 2.0 M glycerol (a loading solution) at 23oC for 30 min. Then shoot tips were treated with Plant Vitrification Solution 2 (PVS2) or PVS3 at 4oC for 90 min and plunged into liquid nitrogen for 1 hour. Following cryopreservation, they were rewarmed at 42oC for 2 min and washed in liquid 1/2 MS medium supplemented with 1.2 M sucrose at 23oC for 30 min. Survival rate was about 80% when shoot tips were treated with PVS2 for 90 min, however, regrowth rate after 3 weeks was about 20%.
• 大腸菌で発現させた氷核タンパク質inaAの氷核活性について

  桑原 慎子, 鈴木 伸吾, 藤川 清三, 荒川 圭太

  低温生物工学会誌 61 2 131 - 134 低温生物工学会 2015年 [査読有り][通常論文]
   

  Some polyphenolic compounds isolated in xylem tissues of woody plant, Katsura tree, showed anti-ice nucleation (supercooling-facilitating) activity in a solution containing ice nucleator such as silver iodide or ice-nucleation bacterial cells, resulting in maintenance of a supercooling state of the solution for a long time and decline of freezing temperature of the solution. Levels of these supercooling-facilitating activities were dependent on types of both the polyphenolic compounds and ice-nucleators used in the assay solution. To study the mechanism of supercooling-facilitating activity by these polyphenolic compounds, characterization of ice-nucleators should be necessary in addition to these polyphenolic compounds resulting in decline of freezing temperature. In the present study, a gene of ice-nucleation inaA protein, of Erwinia ananas was cloned and recombinant inaA protein with histidine-tag was expressed in Escherichia coli cells for further characterization. Freezing temperature of a cell suspension culture of E. coli cells transformed with recombinant inaA gene was higher than that of E. coli cells transformed with recombinant lacZ gene with histidine-tag, a negative control sample, suggesting acquirement of ice-nucleation activity by expression of recombinant inaA gene in transformed E. coli cells.
• Anti-ice nucleating activity of polyphenol compounds against silver iodide

  Toshie Koyama, Takaaki Inada, Chikako Kuwabara, Keita Arakawa, Seizo Fujikawa

  CRYOBIOLOGY 69 2 223 - 228 2014年10月 [査読有り][通常論文]
   

  Freeze-avoiding organisms survive sub-zero temperatures without freezing in several ways, such as removal of ice nucleating agents (INAs), production of polyols, and dehydration. Another way is production of anti-ice nucleating agents (anti-INAs), such as has been reported for several antifreeze proteins (AFPs) and polyphenols, that inhibit ice nucleation by inactivating INAs. In this study, the anti-ice nucleating activity of five polyphenol compounds, including flavonoid and tannin compounds of both biological and synthetic origin, against silver iodide (AgI) was examined by measuring the ice nucleation temperature in emulsified polyphenol solutions containing AgI particles. The emulsified solutions eliminated the influence of contamination by unidentified INAs, thus enabling examination of the anti-ice nucleating activity of the polyphenols against AgI alone. Results showed that all five polyphenol compounds used here have anti-ice nucleating activities that are unique compared with other known anti-INAs, such as fish AFPs (type land III) and synthetic polymers (poly(vinyl alcohol), poly(vinylpyrrolidone) and poly(ethylene glycol)). All five polyphenols completely inactivated the ice nucleating activity of AgI even at relatively low temperatures, and the first ice nucleation event was observed at temperatures between -14.1 and 19.4 degrees C, compared with between -8.6 and 11.8 degrees C for the fish AFPs and three synthetic polymers. These anti-ice nucleating activities of the polyphenols at such low temperatures are promising properties for practical applications where freezing should be prevented. (C) 2014 Elsevier Inc. All rights reserved.
• Consideration of the reasons why dormant buds of trees have evolved extraorgan freezing as an adaptation for winter survival

  Keita Endoh, Chikako Kuwabara, Keita Arakawa, Seizo Fujikawa

  ENVIRONMENTAL AND EXPERIMENTAL BOTANY 106 52 - 59 2014年10月 [査読有り][通常論文]
   

  Dormant buds of larch (Larix kaempferi) exhibit extraorgan freezing, in which extracellular ice crystals are formed in localized areas far from the primordia, as an adaptive response to freezing temperatures. In order to better understand why dormant buds have evolved such a complicated mechanism, the freezing behavior of isolated primordia from dormant buds of larch were examined using cryo-scanning electron microscopy (cryo-SEM) and by freeze-fracture replica electron microscopy (FR-EM). When isolated primordia were slowly frozen (5 degrees C/day), extracellular ice crystals developed within primordia. Thus, isolated primordia serves as a useful model to examine the freezing response of primordia in which extracellular freezing occurs. Slow freezing (5 degrees C/day) of isolated primordia resulted in a much lower survival rate (25% at -30 degrees C) than in intact buds (100% at -30 degrees C). In whole buds, exhibiting extraorgan freezing, cryo-SEM observations indicated that slow freezing to -30 degrees C resulted in deep supercooling with a slight shrinkage of all primordial cells, whereas in isolated primordia, slow freezing to -30 degrees C resulted in intracellular freezing of about half of the cells with other cells exhibiting extracellular freezing with significant shrinkage and deformation. FR-EM of whole buds subjected to slow freezing revealed the formation of intramembrane particle (IMP)-free areas with long rectangular shapes in the plasma membrane of many primordial cells at temperatures lower than -15 degrees C and in a small number of cells in isolated primordia. In isolated primordia, the number of cells exhibiting intracellular freezing, which in most cases did not reveal membrane fracture faces, corresponded to the numbers of cells exhibiting IMP-free areas in whole buds. We speculate that the formation of IMP-free areas in plasma membranes may induce intracellular freezing when extracellular ice crystals have developed within primordial tissues. FR-EM also revealed the formation of fracture-jump lesions only in the plasma membranes of primordial cells of slowly-frozen, isolated primordia. We propose that such fracture-jump lesions are produced not only by osmotic dehydration but also by the deformation of cells due to the presence of extracellular ice crystals within primordial tissues in isolated primordia. The results indicate that the plasma membrane of primordial cells in ecodormant buds of larch are subject to a phase transition that may give rise to intracellular freezing, as well as increased sensitivity to dehydration, when extracellular ice crystals form within primordial tissues. We propose that extraorgan freezing represents a mechanism that prevents the formation of extracellular ice crystals within primordial tissues, thus lowering the risk of intracellular freezing and dehydrative stress. (C) 2014 Elsevier B.V. All rights reserved.
• Analysis of supercooling activities of surfactants

  Chikako Kuwabara, Ryuji Terauchi, Hiroshi Tochigi, Hisao Takaoka, Keita Arakawa, Seizo Fujikawa

  CRYOBIOLOGY 69 1 10 - 16 2014年08月 [査読有り][通常論文]
   

  Supercooling-promoting activities (SCAs) of 25 kinds of surfactants including non-ionic, anionic, cationic and amphoteric types were examined in solutions (buffered Milli-Q water, BMQW) containing the ice nucleation bacterium (INB) Erwinia ananas, silver iodide (AgI) or BMQW alone, which unintentionally contained unidentified ice nucleators, by a droplet freezing assay. Most of the surfactants exhibited SCA in solutions containing AgI but not in solutions containing the INB E. ananas or BMQW alone. SCAs of many surfactants in solutions containing AgI were very high compared with those of previously reported supercooling-promoting substances. Cationic surfactants, hexadecyltrimethylammonium bromide (C16TAB) and hexadecyltrimethylammonium chloride (C16TAC), at concentrations of 0.01% (w/v) exhibited SCA of 11.8 degrees C, which is the highest SCA so far reported. These surfactants also showed high SCAs at very low concentrations in solutions containing AgI. C16TAB exhibited SCA of 5.7 degrees C at a concentration of 0.0005% (w/v). (C) 2014 Elsevier Inc. All rights reserved.
• 樹木の冬芽の自発休眠の解除期における可溶性タンパク質組成変化の分析

  鈴木 伸吾, 高橋 大輔, 桑原 慎子, 上村 松生, 荒川 圭太

  低温生物工学会誌 60 1 59 - 63 低温生物工学会 2014年 [査読有り][通常論文]
   

  In this study, we analyzed soluble proteins in winter bud during endodormancy release. Endodormancy of winter buds of Salix bakko and Larix kaempferi was released clearly in early and late December, respectively. SDS-PAGE analysis showed that soluble protein composition of buds in each species hardly changed in a short period of endodormancy release. Proteome analysis using LC-MS/MS revealed compositional changes in soluble proteins in this period of endodormancy release. However, the proportion of soluble proteins whose levels were changed in this period was under 5% of all detected proteins in each species of winter buds, and the variation was small.
• Analysis of supercooling activity of tannin-related polyphenols

  Chikako Kuwabara, Donghui Wang, Keita Endoh, Yukiharu Fukushi, Keita Arakawa, Seizo Fujikawa

  CRYOBIOLOGY 67 1 40 - 49 2013年08月 [査読有り][通常論文]
   

  Based on the discovery of novel supercooling-promoting hydrolyzable gallotannins from deep supercooling xylem parenchyma cells (XPCs) in Katsura tree (see Wang et al. (2012) [38]), supercooling capability of a wide variety of tannin-related polyphenols (TRPs) was examined in order to find more effective supercooling-promoting substances for their applications. The TRPs examined were single compounds including six kinds of hydrolyzable tannins, 11 kinds of catechin derivatives, two kinds of structural analogs of catechin and six kinds of phenolcarboxylic acid derivatives, 11 kinds of polyphenol mixtures and five kinds of crude plant tannin extracts. The effects of these TRPs on freezing were examined by droplet freezing assays using various solutions containing different kinds of identified ice nucleators such as the ice nucleation bacterium (INB) Erwinia ananas, the INB Xanthomonas campestris, silver iodide and phloroglucinol as well as a solution containing only unintentionally included unidentified airborne ice nucleators. Among the 41 kinds of TRPs examined, all of the hydrolyzable tannins, catechin derivatives, polyphenol mixtures and crude plant tannin extracts as well as a few structural analogs of catechin and phenolcarboxylic acid derivatives exhibited supercooling-promoting activity (SCA) with significant differences (p > 0.05) from at least one of the solutions containing different kinds of ice nucleators. It should be noted that there were no TRPs exhibiting ice nucleation-enhancing activity (INA) in all solutions containing identified ice nucleators, whereas there were many TRPs exhibiting INA with significant differences in solutions containing unidentified ice nucleators alone. An emulsion freezing assay confirmed that these TRPs did not essentially affect homogeneous ice nucleation temperatures. It is thought that not only SCA but also INA in the TRPs are produced by interactions with heterogeneous ice nucleators, not by direct interaction with water molecules. In the present study, several TRPs that might be useful for applications due to their high SCA in many solutions were identified. (C) 2013 Elsevier Inc. All rights reserved.
• Roles of cell walls and intracellular contents in supercooling capability of xylem parenchyma cells of boreal trees

  Jun Kasuga, Keita Endoh, Megumi Yoshiba, Ippei Taido, Keita Arakawa, Matsuo Uemura, Seizo Fujikawa

  PHYSIOLOGIA PLANTARUM 148 1 25 - 35 2013年05月 [査読有り][通常論文]
   

  The supercooling capability of xylem parenchyma cells (XPCs) in boreal hardwood species differs depending not only on species, but also season. In this study, the roles of cell walls and intracellular contents in supercooling capability of XPCs were examined in three boreal hardwood species, Japanese beech, katsura tree and mulberry, whose supercooling capability differs largely depending on species and season. XPCs in these species harvested in winter and summer were treated by rapid freezing and thawing (RFT samples) or by RFT with further washing (RFTW samples) to remove intracellular contents from XPCs in order to examine the roles of cell walls in supercooling. RFT samples were also treated with glucose solution (RFTG samples) to examine roles of intracellular contents in supercooling. The supercooling capabilities of these samples were examined by differential thermal analysis after ultrastructural observation of XPCs by a cryo-scanning electron microscope to confirm effects of the above treatments. XPCs in RFTW samples showed a large reduction in supercooling capability to similar temperatures regardless of species or season. On the other hand, XPCs in RFTG samples showed a large increase in supercooling capability to similar temperatures regardless of species or season. These results indicate that although cell walls have an important role in maintenance of supercooling, change in supercooling capability of XPCs is induced by change in intracellular contents, but not by change in cell wall properties.
• Freezing behavior of cells in evergreen needle leaves of fir (Abies sachalinensis)

  ENDOH Keita, FUJIKAWA Seizo, ARAKAWA Keita

  低温生物工学会誌 58 2 125 - 134 低温生物工学会 2012年10月 [査読有り][通常論文]
   

  Freezing behavior of cells in evergreen needle leaves of fir was observed by cryo-scanning electron microscopy (cryo-SEM) to determine cellular responses to subzero temperatures. Freezing resistance of needle leaves increased from summer to winter, and needle leaves in winter showed a high survival rate after freezing to -30℃ at a cooling rate of 5℃/day. Cryo-SEM observation revealed that intracellular freezing occurred in cells of the epidermis, hypodermis, mesophyll and endodermis in summer needle leaves frozen to -30℃ at a rate of 0.2℃/min. However, extracellular freezing occurred in cells of all tissues in summer needle leaves by freezing to -30℃ at a rate of 5℃/day. In winter, all cells showed extracellular freezing by freezing to -30℃ at both rapid and slow cooling rates. The results suggest that cells in needle leaves of fir adapt to subzero temperatures by extracellular freezing with development of high resistance to freeze-induced dehydration during seasonal cold acclimation.
• Freezing activities of flavonoids in solutions containing different ice nucleators

  Chikako Kuwabara, Donghui Wang, Jun Kasuga, Yukiharu Fukushi, Keita Arakawa, Toshie Koyama, Takaaki Inada, Seizo Fujikawa

  CRYOBIOLOGY 64 3 279 - 285 2012年06月 [査読有り][通常論文]
   

  In this study, we examined the effects on freezing of 26 kinds of flavonoid compounds, which were randomly selected as compounds with structures similar to those of flavonoid compounds existing in deep supercooling xylem parenchyma cells (XPCs) in trees, in solutions containing different kinds of ice nucleators, including the ice nucleation bacterium (INB) Erwinia ananas, INB Xanthomonas campestris, silver iodide, phloroglucinol and unidentified airborne impurities in buffered Milli-Q water (BMQW). Cumulative freezing spectra were obtained in each solution by cooling 2 mu L droplets at 0.2 degrees C/min by a droplet freezing assay. Freezing temperature of 50% droplets (FT50) was obtained from each spectra in a separate analysis with more than 20 droplets and mean FT50 were obtained from more than five separate analyses using more than 100 droplets in total in each flavonoid. Supercooling-promoting activities (SCA) or ice nucleation-enhancing activities (INA) of these flavonoids were determined by the difference in FT50 between control solutions without flavonoids and experimental solutions with flavonoids. In mean values, most of the compounds examined exhibited SCA in solutions containing the INB E. ananas, INB X. campestris, silver iodide, and phloroglucinol although the magnitudes of their activities were different depending on the ice nucleator. In solutions containing the INB E. ananas, 10 compounds exhibited SCAs with significant differences (p < 0.05) in the range of 1.4-4.2 degrees C. In solutions containing silver iodide, 23 compounds exhibited SCAs with significant differences in the range of 2.0-7.1 degrees C. In solutions containing phloroglucinol, six compounds exhibited SCAs with significant differences in the range of 2.4-3.5 degrees C. In solutions containing the INB X. campestris, only three compounds exhibited SCAs with significant differences in the range of 0.9-2.3 degrees C. In solutions containing unidentified airborne impurities (BMQW alone), on the other hand, many compounds exhibited INA rather than SCA. In mean values, only four compounds exhibited SCAs in the range of 2.4-3.2 degrees C (no compounds with significant difference at p < 0.05), whereas 21 compounds exhibited INAs in the range of 0.1-12.3 degrees C (eight compounds with significant difference). It was also shown by an emulsion freezing assay that most flavonoid glycosides examined did not affect homogeneous ice nucleation temperatures, except for a few compounds that become ice nucleators in BMQW alone. These results suggest that most flavonoid compounds affect freezing temperatures by interaction with unidentified ice nucleators in BMQW as examined by a droplet freezing assay. The results of our previous and present studies indicate that flavonoid compounds have very complex effects to regulate freezing of water. (C) 2012 Elsevier Inc. All rights reserved.
• Presence of supercooling-facilitating (anti-ice nucleation) hydrolyzable tannins in deep supercooling xylem parenchyma cells in Cercidiphyllum japonicum

  Donghui Wang, Jun Kasuga, Chikako Kuwabara, Keita Endoh, Yukiharu Fukushi, Seizo Fujikawa, Keita Arakawa

  PLANTA 235 4 747 - 759 2012年04月 [査読有り][通常論文]
   

  Xylem parenchyma cells (XPCs) in trees adapt to subzero temperatures by deep supercooling. Our previous study indicated the possibility of the presence of diverse kinds of supercooling-facilitating (SCF; anti-ice nucleation) substances in XPCs of katsura tree (Cercidiphyllum japonicum), all of which might have an important role in deep supercooling of XPCs. In the previous study, a few kinds of SCF flavonol glycosides were identified. Thus, in the present study, we tried to identify other kinds of SCF substances in XPCs of katsura tree. SCF substances were purified from xylem extracts by silica gel column chromatography and Sephadex LH-20 column chromatography. Then, four SCF substances isolated were identified by UV, mass and nuclear magnetic resonance analyses. The results showed that the four kinds of hydrolyzable gallotannins, 2,2',5-tri-O-galloyl-alpha,beta-d-hamamelose (trigalloyl Ham or kurigalin), 1,2,6-tri-O-galloyl-beta-d-glucopyranoside (trigalloyl Glc), 1,2,3,6-tetra-O-galloyl-beta-d-glucopyranoside (tetragalloyl Glc) and 1,2,3,4,6-penta-O-galloyl-beta-d-glucopyranoside (pentagalloyl Glc), in XPCs exhibited supercooling capabilities in the range of 1.5-4.5A degrees C, at a concentration of 1 mg mL(-1). These SCF substances, including flavonol glycosides and hydrolyzable gallotannins, may contribute to the supercooling in XPCs of katsura tree.
• 種々判定法による過冷却促進(氷核形成阻害)物質の効果の検討

  桑原 慎子, 王 東暉, 春日 純, 森若 元太, 荒川 圭太, 小山 寿恵, 稲田 孝明, 藤川 清三

  低温生物工学会誌 58 1 99 - 103 低温生物工学会 2012年 [査読有り][通常論文]
   

  Supercooling-facilitating (anti-ice nucleation) substances, which are several flavonol glycosides and hydrolyzable tannin, are found in xylem parenchyma cells in trees to keep a liquid state of intracellular water to -40℃ in winter. It is gradually discovered that other associated compounds also have supercooling-facilitating activity in the solution containing ice-nucleating bacteria, Erwinia ananas. These supercooling-facilitating substances are expected to be used in real world application. However, for their applications, judging the supercooling activity not only by standard droplet freezing assay in solutions containing different kinds of ice nucleators but also by more practical methods using a large volume of solutions under shaking may be required. In this study, we measured the supercooling-facilitating activity of several kinds of flavonol glycosides and tannins by droplet freezing assay in solutions with various ice nucleators and by shaking assay in bulk solutions. It was revealed that supercooling activity of substances tested in this study varied according to measurement condition, such as types of ice nucleator, volume of solution, concentration of substances and the presence or absence of shaking.
• カツラ冬芽の細胞の凍結挙動

  遠藤 圭太, 岡田 香織, 鈴木 伸吾, 藤川 清三, 荒川 圭太

  低温生物工学会誌 58 2 179 - 184 低温生物工学会 2012年 [査読有り][通常論文]
   

  Freezing behaviors of cells in dormant buds of katsura (Cercidiphyllum japonicum) tree were examined. Intact dormant buds showed high survival rates after freezing to -30℃ at a rate of 5℃/day (slow freezing). However, flower primordia isolated from dormant buds showed a remarkable decrease in survival rate after slow freezing to -10℃ in the presence of ice crystals adjacent to tissues, while isolated scales showed a high survival rate after slow freezing to -30℃. Katsura buds exhibited a typical pattern of extraorgan freezing, accumulation of large extracellular ice crystals within scale layers, by freezing to -30℃. Cryo-scanning electron microscopic analysis revealed that both extracellular freezing cells without freezable water in the cytoplasm and supercooling cells with freezable water in the cytoplasm of partially dehydrated cells are contained in primordial tissue and extracellular freezing cells existed in scales in dormant buds exhibiting extraorgan freezing. In isolated tissues, intracellular freezing occurred in about 50% of the cells of the flower primordia by freezing to -10℃, but extracellular freezing occurred in all scale cells by freezing to -30℃. These results suggested that katsura dormant buds adapt to subfreezing temperatures by segregation of supercooling primordial cells which are susceptible to extracellular ice crystals, from ice crystals.
• Cold acclimation in the moss Physcomitrella patens involves abscisic acid-dependent signaling

  Salma Begum Bhyan, Anzu Minami, Yasuko Kaneko, Shingo Suzuki, Keita Arakawa, Yoichi Sakata, Daisuke Takezawa

  JOURNAL OF PLANT PHYSIOLOGY 169 2 137 - 145 2012年01月 [査読有り][通常論文]
   

  Overwintering plants develop tolerance to freezing stress through a cold acclimation process by which the cells provoke internal protective mechanisms against freezing. The stress hormone abscisic acid (ABA) is known to increase freezing tolerance of plant cells, but its role in cold acclimation has not been determined. In this study, we used ABA-insensitive lines of the moss Physcomitrella patens to determine whether cold acclimation in bryophytes involves an ABA-dependent process. Two ABA-insensitive lines, both impaired in ABA signaling without showing ABA-induced stress tolerance, were subjected to cold acclimation, and changes in freezing tolerance and accumulation of soluble sugars and proteins were compared to the wild type. The wild-type cells acquired freezing tolerance in response to cold acclimation treatment, but very little increase in freezing tolerance was observed in the ABA-insensitive lines. Analysis of low-molecular-weight soluble sugars indicated that the ABA-insensitive lines accumulated sucrose, a major compatible solute in bryophytes, to levels comparable with those of the wild type during cold acclimation. However, accumulation of the trisaccharide theanderose and of specific LEA-like boiling-soluble proteins was very limited in the ABA-insensitive lines. Furthermore, analysis of cold-induced expression of genes encoding LEA-like proteins revealed that the ABA-insensitive lines accumulate only small amounts of these transcripts during cold acclimation. Our results indicate that cold acclimation of bryophytes requires an ABA-dependent signaling process. The results also suggest that cold-induced sugar accumulation, depending on the sugar species, can either be dependent or independent of the ABA-signaling pathway. (C) 2011 Elsevier GmbH. All rights reserved.
• Change of supercooling capability in solutions containing different kinds of ice nucleators by flavonol glycosides from deep supercooling xylem parenchyma cells in trees

  Chikako Kuwabara, Jun Kasuga, Donghui Wang, Yukiharu Fukushi, Keita Arakawa, Toshie Koyama, Takaaki Inada, Seizo Fujikawa

  CRYOBIOLOGY 63 3 157 - 163 2011年12月 [査読有り][通常論文]
   

  Deep supercooling xylem parenchyma cells (XPCs) in Katsura tree contain flavonol glycosides with high supercooling-facilitating capability in solutions containing the ice nucleation bacterium (INB) Erwinia ananas, which is thought to have an important role in deep supercooling of XPCs. The present study, in order to further clarify the roles of these flavonol glycosides in deep supercooling of XPCs, the effects of these supercooling-facilitating (anti-ice nucleating) flavonol glycosides, kaempferol 3-O-beta-D-glucopyranoside (K3Glc), kaempferol 7-O-beta-D-glucopyranoside (K7Glc) and quercetin 3-O-beta-D-glucopyranoside (Q3Glc), in buffered Milli-Q water (BMQW) containing different kinds of ice nucleators, including INB Xanthomonas campestris, silver iodide and phloroglucinol, were examined by a droplet freezing assay. The results showed that all of the flavonol glycosides promoted supercooling in all solutions containing different kinds of ice nucleators, although the magnitudes of supercooling capability of each flavonol glycoside changed in solutions containing different kinds of ice nucleators. On the other hand, these flavonol glycosides exhibited complicated nucleating reactions in BMQW, which did not contain identified ice nucleators but contained only unidentified airborne impurities. Q3Glc exhibited both supercooling-facilitating and ice nucleating capabilities depending on the concentrations in such water. Both K3Glc and K7Glc exhibited only ice nucleation capability in such water. It was also shown by an emulsion freezing assay in BMQW that K3Glc and Q3Glc had no effect on homogeneous ice nucleation temperature, whereas K7Glc increased ice nucleation temperature. The results indicated that each flavonol glycoside affected ice nucleation by very complicated and varied reactions. More studies are necessary to determine the exact roles of these flavonol glycosides in deep supercooling of XPCs in which unidentified heterogeneous ice nucleators may exist. (C) 2011 Elsevier Inc. All rights reserved.
• 深過冷却するカツラ木部柔細胞由来の加水分解性ガロタンニンの過冷却活性に関する研究

  王 東暉, 春日 純, 桑原 慎子, 福士 幸治, 藤川 清三, 荒川 圭太

  低温生物工学会誌 57 2 147 - 151 低温生物工学会 2011年 [査読有り][通常論文]
   

  Xylem parenchyma cells (XPCs) of boreal trees adapt to subzero temperatures by deep supercooling. Previous studies suggested that intracellular substances were important for maintaining a supercooling state of cellular water at subzero temperatures in XPCs. In our recent studies, four kinds of flavonol glycosides and four kinds of hydrolyzable gallotannins were identified as supercooling-facilitating (SCF; anti-ice nucleation) substances in xylem extracts of katsura tree (Cercidiphyllum japonicum). In the present study, we examined SCF capabilities of the four kinds of hydrolyzable gallotannins in solutions containing different kinds of ice nucleators, including Erwinia ananas, Xanthomonas campestris, silver iodide, and 1,3,5-trihydroxybenzene (phloroglucinol). The results showed that all four gallotannins exhibited SCF capabilities in the solutions containing various kinds of ice nucleators, but capabilities were dependent on their concentrations and the type of ice nucleator used.
• Xylem water-conducting patterns of 34 broadleaved evergreen trees in southern Japan

  Toshihiro Umebayashi, Yasuhiro Utsumi, Shinya Koga, Susumu Inoue, Junji Matsumura, Kazuyuki Oda, Seizo Fujikawa, Keita Arakawa, Kyoichi Otsuki

  TREES-STRUCTURE AND FUNCTION 24 3 571 - 583 2010年06月 [査読有り][通常論文]
   

  A dye injection method was used to elucidate the xylem water-conducting pathways of 34 broadleaved evergreen trees growing in southern Japan: two semi-ring-porous, 26 diffuse-porous, five radial-porous and one non-vessel species. The large earlywood vessels in semi-ring-porous species have a water transport function in only the outermost annual ring, as in deciduous ring-porous species. On the other hand, the small vessels in semi-ring-porous species maintain the water transport function in many outer annual rings. For the other xylem-type species, the many vessels in many outer annual rings have a water transport function. In diffuse-porous species, we categorized the water-conducting pattern within the annual rings into two types: d1 type, where water travels through vessels in the whole region; and d2 type, where water travels mainly through the earlywood vessels. The pattern in radial-porous species is similar to that in the d1 type; the pattern in non-vessels species is similar to that in the d2 type. The vessel diameter in radial-porous species is similar to that of the earlywood vessels of semi-ring-porous species. These results suggest that the conduit diameter size is only one of many factors determining the water-conducting pathways of broadleaved evergreen species.
• Endoplasmic reticulum-localized small heat shock protein that accumulates in mulberry tree (Morus bombycis Koidz.) during seasonal cold acclimation is responsive to abscisic acid

  Norifumi Ukaji, Chikako Kuwabara, Yuri Kanno, Mitsunori Seo, Daisuke Takezawa, Keita Arakawa, Seizo Fujikawa

  TREE PHYSIOLOGY 30 4 502 - 513 2010年04月 [査読有り][通常論文]
   

  With seasonal changes, several proteins accumulate in the endoplasmic reticulum (ER)-enriched fraction in the bark of mulberry tree (Morus bombycis Koidz.). Results of partial amino acid sequence analysis in our previous study suggested that one of these proteins is the ER-localized small heat shock protein (sHSP), designated 20-kD winter-accumulating protein (WAP20). In the present study, molecular and biochemical properties of WAP20 were investigated in detail. The deduced amino acid sequence of the cDNA has the predicted signal sequence to the ER, retention signal to the ER and two consensus regions conserved in sHSPs. Recombinant WAP20 expressed in Escherichia coli also showed typical biochemical features of sHSPs, including the formation of a high-molecular-mass complex between 200 and 300 kD under native conditions, promotion of the renaturation of chemically denaturated citrate synthase and prevention of heat stress-induced aggregation of the enzyme. Transcript levels of WAP20 in the bark tissue were seasonally changed, showing high expression levels from mid-October to mid-December, and the transcript levels were additionally increased and decreased by cold treatment and warm treatment, respectively. WAP20 transcripts were detected abundantly in bark tissue rather than xylem and winter bud tissues during seasonal cold acclimation. The bark tissue specificity of WAP20 accumulation was also observed by exogenous application of phytohormone abscisic acid (ABA) in de-acclimated twigs, whereas WAP20 transcripts were increased in all of these tissues by heat shock treatment at 37 degrees C in summer twigs. The results suggest that ABA may be involved in the expression of the WAP20 gene in bark tissue of the mulberry tree during seasonal cold acclimation.
• Analysis of supercooling-facilitating (anti-ice nucleation) activity of flavonol glycosides

  Jun Kasuga, Yukiharu Fukushi, Chikako Kuwabara, Donghui Wang, Atsushi Nishioka, Emiko Fujikawa, Keita Arakawa, Seizo Fujikawa

  CRYOBIOLOGY 60 2 240 - 243 2010年04月 [査読有り][通常論文]
   

  Deep Supercooling Xylem parenchyma cells (XPCs) of katsura tree (Cercidiphyllum japonicum) contain four kinds of flavonol glycosides with high supercooling-facilitating (anti-ice nucleation) activities These flavonol glycosides have very similar structures, but their supercooling-facilitating activities are very different In this study, we analyzed the supercooling-facilitating activities of 12 kinds of flavonol glycosides in order to determine the chemical structures that might affect supercooling-facilitating activity All of the flavonol glycosides tested showed supercooling-facilitating activity, although the magnitudes of activity differed among the compounds It was clear that the combination of the position of attachment of the glycosyl moiety, the kind of attached glycosyl moiety and the structure of aglycone determined the magnitude of anti-ice nucleation activity However, there is still some ambiguity preventing the exact identification of features that affect the magnitude of supercooling-facilitating activity. (C) 2009 Elsevier Inc. All rights reserved
• DEAR1, a transcriptional repressor of DREB protein that mediates plant defense and freezing stress responses in Arabidopsis

  Tomokazu Tsutsui, Wataru Kato, Yutaka Asada, Kaori Sako, Takeo Sato, Yutaka Sonoda, Satoshi Kidokoro, Kazuko Yamaguchi-Shinozaki, Masanori Tamaoki, Keita Arakawa, Takanari Ichikawa, Miki Nakazawa, Motoaki Seki, Kazuo Shinozaki, Minami Matsui, Akira Ikeda, Junji Yamaguchi

  JOURNAL OF PLANT RESEARCH 122 6 633 - 643 2009年11月 [査読有り][通常論文]
   

  Plants have evolved intricate mechanisms to respond and adapt to a wide variety of biotic and abiotic stresses in their environment. The Arabidopsis DEAR1 (DREB and EAR motif protein 1; At3g50260) gene encodes a protein containing significant homology to the DREB1/CBF (dehydration-responsive element binding protein 1/C-repeat binding factor) domain and the EAR (ethylene response factor-associated amphiphilic repression) motif. We show here that DEAR1 mRNA accumulates in response to both pathogen infection and cold treatment. Transgenic Arabidopsis overexpressing DEAR1 (DEAR1ox) showed a dwarf phenotype and lesion-like cell death, together with constitutive expression of PR genes and accumulation of salicylic acid. DEAR1ox also showed more limited P. syringae pathogen growth compared to wild-type, consistent with an activated defense phenotype. In addition, transient expression experiments revealed that the DEAR1 protein represses DRE/CRT (dehydration-responsive element/C-repeat)-dependent transcription, which is regulated by low temperature. Furthermore, the induction of DREB1/CBF family genes by cold treatment was suppressed in DEAR1ox, leading to a reduction in freezing tolerance. These results suggest that DEAR1 has an upstream regulatory role in mediating crosstalk between signaling pathways for biotic and abiotic stress responses.
• Cryo-scanning electron microscopic study on freezing behaviors of tissue cells in dormant buds of larch (Larix kaempferi)

  Keita Endoh, Jun Kasuga, Keita Arakawa, Toshiaki Ito, Seizo Fujikawa

  CRYOBIOLOGY 59 2 214 - 222 2009年10月 [査読有り][通常論文]
   

  The freezing behavior of dormant buds in larch, especially at the cellular level, was examined by a Cryo-SEM. The dormant buds exhibited typical extraorgan freezing. Extracellular ice crystals accumulated only in basal areas of scales and beneath crown tissues, areas in which only these living cells had thick walls unlike other tissue cells. By slow cooling (5 degrees C/day) of dormant buds to -50 degrees C, all living cells in bud tissues exhibited distinct shrinkage without intracellular ice formation detectable by Cryo-SEM. However, the recrystallization experiment of these slowly cooled tissue cells. which was done by further freezing of slowly cooled buds with LN and then rewarming to -20 degrees C, confirmed that some of the cells in the leaf primordia, shoot primordia and apical meristem, areas in which cells had thin walls and in which no extracellular ice accumulated, lost freezable water with slow cooling to -30 degrees C, indicating ability of these cells to adapt by extracellular freezing, whereas other cells in these tissues retained freezable water with slow cooling even to -50 degrees C, indicating adaptation of these cells by deep supercooling. On the other hand, all cells in crown tissues and in basal areas of scales, areas in which cells had thick walls and in which large masses of ice accumulated, had the ability to adapt by extracellular freezing. It is thought that the presence of two types of cells exhibiting different freezing adaptation abilities within a bud tissue is quite unique and may reflect sophisticated freezing adaptation mechanisms in dormant buds. (C) 2009 Elsevier Inc. All rights reserved.
• Factors related to change of deep supercooling capability in xylem parenchyma cells of trees

  Seizo Fujikawa, Jun Kasuga, Naoki Takata, Keita Arakawa

  Plant Cold Hardiness: From the Laboratory to the Field 29 - 42 2009年07月15日 [査読無し][通常論文]
  
• Impact of simulated acid snow stress on leaves of cold-acclimated winter wheat

  Keita Arakawa, Hidetoshi Inada, Seizo Fujikawa

  Plant Cold Hardiness: From the Laboratory to the Field 173 - 182 2009年07月15日 [査読無し][通常論文]
  
• 樹木の凍結抵抗性 : 凍らない水

  藤川 清三, 春日 純, 荒川 圭太

  低温生物工学会誌 55 1 37 - 41 低温生物工学会 2009年 [査読無し][通常論文]
   

  Xylem parenchyma cells of trees adapt to subfreezing temperatures by deep supercooling. The mechanisms of deep supercooling, except for physical isolation of water, have been reviewed especially in relation with existence of diverse kinds of supercooling-facilitating substances in the xylem parenchyma cells. Present review also suggested possibility on application of such supercooling-facilitating substances to make unfrozen water in purpose of low-temperature preservation as well as to regulate freezing conditions in purpose of cryopreservation for biological materials.
• Improved cryopreservation by diluted vitrification solution with supercooling-facilitating flavonol glycoside

  Daisuke Kami, Jun Kasuga, Keita Arakawa, Seizo Fujikawa

  CRYOBIOLOGY 57 3 242 - 245 2008年12月 [査読有り][通常論文]
   

  The effect of kaempferol-7-O-glucoside (KF7G), one of the supercooling-facilitating flavonol glycosides which was originally found in deep supercooling xylem parenchyma cells of the katsura tree and was found to exhibit the highest level of supercooling-facilitating activity among reported substances, was examined for successful cryopreservation by vitrification procedures, with the aim of determining the possibility of using diluted vitrification solution (VS) to reduce cryoprotectant toxicity and also to inhibit nucleation at practical cooling and rewarming by the effect of supplemental KF7G. Examination was performed using shoot apices of cranberry and plant vitrification solution 2 (PVS2) with dilution. Vitrification procedures using the original concentration (100%) of PVS2 caused serious injury during treatment with PVS2 and resulted in no regrowth after cooling and rewarming (cryopreservation). Dilution of the concentration of PVS2 to 75% or 50% (with the same proportions of constituents) significantly reduced injury by PVS2 treatment, but regrowth was poor after cryopreservation. It is thought that dilution of PVS2 reduced injury by cryoprotectant toxicity, but such dilution caused nucleation during cooling and/or rewarming, resulting in poor survival. On the other hand, addition of 0.5 mg/ml (0.05% w/v) KF7G to the diluted PVS2 resulted in significantly (p < 0.05) higher regrowth rates after cryopreservation. It is thought that addition of supercooling-facilitating KF7G induced vitrification even in diluted PVS2 probably due to inhibition of ice nucleation during cooling and rewarming and consequently resulted in higher regrowth. The results of the present study indicate the possibility that concentrations of routinely used VSs can be reduced by adding supercooling-facilitating KF7G, by which more successful cryopreservation might be achieved for a wide variety of biological materials. (C) 2008 Elsevier Inc. All rights reserved.
• Deep supercooling xylem parenchyma cells of katsura tree (Cercidiphyllum japonicum) contain flavonol glycosides exhibiting high anti-ice nucleation activity

  Jun Kasuga, Yasuyuki Hashidoko, Atsushi Nishioka, Megumi Yoshiba, Keita Arakawa, Seizo Fujikawa

  PLANT CELL AND ENVIRONMENT 31 9 1335 - 1348 2008年09月 [査読有り][通常論文]
   

  Xylem parenchyma cells (XPCs) of boreal hardwood species adapt to sub-freezing temperatures by deep supercooling to maintain a liquid state of intracellular water near -40 degrees C. Our previous study found that crude xylem extracts from such tree species exhibited anti-ice nucleation activity to promote supercooling of water. In the present study, thus, we attempted to identify the causative substances of supercooling. Crude xylem extracts from katsura tree (Cercidiphyllum japonicum), of which XPCs exhibited deep supercooling to -40 degrees C, were prepared by methanol extraction. The crude extracts were purified by liquid-liquid extraction and then by silica gel column chromatography. Although all the fractions obtained after each purification step exhibited some levels of anti-ice nucleation activity, only the most active fraction was retained to proceed to the subsequent level of purification. High-performance liquid chromatography (HPLC) analysis of a fraction with the highest level of activity revealed four peaks with high levels of anti-ice nucleation activity in the range of 2.8-9.0 degrees C. Ultraviolet (UV), mass and nuclear magnetic resonance (NMR) spectra revealed that these four peaks corresponded to quercetin-3-O-beta-glucoside (Q3G), kaempferol-7-O-beta-glucoside (K7G), 8-methoxykaempferol-3-O-beta-glucoside (8MK3G) and kaempferol-3-O-beta-glucoside (K3G). Microscopic observations confirmed the presence of flavonoids in cytoplasms of XPCs. These results suggest that diverse kinds of anti-ice nucleation substances, including flavonol glycosides, may have important roles in deep supercooling of XPCs.
• 凍結過程に存在する酸性物質が植物細胞の生存に及ぼす影響

  稲田 秀俊, 藤川 清三, 荒川 圭太

  日本冷凍空調学会論文集 25 2 233 - 238 公益社団法人 日本冷凍空調学会 2008年 [査読有り][通常論文]
   

  The objective of this study was to clarify the mechanism of injury caused by acid snow stress in wintering plants. In this study, influence of simulated acid snow stress on leaf tissues of wintering plant was conveniently estimated by extracellular freezing tests under acid conditions in vitro. The survival rates of leaf tissues after freeze-thawing with 0.3 ml of sulfuric acid solution of pH 2.0 were significantly decreased, compared with the survival rates under acid condition of pH 3.0 or pure water. In this study, the initial volume of sulfuric acid solutions was the same in the treatments. Therefore, it is thought that the more acidic the initial pH of sulfuric acid solution becomes, the greater the volume of residual unfrozen solution with concentrated sulfuric acid in the extracellular part at a subzero temperature would be, consequently, the survival rate of leaf tissues were decreased. When leaf tissues were freeze-thawed with a large volume of sulfuric acid solution of pH 3.0, the survival rate of leaf tissues was comparable to the survival rate using pH 2.0. These results suggest that an increase in the volume of acid meltwater derived from snow cover will enhance the damage to wintering plants even the mild acidity of the acid snow.
• Long- and short-term freezing induce different types of injury in Arabidopsis thaliana leaf cells

  M. Nagao, K. Arakawa, D. Takezawa, S. Fujikawa

  PLANTA 227 2 477 - 489 2008年01月 [査読有り][通常論文]
   

  In nature, intact plant cells are subjected to freezing and can remain frozen for prolonged periods. We assayed the survival of Arabidopsis thaliana leaf cells following freezing and found that short- and long-term exposures produced different types of cellular injury. To identify the cause of these injuries, we examined the ultrastructure of the cell plasma membranes. Our results demonstrate that ultrastructural changes in the plasma membrane due to short-term freezing are associated with interbilayer events, including close apposition of the membranes. In both acclimated and non-acclimated leaf cells, these interbilayer events resulted in "fracture-jump lesions" in the plasma membrane. On the other hand, long-term freezing was associated with the development of extensive protein-free areas caused by the aggregation of intramembrane proteins with consequent vesiculation of the affected membrane regions; this effect was clearly different from the ultrastructural changes induced by interbilayer events. We also found that prolonged exposure of non-acclimated leaf cells to a concentrated electrolyte solution produced effects that were similar to those caused by long-term freezing, suggesting that the ultrastructural changes observed in the plasma membrane following long-term freezing are produced by exposure of the leaf cells to a concentrated electrolyte solution. This study illustrates multiple causes of freezing-induced injury in plant cells and may provide useful information regarding the functional role of the diverse changes that occur during cold acclimation.
• Conducting pathways in north temperate deciduous broadleaved trees

  Toshihiro Umebayashi, Yasuhiro Utsumi, Shinya Koga, Susumu Inoue, Seizo Fujikawa, Keita Arakawa, Junji Matsumura, Kazuyuki Oda

  IAWA JOURNAL 29 3 247 - 263 2008年 [査読有り][通常論文]
   

  The interspecific variation of dye ascent in the stems of 44 broadleaved deciduous species growing in Japan was studied using freeze-dried samples after dye injection. The dye ascending pattern differed both within and between ring-porous and diffuse-porous species. In large earlywood vessels of all ring-porous species, the dye ascended only in the outermost annual ring, and the inner annual rings had lost their water transport function. The dye ascending pattern within the inner annual rings in the ring-porous species was categorized into three types: i) the dye ascended both in the many latewood vessels throughout the latewood and small earlywood vessels; ii) the dye ascended in the many vessels throughout the latewood; and iii) the dye ascended mainly in the late latewood vessels. In diffuse-porous species, the dye ascending pattern within the annual rings also was categorized into three types: i) the dye ascended throughout the annual rings; ii) the dye ascended mainly in the earlywood vessels, and iii) the dye ascended mainly in the latewood vessels. Xylem water distribution was also examined by cryo-SEM in three ring-porous and three diffuse-porous species that had different dye ascending patterns. The water distribution pattern within annual rings was correlated with the dye ascending pattern except for one diffuseporous species (Salix gracilistyla). In this case, water was distributed in the whole region of the annual rings although dye was mainly distributed in the earlywood. These results showed that the functional area of water transport within annual rings differed among ring-porous species and diffuse-porous species.
• Anti-ice nucleation activity in xylem extracts from trees that contain deep supercooling xylem parenchyma cells

  Jun Kasuga, Kaoru Mizuno, Keita Arakawa, Seizo Fujikawa

  CRYOBIOLOGY 55 3 305 - 314 2007年12月 [査読有り][通常論文]
   

  Boreal hardwood species, including Japanese white birch (Betula platyphylla Sukat. var. japonica Hara), Japanese chestnut (Castanea crenata Sieb. et Zucc.), katsura tree (Cercidiphyllum japonicum Sieb. et Zucc.), Siebold's beech (Fagus crenata Blume), mulberry (Morus bombycis Koidz.), and Japanese rowan (Sorbus commixta Hedl.), had xylem parenchyma cells (XPCs) that adapt to subfreezing temperatures by deep supercooling. Crude extracts from xylem in all these trees were found to have anti-ice nucleation activity that promoted supercooling capability of water as measured by a droplet freezing assay. The magnitude of increase in supercooling capability of water droplets in the presence of ice-nucleation bacteria, Erwinia ananas, was higher in the ranges from 0.1 to 1.7 degrees C on addition of crude xylem extracts than freezing temperature of water droplets on addition of glucose in the same concentration (100 mosmol/kg). Crude xylem extracts from C japonicum provided the highest supercooling capability of water droplets. Our additional examination showed that crude xylem extracts from C. japonicum exhibited anti-ice nucleation activity toward water droplets containing a variety of heterogeneous ice nucleators, including ice-nucleation bacteria, not only E ananas but also Pseudomonas syringae (NBRC3310) or Xanthomonas campestris, silver iodide or airborne impurities. However, crude xylem extracts from C. japonicum did not affect homogeneous ice nucleation temperature as analyzed by emulsified micro-water droplets. The possible role of such anti-ice nucleation activity in crude xylem extracts in deep supercooling of XPCs is discussed. (C) 2007 Elsevier Inc. All rights reserved.
• Gene expression associated with increased supercooling capability in xylem parenchyma cells of larch (Larix kaempferi)

  Naoki Takata, Jun Kasuga, Daisuke Takezawa, Keita Arakawa, Seizo Fujikawa

  JOURNAL OF EXPERIMENTAL BOTANY 58 13 3731 - 3742 2007年10月 [査読有り][通常論文]
   

  Xylem parenchyma cells (XPCs) in larch adapt to subfreezing temperatures by deep supercooling, while cortical parenchyma cells (CPCs) undergo extracellular freezing. The temperature limits of supercooling in XPCs changed seasonally from -30 degrees C during summer to -60 degrees C during winter as measured by freezing resistance. Artificial deacclimation of larch twigs collected in winter reduced the supercooling capability from -60 degrees C to -30 degrees C. As an approach to clarify the mechanisms underlying the change in supercooling capability of larch XPCs, genes expressed in association with increased supercooling capability were examined. By differential screening and differential display analysis, 30 genes were found to be expressed in association with increased supercooling capability in XPCs. These 30 genes were categorized into several groups according to their functions: signal transduction factors, metabolic enzymes, late embryogenesis abundant proteins, heat shock proteins, protein synthesis and chromatin constructed proteins, defence response proteins, membrane transporters, metal-binding proteins, and functionally unknown proteins. All of these genes were expressed most abundantly during winter, and their expression was reduced or disappeared during summer. The expression of all of the genes was significantly reduced or disappeared with deacclimation of winter twigs. Interestingly, all but one of the genes were expressed more abundantly in the xylem than in the cortex. Eleven of the 30 genes were thought to be novel cold-induced genes. The results suggest that change in the supercooling capability of XPCs is associated with expression of genes, including genes whose functions have not been identified, and also indicate that gene products that have been thought to play a role in dehydration tolerance by extracellular freezing also have a function by deep supercooling.
• Larch (Larix kaempferi) xylem parenchyma cells respond to subfreezing temperature by deep supercooling

  Jun Kasuga, Naoki Takata, Kenichi Yamane, Katsushi Kuroda, Keita Arakawa, Seizo Fujikawa

  CRYOLETTERS 28 2 77 - 81 2007年03月 [査読有り][通常論文]
   

  In previous studies, xylem parenchyma cells (XPCs) in the boreal softwood species larch, which has thick and rigid walls similar to those of XPCs in boreal hardwood species, were shown to respond to subfreezing temperature by deep supercooling during summer but change their freezing behavior to extracellular freezing during winter (4). In this study, we reexamined freezing behavior of XPCs in larch by observation of deep etching of frozen samples as well as observation of re-warmed samples after freezing using a cryo-scanning electron microscope. The results showed that XPCs in larch adapts to subfreezing temperature by deep supercooling throughout all seasons. Such freezing behavior is the same as that of XPCs in boreal hardwood species.
• High accumulation of soluble sugars in deep supercooling Japanese white birch xylem parenchyma cells

  Jun Kasuga, Keita Arakawa, Seizo Fujikawa

  NEW PHYTOLOGIST 174 3 569 - 579 2007年 [査読有り][通常論文]
   

  Seasonal changes in the accumulation of soluble sugars in extracellular freezing cortical parenchyma cells and deep supercooling xylem parenchyma cells in Japanese white birch (Betula platyphylla var. japonica) were compared to identify the effects of soluble sugars on the mechanism of deep supercooling, which keeps the liquid state of water in cells under extremely low temperatures for long periods. Soluble sugars in both tissues were analyzed by high-performance liquid chromatography (HPLC), and the concentrations of sugars in cells were estimated by histological observation of occupancy rates of parenchyma cells in each tissue. Relative and equilibrium melting points of parenchyma cells were measured by differential thermal analysis and cryoscanning electron microscopy, respectively. In both xylem and cortical parenchyma cells, amounts of sucrose, raffinose and stachyose increased in winter, but amounts of fructose and glucose exhibited little change throughout the entire year. In addition, no sugars were found to be specific for either tissue. Combined results of HPLC analyses, histological observation and melting point analyses confirmed that the concentration of sugars was much higher in xylem cells than in cortical cells. It is thought that the higher concentration of soluble sugars in xylem cells may contribute to facilitation of deep supercooling in xylem cells by depressing the nucleation temperature.
• Effects of Light Condition after Simulated Acid Snow Stress on Leaves of Winter Wheat

  INADA Hidetoshi, FUJIKAWA Seizo, SAITO Hideyuki, ARAKAWA Keita

  Environmental Sciences (Tokyo) 14 Supplement 53 - 71 2007年 [査読有り][通常論文]
  
• Optimal conditions for visualizing water-conducting pathways in a living tree by the dye injection method

  Toshihiro Umebayashi, Yasuhiro Utsumi, Shinya Koga, Susumu Inoue, Yasuki Shiiba, Keita Arakawa, Junji Matsumura, Kazuyuki Oda

  Tree Physiology 27 7 993 - 999 2007年 [査読有り][通常論文]
   

  To elucidate the water-conducting pathways in living trees by the dye injection method, suitable sample preparation procedures are needed. We evaluated quantitatively the properties and concentrations of three dyes (acid fuchsin, basic fuchsin and safranin) widely used for this purpose, and determined the optimal conditions required to avoid artifacts after dye injection into the sap stream of Pieris japonica D. Don. Among the dyes tested, an aqueous solution of acid fuchsin at a concentration of 0.1% or more was the most useful for delineating water movement. In non-transpiring stem segments, the vertical movement of acid fuchsin by capillarity and diffusion from the dye injection site was limited. However, acid fuchsin moved rapidly in the horizontal direction by capillarity and diffusion, and most xylem cells were stained within 2 h. A delay of more than 2 h between dye injection and examination of the tissues greatly reduces the precision of the method. Use of the dye injection method without appropriate, well-defined experimental procedures may give rise to misleading information about the functional water-conducting pathway in living trees. © 2007 Heron Publishing.
• Role of intracellular contents to facilitate supercooling capability in beech (Fagus crenata) xylem parenchyma cells

  Jun Kasuga, Kaoru Mizuno, Natsuko Miyaji, Keita Arakawa, Seizo Fujikawa

  CRYOLETTERS 27 5 305 - 310 2006年09月 [査読有り][通常論文]
   

  In order to find the possible role of intracellular contents in facilitating the supercooling capability of xylem parenchyma cells, changes in the temperature of supercooling levels were compared before and after the release of intracellular substances from beech xylem parenchyma cells by DTA. Various methods were employed to release intracellular substances from xylem parenchyma cells and all resulted in a reduction of supercooling ability. It was concluded that the reduction of supercooling ability primarily resulted from changes of intracellular conditions, including the release of intracellular contents or their mixing with extracellular solutions, rather than due to changes of cell wall structures. It is therefore suggested that any unidentified intracellular contents may function to facilitate supercooling capability in xylem parenchyma cells.
• Influence of simulated acid snow stress on leaf tissue of wintering herbaceous plants.

  Hidetoshi Inada, Manabu Nagao, Seizo Fujikawa, Keita Arakawa

  Plant & cell physiology 47 4 504 - 12 2006年04月 [査読有り][通常論文]
   

  Acid snow might be an environmental stress factor for wintering plants since acid precipitates are locally concentrated in snow and the period in which ice crystals are in contact with shoots might be longer than that of acid precipitates in rain. In this study, 'equilibrium' and 'prolonged' freezing tests with sulfuric acid, which simulate situations of temperature depression and chronic freezing at a subzero temperature with acid precipitate as acid snow stress, respectively, were carried out using leaf segments of cold-acclimated winter wheat. When leaf segments were frozen in the presence of sulfuric acid solution (pH 4.0, 3.0 or 2.0) by equilibrium freezing with ice seeding, the survival rate of leaf samples treated with sulfuric acid solution of pH 2.0 decreased markedly. Leaf samples after supercooling to -4 and -8 degrees C in the presence of sulfuric acid solution (pH 2.0) without ice seeding were less damaged. When leaf samples were subjected to prolonged freezing at -4 and -8 degrees C for 7 d with sulfuric acid (pH 2.0), the survival rates of leaf samples exposed to sulfuric acid decreased more than those of leaf samples treated with water. On the other hand, leaf samples were less damaged by prolonged supercooling at -4 and -8 degrees C for 7 d with sulfuric acid (pH 2.0). The results suggest that an acid condition (pH 2.0) in the process of extracellular freezing and/or thawing promotes freezing injury of wheat leaves.
• Accumulation of theanderose in association with development of freezing tolerance in the moss Physcomitrella patens

  M Nagao, K Oku, A Minami, K Mizuno, M Sakurai, K Arakawa, S Fujikawa, D Takezawa

  PHYTOCHEMISTRY 67 7 702 - 709 2006年04月 [査読有り][通常論文]
   

  Mosses are known to have the ability to develop high degrees of resistance to desiccation and freezing stress at cellular levels. However, underlying cellular mechanisms leading to the development of stress resistance in mosses are not understood. We previously showed that freezing tolerance in protonema cells of the moss Physcomitrella patens was rapidly increased by exogenous application of the stress hormone abscisic acid (ABA) [Minami, A., Nagao, M., Arakawa, K., Fujikawa, S., Takezawa, D., 2003a. Abscisic acid-induced freezing tolerance in the moss Physcomitrella patens is accompanied by increased expression of stress-related genes. J. Plant Physiol. 160, 475483]. Herein it is shown that protonema cells with acquired freezing tolerance specifically accumulate low-molecular-weight soluble sugars. Analysis of the most abundant trisaccharide revealed that the cells accumulated theanderose (G(6)-alpha-glucosyl sucrose) in close association with enhancement of freezing tolerance by ABA treatment. The accumulation of theanderose was inhibited by cycloheximide, an inhibitor of nuclear-encoded protein synthesis, coinciding with a remarkable decrease in freezing tolerance. Furthermore, theanderose accumulation was promoted by cold acclimation and treatment with hyperosmotic solutes, both of which had been shown to enhance cellular freezing tolerance. These results reveal a novel role for theanderose, whose biological function has been obscure, in high freezing tolerance in moss cells. (c) 2006 Elsevier Ltd. All rights reserved.
• Functional role of winteraccumulating proteins from mulberry tree in adaptation to winter-induced stresses

  S. Fujikawa, N. Ukaji, M. Nagao, K. Yamane, D. Takezawa, K. Arakawa

  Cold Hardiness in Plants: Molecular Genetics, Cell Biology, and Physiology 181 - 202 2006年01月10日 [査読無し][通常論文]
  
• Physiological and morphological alterations associated with development of freezing tolerance in the moss physcomitrella patens

  A. Minami, M. Nagao, K. Arakawa, S. Fujikawa, D. Takezawa

  Cold Hardiness in Plants: Molecular Genetics, Cell Biology, and Physiology 138 - 152 2006年01月10日 [査読無し][通常論文]
  
• 酸性雪ストレスに対する越冬性植物の生理応答

  稲田 秀俊, 藤川 清三, 荒川 圭太

  低温生物工学会誌 52 2 163 - 168 低温生物工学会 2006年 [査読有り][通常論文]
   

  The object of this study is to clarify the responses of wintering plants that were re-grown under light / dark condition after treatment of simulated acid snow (SAS) stress with sulfuric acid solution. Fresh weight and relative water content of mature leaves were markedly decreased compared with those of younger leaves during a re-growth period of wheat pretreated by SAS (pH 2.0). Photochemical efficiency of mature leaves was decreased during a re-growth period over 24 hours, but recovered in younger leaves. The level of lipid peroxidation in mature leaves was higher than that of younger leaves during a re-growth period within 24 hours.
• Physiological changes in relation to the development of freezing resistance in xylem tissue of Betula platyphylla during seasonal cold acclimation

  Arakawa K, Kasuga J, Takashima H, Fujikawa S

  Tree Sap III 93 - 97 2006年 [査読無し][通常論文]
  
• 凍らない水 : 深過冷却する樹木木部柔細胞から水を凍結させない物質を探す

  藤川 清三, 春日 純, 荒川 圭太

  化学と生物 43 5 280 - 282 日本農芸化学会 2005年05月01日 [査読無し][通常論文]
  
• Rapid degradation of starch in chloroplasts and concomitant accumulation of soluble sugars associated with ABA-induced freezing tolerance in the moss Physcomitrefla patens

  M Nagao, A Minami, K Arakawa, S Fujikawa, D Takezawa

  JOURNAL OF PLANT PHYSIOLOGY 162 2 169 - 180 2005年02月 [査読有り][通常論文]
   

  Abscisic acid (ABA) has been postulated to play a role in the development of freezing tolerance during the cold acclimation process in higher plants, but its role in cold tolerance in lower land plants has not been elucidated. The moss Physcomitrella patens rapidly developed freezing tolerance when its protonemata were grown in a medium containing ABA, with dramatic changes in the LT50 value from -2 degrees C to over -10 degrees C. We examined physiological and morphological. alterations in protonema cells caused by ABA treatment to elucidate early cellular events responsible for rapid enhancement of freezing tolerance. Microscopic observations revealed that ABA treatment for 1 day resulted in a dramatic alteration in the appearance of intracellular organelles. ABA-treated cells had slender chloroplasts, with a reduced amount of starch grains, in comparison with those of non-treated cells. The ABA-treated cells also had several segmented vacuoles while many of non-treated cells had one central vacuole. When frozen to -4 degrees C, freezing injury-associated ultrastructural. changes such as formation of aparticulate domains and fracture-jump lesions were frequently observed in the plasma membrane of non-treated protonema cells but not in that of ABA-treated cells. The ABA treatment increased the osmotic concentration of the protonema cells, in correlation with accumulation of free soluble sugars. These results suggest that ABA-induced accumulation of soluble sugars, associated with morphological changes in organelles, mitigated freezing-induced structural damage in the plasma membrane, eventually leading to enhancement of freezing tolerance in the protonema cells. (c) 2004 Elsevier GmbH. All rights reserved.
• 針葉樹木部からの氷核形成阻害物質の検出

  水野 薫, 春日 純, 荒川 圭太, 藤川 清三

  低温生物工学会誌 51 2 111 - 114 低温生物工学会 2005年 [査読有り][通常論文]
   

  Xylem parenchyma cells of most boreal trees including conifers adapt to subfreezing temperature by deep supercooling. Present study showed that crude extracts from xylem of several conifers had effect to promote supercooling of small water droplets with AgI as an ice-nucleating substance. Fractionation of the crude extracts from xylem of Larix kaempferi indicated that any substances except for soluble sugars were responsible for the promotion of supercooling, possibly by inhibiting or reducing ice nucleation. We also showed that the extracts from xylem of L. kaempferi had effect to promote supercooling, not only of small water droplets (2μl) but also of bulk water (1ml).
• 凍結融解過程における酸性条件が越冬性植物の生存に及ぼす影響

  稲田 秀俊, 藤川 清三, 荒川 圭太

  低温生物工学会誌 51 2 115 - 119 低温生物工学会 2005年 [査読有り][通常論文]
   

  Acid precipitates in snow may be a stress factor that affects the growth of wintering plants. The objective of this study was to determine the influence of acid-snow stress on leaves of wintering plants by in vitro experiments. Three different experiments, equilibrium freezing, prolonged freezing and repeated freeze-thawing, were carried out in order to simulate freeze-thawing of acid snow during winter. The results showed that acidification in the process of freeze-thawing caused enhancement of freezing injury of wintering plants.
• ブナ木部組織の深過冷却能に関与する蛋白質因子の探索

  高橋 宏和, 荒川 圭太, 藤川 清三

  低温生物工学会誌 51 2 105 - 109 低温生物工学会 2005年 [査読有り][通常論文]
   

  Change in the deep supercooling (DSC) ability of xylem tissues of beech (Fagus crenata L.) twigs was detected by a differential thermal analysis (DTA). DSC abilities of xylem tissue shown as peaks of low temperature exotherms in DTA were about -30℃ in summer and -40℃ in winter. DSC ability of beech twigs in winter was lowered to about -30℃ by heat treatment at 60℃ for 10 min but was not lowered by heat treatment at 50℃ for 10 min. In order to find protein factors related to DSC ability, changes in the protein composition of xylem tissues caused by seasonal cold acclimation and heat treatments were analyzed by two-dimensional gel electrophoresis. About 350 protein spots were detected in the crude soluble fraction of xylem tissues in winter. Seventy-five protein spots were induced during seasonal cold acclimation. Fifty-six of the cold acclimation-induced proteins remained after heat treatment of xylem tissues at 50℃ for 10 min, and 12 of the 56 proteins were decreased by heat treatment of xylem tissues at 60℃ for 10 min.
• Cold acclimation in bryophytes: low-temperature-induced freezing tolerance in Physcomitrella patens is associated with increases in expression levels of stress-related genes but not with increase in level of endogenous abscisic acid

  A Minami, M Nagao, K Ikegami, T Koshiba, K Arakawa, S Fujikawa, D Takezawa

  PLANTA 220 3 414 - 423 2005年01月 [査読有り][通常論文]
   

  Bryophyte species growing in areas in which temperatures fall below zero in winter are likely to have tolerance to freezing stress. It is well established in higher plants that freezing tolerance is acquired by exposure to non-freezing low temperatures, accompanied by expression of various genes and increases in levels of the stress hormone abscisic acid (ABA). However, little is known about the physiological changes induced by cold acclimation in non-vascular plants such as bryophytes. We examined the effects of low temperatures on protonema cells of the moss Physcomitrella patens (Hedw.) Bruch & Schimp. The freezing tolerance of protonema cells was clearly increased by incubation at low temperatures ranging from 10degreesC to 0degreesC, with maximum tolerance achieved by incubation at 0degreesC for several days. The enhancement of freezing tolerance by low temperatures occurred in both light and dark conditions and was accompanied by accumulation of several transcripts for late-embryogenesis-abundant (LEA) proteins and boiling-soluble proteins. By de-acclimation, low-temperature-induced expression of these transcripts and proteins, as well as the freezing tolerance, was reduced. Interestingly, endogenous levels of ABA in tissues or that secreted into the culture medium were not specifically increased by low-temperature treatment. Furthermore, removal of ABA from the medium by addition of activated charcoal did not affect low-temperature-induced freezing tolerance of the protonema cells. Our results provide evidence that bryophytes have an ABA-independent cold-signaling pathway leading to expression of stress-related genes and resultant acquisition of freezing tolerance.
• Accumulation of pathogenesis-related (PR) 10/Bet v 1 protein homologues in mulberry (Morus bombycis Koidz.) tree during winter

  N. UKAJI, C. KUWABARA, D. TAKEZAWA, K. ARAKAWA, S. FUJIKAWA

  Plant, Cell and Environment 27 9 1112 - 1121 2004年09月 [査読有り][通常論文]
  
• 植物細胞の深過冷却現象およびそのメカニズム

  藤川 清三, 荒川 圭太

  低温生物工学会誌 50 1 33 - 36 低温生物工学会 2004年 [査読無し][通常論文]
   

  Xylem ray parenchyma cells in woody plants adapt to freezing of apoplast water by deep supercooling. The mechanism of deep supercooling is proved not due to merely cell wall property that water in micro-capillaries within the cell walls are sufficiently small to keep supercooling by which inhibits ice seeding of protoplasts through the cell walls. The mechanism of supercooling, thus, may respond to intracellular factors, such as accumulation of any substances that facilitate supercooling. Our approach to identify these intracellular substances from xylem ray parenchyma cells of woody plants that exhibit deep supercooling was introduced in this review.
• A methyl viologen-resistant mutant of Arabidopsis, which is allelic to ozone-sensitive rcd1, is tolerant to supplemental ultraviolet-B irradiation

  T Fujibe, H Saji, K Arakawa, N Yabe, Y Takeuchi, KT Yamamoto

  PLANT PHYSIOLOGY 134 1 275 - 285 2004年01月 [査読有り][通常論文]
   

  To better understand the role of active oxygen species (AOS) in acquired resistance to increased levels of ultraviolet (UV)-B irradiation in plants, we isolated an Arabidopsis mutant that is resistant to methyl viologen, and its sensitivity to UV-B was investigated. A complementation test revealed that the obtained mutant was allelic to the ozone-sensitive radical-induced. cell death1-1 (rcd1-1). Therefore, this mutant was named rcd1-2. rcd1-2 was recessive and nearly 4-fold more resistant to methyl viologen than wild type. It exhibited a higher tolerance to short-term UV-B supplementation treatments than the wild type: UV-B-induced formation of cyclobutane pyrimidine dimers was reduced by one-half after 24 h of exposure; the decrease in quantum yield of photosystem II was also diminished by 40% after 12 h of treatment. Furthermore, rcd1-2 was tolerant to freezing. Steady-state mRNA levels of plastidic Cu/Zn superoxide dismutase and stromal ascorbate peroxidase were higher in rcd1-2 than in wild type, and the mRNA level of the latter enzyme was enhanced by UV-13 exposure more effectively in rcd1-2. UV-B-absorbing compounds were more accumulated in rcd1-2 than in wild type after UV-B exposure for 24 h. These findings suggest that rcd1-2 methyl viologen resistance is due to the enhanced activities of the AOS-scavenging enzymes in chloroplasts and that the acquired tolerance to the short-term UV-B exposure results from a higher accumulation of sunscreen pigments. rcd1 appears to be a mutant that constitutively shows stress responses, leading to accumulation of more pigments and AOS-scavenging enzymes without any stresses.
• Frost-susceptible protein in plasma membranes in tubers of Helianthus tuberosus L.

  K Arakawa, M Hanazaki, S Yoshida

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 68 1 175 - 182 2004年01月 [査読有り][通常論文]
   

  When plasma membranes were prepared from tubers of Helianthus tuberosus L. (Jerusalem artichoke) frozen at a sublethal temperature (-10degreesC), the levels of some plasma membrane proteins, named frost-susceptible proteins (FSPs), decreased [Uemura, M., et al., Plant Physiol., 80, 187-195 (1986)]. The aim of this study was to characterize the response of FSP120, which is named FSP-3 in a previous report, to freezing treatment by immunoblotting. Levels of FSP120 in the plasma membranes of tubers decreased after sublethal freezing, whereas no degraded products were detected in the microsomes or the soluble fraction. The amount of FSP120 in the crude extract of frozen tubers remained at a comparable level to that of the unfrozen tubers. These results suggest that FSP120 might be released from plasma membranes during freezing treatment of the tubers of Jerusalem artichoke.
• Abscisic acid-induced freezing tolerance in the moss Physcomitrella patens is accompanied by increased expression of stress-related genes

  A Minami, M Nagao, K Arakawa, S Fujikawa, D Takezawa

  JOURNAL OF PLANT PHYSIOLOGY 160 5 475 - 483 2003年05月 [査読有り][通常論文]
   

  Abscisic acid (ABA)-induced genes are implicated in the development of freezing tolerance during cold acclimation in higher plants, but their roles in lower land plants have not been determined. We examined ABA- and cold-induced changes in freezing tolerance and gene expression in the moss Physcomitrella patens. Slow equilibrium freezing to -4degreesC of R patens protonemata grown under normal growth conditions killed more than 90% of the cells, indicating that the protonema cells are freezing-sensitive. ABA treatment for 24h dramatically increased the freezing tolerance of the protonemata, while cold treatment only slightly increased the freezing tolerance within the same period. We examined the expressions of fourteen Physcomitrella patens ABA-responsive genes (PPARs), isolated from ABA-treated protonemata. ABA treatment resulted in a remarkable increase in the expression of all the PPAR genes within 24 h. Several of the PPAR genes (PPAR 1 to 8, and 14) were also responsive to cold, but the response was much slower than that to ABA. Treatment with hyperosmotic concentrations of NaCl and mannitol increased freezing tolerance of protonemata and also increased the expression levels of eleven PPAR genes (PPAR2, 3, 5 to 8, and 10 to 14). These results suggest that ABA and environmental stresses positively affect the expression of common genes that participate in protection of protonema cells leading to the development of freezing tolerance.
• Xylem ray parenchyma cells in boreal hardwood species respond to subfreezing temperatures by deep supercooling that is accompanied by incomplete desiccation

  K Kuroda, J Kasuga, K Arakawa, S Fujikawa

  PLANT PHYSIOLOGY 131 2 736 - 744 2003年02月 [査読有り][通常論文]
   

  It has been accepted that xylem ray parenchyma cells (XRPCs) in hardwood species respond to subfreezing temperatures either by deep supercooling or by extracellular freezing. Present study by cryo-scanning electron microscopy examined the freezing responses of XRPCs in five boreal hardwoods: Salix sachalinensis Fr. Schmit, Populus sieboldii Miq., Betula platyphylla Sukat. var japonica Hara, Betula pubescens Ehrh., and red osier dogwood (Cornus sericea), in which XRPCs have been reported to respond by extracellular freezing. Cryo-scanning electron microscopy observations revealed that slow cooling of xylem to -80degreesC resulted in intracellular freezing in the majority of XRPCs in S. sachalinensis, an indication that these XRPCs had been deep supercooled. In contrast, in the majority of XRPCs in P. sieboldii, B. platyphylla, B. pubescens, and red osier dogwood, slow cooling to -80degreesC produced slight cytorrhysis without clear evidence of intracellular freezing, suggesting that these XRPCs might respond by extracellular freezing. In these XRPCs exhibited putative extracellular freezing; however, deep etching revealed the apparent formation of intracellular ice crystals in restricted local areas. To confirm the occurrence of intracellular freezing, we rewarmed these XRPCs after cooling and observed very large intracellular ice crystals as a result of the recrystallization. Thus, the XRPCs in all the boreal hardwoods that we examined responded by deep supercooling that was accompanied with incomplete desiccation. From these results, it seems possible that limitations to the deep-supercooling ability of XRPCs might be a limiting factor for adaptation of hardwoods to cold climates.
• アブシジン酸による耐凍性上昇に伴うヒメツリガネゴケの生理的変化の解析

  南 杏鶴, 長尾 学, 荒川 圭太, 藤川 清三, 竹澤 大輔

  低温生物工学会誌 49 2 179 - 183 低温生物工学会 2003年 [査読有り][通常論文]
   

  The phytohormone abscisic acid (ABA) plays an important role in freezing tolerance in plants. Treatment with ABA increased freezing tolerance of protonema cells of the moss Physcomitrella patens. ABA triggered morphological changes in cellular organelles, such as reduction in sizes of chloroplasts and vacuoles, and physiological changes such as accumulation of soluble sugars, especially that of sucrose, and boiling-stable proteins. We used protein synthesis inhibitors, protein serine/threonine kinase inhibitors and protein serine/threonine phosphatase inhibitors to examine cellular events associated with ABA-induced freezing tolerance. The results indicated that a protein synthesis inhibitor cycloheximide dramatically decreased freezing tolerance of the ABA-treated cells and accumulation of boiling-stable proteins. A phosphatase inhibitor okadaic acid also decreased ABA-induced freezing tolerance in P. patens. These inhibitors, however, did not have any effect on ABA-induced accumulation of sucrose. These results suggest critical roles of de novo synthesis of nuclear encoded protein and phosphatase-mediated signal transduction in ABA-induced cellular processes leading to freezing tolerance, and that sucrose only plays a limited role in these processes.
• シラカンバ木部組織に蓄積する可溶性糖の季節変化

  春日 純, 藤川 清三, 荒川 圭太

  低温生物工学会誌 49 2 185 - 189 低温生物工学会 2003年 [査読有り][通常論文]
   

  Most of xylem parenchyma cells of boreal hard wood species respond to subfreezing temperature by deep supercooling. Because xylem tissue cells that adapt by deep supercooling have the least resistance to subfreezing temperature among woody tissues, the limit of supercooling ability of xylem parenchyma cells is an essential factor for the survival of woody plants in cold areas. However, the deep supercooling mechanism of xylem parenchyma cell has not yet been clarified. In this study, accumulation of soluble sugars during seasonal cold acclimation in birch (Betula platyphylla var. japonica Kara) xylem tissue has been examined to discuss the influences of the intracellular sugars to the supercooling ability of xylem parenchyma cells. The supercooling ability of xylem parenchyma cells of birch seasonally changed. Fructose, glucose, sucrose, raffinose and stachyose were mainly accumulated in birch xylem parenchyma cells. Seasonal change in the amount of sucrose, raffinose and stachyose was positively correlated with the supercooling ability in xylem parenchyma cells. Although the proportion of each sugar composition was different between xylem parenchyma cells that undergo deep supercooling and bark parenchyma cells that undergo extracellular freezing, the major components of soluble sugars which accumulated in xylem cells were same with those of bark cells. However, the concentration of soluble sugars in xylem parenchyma cells might be higher than that of bark cells or cambial cells. It seems that the intracellular soluble sugars participate, at least in part, to the increase of supercooling ability of xylem parenchyma cells via increasing intracellular osmotic concentration. However, such a freezing temperature depression alone cannot explain high supercooling ability of xylem parenchyma cells, suggesting presence of other factors which may promote supercooling.
• 深過冷却で寒冷環境に適応するカラマツ木部柔細胞の遺伝子発現

  高田 直樹, 南 杏鶴, 荒川 圭太, 竹澤 大輔, 藤川 清三

  低温生物工学会誌 49 2 191 - 194 低温生物工学会 2003年 [査読有り][通常論文]
   

  Xylem ray parenchyma cells (XRPCs) of Larix kaempferi respond to subfreezing temperatures by deep supercooling in winter. We carried out differential display to isolate genes expressed in association with increase in the magnitude of deep supercooling ability in XRPCs of L. kaempferi, and identified ten genes preferentially expressed during winter seasons. These genes encoded proteins with sequence similarity to NBS/LRR protein (WXL1), organic anion transporter (WXL2), H-pyrophosphatase (WXL3), aminoalcoholphosphotransferase (WXL4), flavonol 3-O-glucosyltransferase (WXL5), histone H2A (WXL6), LRR-containing F-box protein (WXL7), reverse transcriprase (WXL8), a protein similar to Arabidopsis protein T20 L15, 20 (WXL9) and an unknown protein (WXL10). These genes were differentially expressed during fall and winter seasons. Expression of transcripts of WXL4 and 9 increased from October, while that of WXL1, 2, 3, 5, 6, 7, 8 and 10 dramatically increased in November. Transcripts of WXL3 and 9 were similarly expressed in xylem and cortex tissues, while those of WXL1, 2, 4, 5, 6, 7, 8 and 10 were preferentially expressed in xylem tissue.
• 樹木木部柔細胞の深過冷却をもたらすと考えられている細胞壁マイクロキャピラリーサイズを解析する試み

  田中 聡子, 長尾 学, 船田 良, 藤川 清三, 荒川 圭太

  低温生物工学会誌 49 2 209 - 213 低温生物工学会 2003年 [査読有り][通常論文]
   

  The size of micro-capillaries within cell walls of xylem parenchyma cells of woody plants has been estimated by the plasmolysis method using hypertonic solutions consisted of various size of molecules ranging from 0.8 nm to 5.2 nm. All xylem parenchyma cells examined, including xylem ray parenchyma cells exhibiting typical deep supercooling to -40℃, produced distinct plasmolysis by treatment with these all hypertonic solutions, indicating that size of cell wall micro-capillaries in xylem cells exceeded more than 5.2 nm. Water located in cell wall micro-capillaries in size larger than 5.2 nm cannot supercool at least below -20℃. Thus, it is suggested that the cause of deep supercooling of xylem parenchyma cells in woody plants cannot be explained solely by structural property of cell walls in relation with the size of micro-capillaries.
• キクイモ塊茎の凍結傷害過程で生じる細胞膜蛋白質の組成変化に関する研究

  荒川 圭太, 稲田 秀俊

  低温生物工学会誌 49 2 203 - 207 低温生物工学会 2003年 [査読有り][通常論文]
   

  When tubers of Helianthus tuberosus L. (Jerusalem artichoke) were frozen at -10℃ by equilibrium freezing, the tubers were injured lethally after thawing. In plasma membrane fraction prepared from sublethally frozen tubers, the levels of some plasma membrane proteins, named frost-susceptible proteins (FSPs), decreased as compared with that from unfrozen tubers. Response of FSP120, one of the major FSPs with a molecular mass of 120 kDa, to freezing treatment were characterized using the antibody to FSP120 in this study. The total amount of FSP120 in the crude extract scarcely declined after sublethal freezing treatment of tubers although the levels of FSP120 in the crude microsomes decreased after freezing treatment. These results suggest that FSP120 might be released from plasma membranes during freezing treatment of the tubers of Jerusalem artichoke.
• シラカンバ木部組織において季節的低温馴化によって誘導される細胞外蛋白質の解析

  荒川 圭太, 春日 純, 高島 寛, 藤川 清三

  低温生物工学会誌 49 2 195 - 201 低温生物工学会 2003年 [査読有り][通常論文]
   

  Freezing resistance of xylem ray parenchyma cells of birch (Betula platyphylla L.) by deep supercooling increases during seasonal cold acclimation. When apoplast substances were partially eluted by an acidic solution from winter xylem tissue, freezing resistance by deep supercooling of the tissue was decreased. Several major proteins were detected in the acid-extractable fraction of xylem tissue, and the amounts of those proteins were increased in winter. Immunoblot analysis showed that some of the major proteins are similar. N-terminal amino acid sequencing revealed that they are similar to the pathogenesis-related proteins.
• Roles of the plasma membrane and the cell wall in the responses of plant cells to freezing

  T Yamada, K Kuroda, Y Jitsuyama, D Takezawa, K Arakawa, S Fujikawa

  PLANTA 215 5 770 - 778 2002年09月 [査読無し][通常論文]
   

  In an effort to clarify the responses of a wide range of plant cells to freezing, we examined the responses to freezing of the cells of chilling-sensitive and chilling-resistant tropical and subtropical plants. Among the cells of the plants that we examined, those of African violet (Saintpaulia grotei Engl.) leaves were most chilling-sensitive, those of hypocotyls in mungbean [Vigna radiata (L.) R. Wilcz] seedlings were moderately chilling-sensitive, and those of orchid [Paphiopedilum insigne (Wallich ex Lindl.) Pfitz.] leaves were chilling-resistant, when all were chilled at -2 degreesC. By contrast, all these plant cells were freezing-sensitive and suffered extensive damage when they were frozen at -2 degreesC. Cryo-scanning electron microscopy (Cryo-SEM) confirmed that, upon chilling at -2 degreesC, both chilling-sensitive and chilling-resistant plant cells were supercooled. Upon freezing at -2 degreesC, by contrast, intracellular freezing occurred in Saintpaulia leaf cells, frost plasmolysis followed by intracellular freezing occurred in mungbean seedling cells, and extracellular freezing (cytorrhysis) occurred in orchid leaf cells. We postulate that chilling-related destabilization of membranes might result in the loss of the ability of the plasma membrane to act as a barrier against the propagation of extracellular ice in chilling-sensitive plant cells. We also examined the role of cell-walls in the response to freezing using cells in which the plasma membrane had been disrupted by repeated freezing and thawing. In chilling-sensitive Saintpaulia and mungbean cells, the cells with a disrupted plasma membrane responded to freezing at -2 degreesC by intracellular freezing. By contrast, in chilling-resistant orchid cells, as well as in other cells of chilling-resistant and freezing-resistant plant tissues, including leaves of orchard grass (Dactylis glomerata L.), leaves of Arabidopsis thaliana (L.) Heynh. and cortical tissues of mulberry (Morus bombycis Koids.). cells with a disrupted plasma membrane responded to freezing by extracellular freezing. Our results indicate that, in the chilling-sensitive plants cells that we examined. not only the plasma membrane but also the cell wall lacked the ability to serve as a barrier against the propagation of extracellular ice, whereas in the chilling-resistant plant cells that we examined, not only the plasma membrane but also the cell wall acted as a barrier against the propagation of extracellular ice. It appears. therefore, that not only the plasma membrane but also the cell wall greatly influences the freezing behavior of plant cells.
• Abscisic acid- and cold-induced thaumatin-like protein in winter wheat has an antifungal activity against snow mould, Microdochium nivale

  C Kuwabara, D Takezawa, T Shimada, T Hamada, S Fujikawa, K Arakawa

  PHYSIOLOGIA PLANTARUM 115 1 101 - 110 2002年05月 [査読有り][通常論文]
   

  Cold acclimation of winter wheat (Triticum aestivum L.) seedlings induces accumulation in the apoplast of taTLPs that are similar to thaumatin-like proteins (TLPs), which are pathogenesis-related proteins. We characterized a cDNA of WAS-3a encoding the major isoform of taTLPs from winter wheat cells and showed that WAS-3a transcripts were markedly increased by treatment with ABA and by treatment with elicitors (chitosan, beta-glucan and cell wall fractions of Fusarium oxysporum and Microdochium nivale) in wheat cells. To analyse the function of WAS-3a, a highly efficient expression system using wheat cells was established, and a large amount of recombinant WAS-3a protein (rWAS-3a) was obtained with near homogeneity. Antifungal assays using various fungi grown on agar plates revealed that rWAS-3a inhibits hyphal growth of pink snow mould, Microdochium nivale, at a low temperature. The results suggest that cold-induced taTLPs that accumulate in the apoplast contribute to snow mould resistance of winter wheat.
• Cold acclimation-induced WAP27 localized in endoplasmic reticulum in cortical parenchyma cells of mulberry tree was homologous to group 3 late-embryogenesis abundant proteins

  N Ukaji, C Kuwabara, D Takezawa, K Arakawa, S Fujikawa

  PLANT PHYSIOLOGY 126 4 1588 - 1597 2001年08月 [査読有り][通常論文]
   

  We have shown that two 27-kD proteins, designated as WAP27A and WAP27B, were abundantly accumulated in endoplasmic reticulum-enriched fractions isolated from cortical parenchyma cells of mulberry tree (Mortis bombycis Koidz.) during winter (N. Ukaji, C. Kuwabara, D. Takezawa, K. Arakawa, S. Yoshida, S. Fujikawa [1999] Plant Physiol 120: 480-489). In the present study, cDNA clones encoding WAP27A and WAP27B were isolated and characterized. The deduced amino acid sequences of WAP27A and WAP27B cDNAs had 12 repeats of an 11-mer amino acid motif that was the common feature of group 3 late-embryogenesis-abundant proteins. Under field conditions, transcripts of WAP27 genes were initially detected in mid-October, reached maximum level from mid-November to mid-December, and then gradually decreased. The transcript levels of WAP27 genes in cortical parenchyma cells harvested in October was drastically induced by cold treatment within a few days, whereas those in cortical parenchyma cells harvested in August were low even by cold treatment for 3 weeks. Immunocytochemical analysis by electron microscopy confirmed that WAP27 was localized specifically in vesicular-form ER and also localized in dehydration-induced multiplex lamellae-form ER. The role of WAP27 in the ER is discussed in relation to acquisition of freezing tolerance of cortical parenchyma cells in mulberry tree during winter.
• ATP analogue binding to the A subunit induces conformational changes in the E subunit that involves a disulfide bond formation in plant V-ATPase

  Y Kawamura, K Arakawa, M Maeshima, S Yoshida

  EUROPEAN JOURNAL OF BIOCHEMISTRY 268 10 2801 - 2809 2001年05月 [査読有り][通常論文]
   

  Vacuolar H+-ATPase (V-ATPase) consists of a catalytic head, a stalk part and a membrane domain. We indirectly investigated the interaction between the A subunit (catalytic head) and the E subunit (stalk part) using an ATP analogue, adenosine 5'-[beta,gamma -imino]triphosphate (AMP-PNP), which holds the enzyme in the substrate-binding state. AMP-PNP treatment caused a mobility shift of the E subunit with a faster migration in SDS/polyacrylamide gel electrophoresis without a reductant, while ATP treatment did not. A mobility shift of the E subunit has been detected in several plants. As polypeptides with intramolecular disulfide bonds migrate faster than those without disulfide bonds, the mobility shift may be due to the formation of an intramolecular disulfide bond by two cysteine residues conserved among several plant species. The mobility shift may be involved in the binding of AMP-PNP to the ATP-binding site, which exists in the A and B subunits, as it was inhibited by the addition of ATP. Pretreatment with 2'-3'-O-(4-benzoylbenzoyl)-ATP (Bz-ATP), which modifies the ATP-binding site of the B subunit under UV illumination, did not inhibit the mobility shift of the E subunit caused by AMP-PNP treatment. The response of V-ATPase following the AMP-PNP binding may cause a conformational change in the E subunit into a form that is susceptible to oxidation of cysteine residues. This is the first demonstration of interaction between the A and E subunits in the substrate-binding state of a plant V-ATPase.
• Tissue specificity of E subunit isoforms of plant vacuolar H+-ATPase and existence of isotype enzymes

  Y Kawamura, K Arakawa, M Maeshima, S Yoshida

  JOURNAL OF BIOLOGICAL CHEMISTRY 275 9 6515 - 6522 2000年03月 [査読有り][通常論文]
   

  Immunoblot analyses and partial amino acid sequencings revealed that both the 40- (E1) and 37-kDa (E2) subunits of V-ATPase in the pea epicotyl were E subunit isoforms. Similarly, both the 35- (D1) and 29-kDa (D2) subunits were D subunit isoforms, although the similarity of the amino acid sequences is still unknown. In immunoblot analyses, two or three E subunit isoforms with molecular masses ranging from 29 to 40 kDa were detected in other plants. Two isotypes of V-ATPase from the pea epicotyl were separated by ion exchange chromatography and had subunit compositions differing only in the ratio of E1 and E2. There was a difference in the V-max and K-m of ATP hydrolysis between the two isotypes. E1 was scarcely detected in crude membrane fractions from the leaf and cotyledon, while E2 was detected in fractions from all of the tissues examined. The compositions of D subunit isoforms in the leaf and epicotyl were different, and the vacuolar membrane in the leaf did not contain D2. The efficiency of H+ pumping activity in the vacuolar membrane of the leaf was higher than that of the epicotyl. The results suggest that the presence of the isoforms of D and E subunits is characteristic to plants and that the isoforms are closely related to the enzymatic properties.
• Freezing adaptation mechanisms of living tissue cells in Betula platyphylla var. japonica Hara and their relation to cell wall properties.

  Fujikawa S, Kubota M, Kuroda K, Takezawa D, Arakawa K

  Tree Sap II 93 - 96 2000年 [査読無し][通常論文]
  
• Alterations of intracellular pH in response to low temperature stresses

  S Yoshida, K Hotsubo, Y Kawamura, M Murai, K Arakawa, D Takezawa

  JOURNAL OF PLANT RESEARCH 112 1106 225 - 236 1999年06月 [査読有り][通常論文]
   

  In chilling-sensitive plants, the inactivation of the vacuolar H+-ATPase is one of the primary cellular events directly resulting from cold exposure. We demonstrate here that cold-induced inactivation of the proton translocating enzyme is closely linked to the rapid acidification of the cytoplasm and the concomitant alkalization of the vacuoles, suggesting an important role of the enzyme in maintaining homeostasis of the cellular pH in a cold environment. The stability of the vacuolar H+-ATPase to cold both in vivo and in vitro is distinctly different between species sensitive and insensitive to cold. These findings provide further insight into the way in which the vacuolar H+-ATPase is involved in cold adaptation of plants. In addition, the temperature reduction and the concentration of the cytoplasm as a consequence of freeze-induced dehydration may also result in changes in the cellular pH. In fact, we demonstrate here that the cytoplasm is markedly acidified upon freezing; in particular, in cells of less hardy plants. Freeze-induced acidification is presumably due to changes in the physicochemical properties of the cytoplasm and the changes in the permeability of the vacuolar membrane both of which result from severe dehydration. The physiological significance of freeze-induced acidification of the cytoplasm is discussed.
• Akinete formation in Tribonema bombycinum derbes et solier (Xanthophyceae) in relation to freezing tolerance

  M Nagao, K Arakawa, D Takezawa, S Yoshida, S Fujikawa

  JOURNAL OF PLANT RESEARCH 112 1106 163 - 174 1999年06月 [査読有り][通常論文]
   

  The process of akinete formation in relation to the acquirement of freezing tolerance in a fresh water alga, Tribonema bombycinum (Xanthophyceae), was examined, T. bombycinum shifted from vegetative cells to akinetes with starving by a prolonged batch culture, by culture with a diluted medium, or by culture with a single nutrient-deficient medium. In addition, akinetes developed by desiccation, but cold treatment at 4 C did not facilitate akinete formation. During starving, the vegetative cells, which had a large central vacuole in the protoplasm and thin cell walls, finally changed to akinetes, which had many small vacuoles and oil droplets in the protoplasm and thick cell walls. During akinete formation by starving, the freezing tolerance (LT50) increased gradually from -3C in vegetative cells to far below -30C in akinetes. When vegetative cells were subjected to equilibrium freezing, their size shrank greatly and aparticulate domains accompanied by fracture-jump lesions developed in the plasma membranes. Akinetes subjected to equilibrium freezing showed little shrinkage, and freezing-induced ultrastructural changes did not occur in the plasma membranes. The morphological changes in the process of akinete formation and the responses to equilibrium freezing resembled those of cold-acclimated terrestrial plants.
• Accumulation of small heat-shock protein homologs in the endoplasmic reticulum of cortical parenchyma cells in mulberry in association with seasonal cold acclimation

  N Ukaji, C Kuwabara, D Takezawa, K Arakawa, S Yoshida, S Fujikawa

  PLANT PHYSIOLOGY 120 2 481 - 489 1999年06月 [査読有り][通常論文]
   

  Cortical parenchyma cells of mulberry (Morus bombycis Koidz.) trees acquire extremely high freezing tolerance in winter as a result of seasonal cold acclimation. The amount of total proteins in endoplasmic reticulum (ER)-enriched fractions isolated from these cells increased in parallel with the process of cold acclimation. Protein compositions in the ER-enriched fraction also changed seasonally, with a prominent accumulation of 20-kD (WAP20) and 27-kD (WAP27) proteins in winter. The N-terminal amino acid sequence of WAP20 exhibited homology to ER-localized small heat-shock proteins (smHSPs), whereas that of WAP27 did not exhibit homology to any known proteins. Like other smHSPs, WAP20 formed a complex of high molecular mass in native-polyacrylamide gel electrophoresis. Furthermore, not only WAP20 but also 21-kD proteins reacted with antibodies against WAP20. Fractionation of the crude microsomes by isopycnic sucrose-gradient centrifugation revealed that both WAP27 and WAP20 were distributed on a density corresponding to the fractions with higher activity of ER marker enzyme, suggesting localization of these proteins in the ER. When ER-enriched fractions were treated with trypsin in the absence of detergent, WAP20 and WAP27 were undigested, suggesting localization of these proteins inside the ER vesicle. The accumulation of a large quantity of smHSPs in the ER in winter as a result of seasonal cold acclimation indicates that these proteins may play a significant role in the acquisition of freezing tolerance in cortical parenchyma cells of mulberry trees.
• Abscisic acid-induced secretory proteins in suspension-cultured cells of winter wheat

  C Kuwabara, K Arakawa, S Yoshida

  PLANT AND CELL PHYSIOLOGY 40 2 184 - 191 1999年02月 [査読有り][通常論文]
   

  In suspension-cultured cells of winter wheat (Triticum aestivum L. cv. Chihokukomugi), the accumulation of soluble secretory proteins in the culture medium was promoted by ABA treatment in comparison with non-treated cells. The total amount of secretory proteins in ABA-treated cells was 1.7-fold higher than that in nontreated cells. The analysis of two-dimensional electrophoresis revealed that at least twelve secretory proteins were induced by ABA, and these were named WAS (wheat ABA-induced secretory) proteins 1 to 12. N-terminal amino acid sequence analysts of WAS proteins revealed the sequences of WAS-2 and WAS-3. Homology searches showed that WAS-2 had 55% identity with the N-terminus of the wheat chemically induced gene (WCI-5 gene) product. WAS-8 was also shown to have 93% identify with the N-terminus of the barley protein R, a typical member of thaumatin-like proteins (TLPs). Immunoblot analysis also suggested that WAS3 was related to protein R. These results suggest that exogenous ABA induces some basic secretory proteins that are related to the plant defense system in wheat.
• 低温馴化によるシラカンバ木部放射柔細胞の細胞壁の変化

  久保田 勝利, 荒川 圭太, 黒田 克史, 藤川 清三

  低温生物工学会誌 45 2 124 - 127 低温生物工学会 1999年 [査読有り][通常論文]
   

  In this study, we examined freezing behavior of birch xylem ray parenchyma cells. In fresh samples, summer cells exhibited supercooling to around -15℃ and winter cells exhibited it to around -50℃. In samples where the plasma membranes were destructed by freeze-thawing, summer cells still exhibited supercooling to around -10℃ and winter cells exhibited it to around -30℃, showing that cell walls are responsible, at least partially, for supercooling and that cold acclimation alters the property of supercooling ability of the cell walls. As one of possible causes of the cell wall changes, we examined effects of cell wall-bounding proteins. The result showed that extraction of proteins from cell walls altered the freezing behavior of xylem ray parenchyma cells.
• Accumulation of 19-kDa plasma membrane polypeptide during induction of freezing tolerance in wheat suspension-cultured cells by abscisic acid

  M Koike, D Takezawa, K Arakawa, S Yoshida

  PLANT AND CELL PHYSIOLOGY 38 6 707 - 716 1997年06月 [査読有り][通常論文]
   

  Suspension-cultured cells derived from immature embryos of winter wheat (Triticum aestivum L. cv. Chihoku) were used in experiments designed to obtain clues to the mechanism of the ABA-induced development of freezing tolerance. Cultured cells treated with 50 mu M ABA for 5 d at 23 degrees C acquired the maximum level of freezing tolerance (LT50; -21.6 degrees C). The increased freezing tolerance of ABA-treated cells was closely associated with the remarkable accumulation of 19-kDa polypeptides in the plasma membrane. The 19-kDa polypeptide components were isolated by preparative gel electrophoresis and were further separated into one major (AWPM-19) and other minor polypeptide components by Tricine-SDS-PAGE, N-terminal amino acid sequence of AWPM-19 was determined, and a cDNA clone encoding AWPM-19 was isolated by PCR from the library prepared from the ABA-treated cultured cells. The cDNA clone (WPM-I) encoded a 18.9 kDa hydrophobic polypeptide with four putative membrane spanning domains and with a high pi value (10.2). Expression of WPM-I mRNA was dramatically induced by 50 mu M ABA within a few hours. These results suggest that the AWPM-19 might be closely associated with the ABA-induced increase in freezing tolerance in wheat cultured cells.
• Characterization of vacuolar H+-ATPases that are sensitive and tolerant to cold

  K Hotsubo, Y Kawamura, D Takezawa, K Arakawa, S Yoshida

  PLANT COLD HARDINESS 237 - 244 1997年 [査読有り][通常論文]
   

  The susceptibility of the vacuolar H+-ATPase to cold in vivo differed markedly between leguminous species sensitive and tolerant to cold. In chilling-sensitive species such as mung bean (Vigna radiata L.), the vacuolar H+-ATPase exhibited a marked decline in activity during the early process of cold treatment. In chilling-tolerant species such as pea (Pisum sativm L.), by contrast, the H+-ATPase remained to be highly active for long periods of cold treatment. The obvious difference in the cold susceptibility of the vacuolar H+-ATPases in vivo between the two species was also reflected in the sensitivity to the MgATP-dependent cold inactivation in vitro, in particular, with respect to the sensitivity to chaotropic anions, such as Cl-, NO2-, and NO3-. The H+-ATPase from chilling-sensitive species is more susceptible to the anions, especially to the NO2-, than the enzyme from chilling-tolerant species. The vacuolar H+-ATPases were categorized into two types, namely, the cold-sensitive "mung bean-type" and the cold-stable "pea-type" enzymes, in terms of the susceptibility to the MgATP-dependent cold inactivation in the presence of NO2- ions. Immunoblot analyses using anti-mung bean H+-ATPase antibodies have revealed that the antigenicity of the 16 kD proteolipids is distinctly different between the two types of enzyme.
• PROPERTIES OF 2 N-LINKED GLYCOPROTEINS ASSOCIATED WITH THE NUCLEAR-ENVELOPE IN MUNG BEAN HYPOCOTYLS

  M ODAIRA, K ARAKAWA, S YOSHIDA, M MAESHIMA

  PLANT AND CELL PHYSIOLOGY 36 6 945 - 953 1995年09月 [査読有り][通常論文]
   

  Nuclei from mung bean (Vigna radiata) hypocotyls contained two glycoproteins of 50 and 49 kDa, respectively, that reacted with concanavalin A. The glycoproteins were released from the nuclear envelope by treatment with 2 M KCI but not with nucleases. The glycoproteins, tentatively named gp50 and gp49, were isolated and characterized. Gel-permeation chromatography suggested that gp50 and gp49 seem to exist as a complex with other components. The glycoproteins could be detected only in the nuclear fraction by immunoblot analysis with specific antibodies, and they were not detected in endoplasmic reticulum, plasma membrane, vacuolar membrane or mitochondria. Agglutinin I from Ulex europeaus, peanut agglutinin, soybean agglutinin and wheat germ agglutinin all failed to bind to the glycoproteins. Treatment with glycopeptidase F removed all oligosaccharides from the glycoproteins and decreased their molecular masses by about one thousand daltons each. These results suggest that the glycoproteins contained N-linked, high-mannose-type oligosaccharides with six or seven hexose residues. gp50 and gp49 seemed to be isoforms of a single glycoprotein because the two proteins had some common properties. Nuclear fractions from azuki bean (Phaseolus angularis) and soybean (Glycine max) contained proteins that were immunologically similar to gp50 and gp49.
• COLD-INDUCED ALTERATIONS IN PLASMA-MEMBRANE PROTEINS THAT ARE SPECIFICALLY RELATED TO THE DEVELOPMENT OF FREEZING TOLERANCE IN COLD-HARDY WINTER-WHEAT

  BL ZHOU, K ARAKAWA, S FUJIKAWA, S YOSHIDA

  PLANT AND CELL PHYSIOLOGY 35 2 175 - 182 1994年03月 [査読有り][通常論文]
   

  The objective of this study was to identify plasma membrane proteins that are specifically induced by cold acclimation in wheat (Triticum aestivum L.). Two cultivars with a marked difference in the genetic ability to cold-acclimate, namely, spring wheat (cv. Chinese Spring) and winter wheat (cv. Norstar), were used as the experimental material. After four weeks of growth in a cold chamber, the freezing tolerance in the shoots of winter wheat increased to -18-degrees-C, whereas it increased only to - 8-degrees-C in the shoots of spring wheat. In the case of roots from both cultivars, freezing tolerance increased only slightly after the growth in the cold environment. Cold acclimation induced remarkable changes in the electrophoretic patterns of plasma membrane proteins which depended on both the cultivar and the tissue examined. Levels of polypeptides with molecular masses from 22 to 31 kDa decreased in both the root and shoot plasma membranes from both cultivars. Among these polypeptides, levels of those of 28 and 26 kDa decreased abruptly after one week of cold acclimation. By contrast, levels of polypeptides of 89, 83, 52, 23, 18 and 17 kDa increased specifically in the shoots of winter wheat. The increases in the levels of the 23-, 18- and 17-kDa polypeptides were proportional to the development of freezing tolerance. Freeze-fracture electron microscopy of plasma membranes from shoot cells revealed that the number of intramembrane particles on the fracture faces decreased markedly in winter wheat after cold acclimation, but to a lesser extent in spring wheat. These results suggest that the plasma membranes might undergo molecular reorganization during cold acclimation.
• Accumulation of glycinebetaine during cold acclimation and freezing tolerance in leaves of winter and spring barley plants

  S. KISHITANI, K. WATANABE, S. YASUDA, K. ARAKAWA, T. TAKABE

  Plant, Cell and Environment 17 1 89 - 95 1994年01月 [査読有り][通常論文]
  
• Betaine Aldehyde Dehydrogenase in the Gramineae : Levels in Leaves of Both Betaine-Accumulating and Nonaccumulating Cereal Plants :

  ISHITANI Manabu, ARAKAWA Keita, MIZUNO Katsuhiko, KISHITANI Sachie, TAKABE Tetsuko

  Plant and Cell Physiology 34 3 493 - 495 Japanese Society of Plant Physiologists 1993年04月 [査読有り][通常論文]
   

  The accumulation of betaine and the distribution of betaine aldehyde dehydrogenase, which catalyzes the last step in the synthesis of betaine, were analyzed in leaves of control and salt-stressed cereal plants of the Gramineae. BADH protein was present in both betaine-accumulating and nonaccumulating leaves.
• IMMUNOLOGICAL STUDIES OF BETAINE ALDEHYDE DEHYDROGENASE IN BARLEY

  ARAKAWA, K, MIZUNO, K, KISHITANI, S, TAKABE, T

  PLANT AND CELL PHYSIOLOGY 33 7 833 - 840 1992年10月 [査読有り][通常論文]
  
• Levels of betaine and betaine aldehyde dehydrogenase activity in the green leaves, and atiolated leaves and roots of barley.

  Arakawa K, Katayama M, Takabe T

  Plant and Cell Physiology 31 6 797 - 803 1990年09月 [査読有り][通常論文]
  
• 植物耐塩性の分子機構

  石谷学, 荒川圭太, 高倍鉄子

  植物の化学調節 25 149 - 162 植物化学調節学会 1990年 [査読無し][通常論文]
  
• Betaine accumulation and osmoregulation in higher plants and cyanobacteria

  Takabe T, Arakawa K

  Plant water relations and growth under stress 151 - 158 1989年 [査読無し][通常論文]
  
• CO2 Fixation Rate and RuBisCO Content Increase in the Halotolerant Cyanobacterium, Aphanothece halophytica, Grown in High Salinities

  Tetsuko Takabe, Aran Incharoensakdi, Keita Arakawa, Sadaki Yokota

  Plant Physiology 88 4 1120 - 1124 1988年12月01日 [査読有り][通常論文]
  
• Purification of betaine-aldehyde dehydrogenase from spinach leaves and preparation of its antibody

  Keita Arakawa, Tetsuko Takabe, Tatsuo Sugiyama, Takashi Akazawa

  Journal of Biochemistry 101 6 1485 - 1488 1987年 [査読有り][通常論文]
   

  Betaine-aldehyde dehydrogenase was purified from spinach leaves and characterized. The Molecular weight of the enzyme was estimated to be 120 kDa by a gel filtration chromatorgraphy. The enzyme was judged to consist of two identical pieces of the monomeric subunit with molecular weight of 60 kDa. A specific polyclonal antibody was raised against the enzyme subunit. © 1987 Oxford University Press.

MISC

• ブナ種子の乾燥耐性機構に関する研究

  齋藤拓哉, 米岡宏喜, 花岡創, 遠藤圭太, 木下剛志, 鈴木伸吾, 荒川圭太 低温生物工学会セミナー及び年会講演要旨集 68th (CD-ROM) 2023年
• カツラ樹皮に検出された氷核活性の性質

  古賀泰雅, 鉄穴口晃, 鈴木伸吾, 鈴木伸吾, 重冨顕吾, 荒川圭太 日本木材学会北海道支部講演集(Web) (53) 2021年
• 高オゾン環境下でのヤマナラシ属2種の成長と病虫害の観察-中間報告-

  増井昇, 菅井徹人, 渡邊陽子, 渡部敏裕, 塩尻かおり, 佐々木圭子, 藤戸永司, 荒川圭太, 佐藤冬樹, 小池孝良 北方森林保全技術(Web) (38) 2021年
• ブナ種子の乾燥耐性

  米岡宏喜, 花岡創, 遠藤圭太, 荒川圭太 日本木材学会北海道支部講演集(Web) (52) 2020年
• 交雑ポプラ培養細胞へのGFP-TUA6およびLifeact-mCherryの導入による細胞骨格の可視化

  山岸祐介, 高田直樹, 渡辺宇外, 荒川圭太, 佐野雄三, 半智史, 船田良 日本木材学会大会研究発表要旨集(完全版)(CD-ROM) 70th 2020年
• 都市生態系へのオゾン汚染の影響：実験的研究によるＪＳＴへのとり組み

  小池 孝良, 菅井 徹人, 渡部 敏裕, 市川 一, 藤戸 永志, 佐々木 圭子, 曲 来葉, 渡辺 誠, 荒川 圭太, 山﨑 友紀, 佐藤 冬樹 北方森林保全技術 (37) 11 -17 2019年12月 [査読無し][通常論文]
  
• 日本木材学会北海道支部 第49回研究会 開催報告

  山岸 祐介, 宮﨑 淳子, 荒川 圭太, 河原﨑 政行 木材工業 = Wood industry 73 (10) 406 -408 2018年10月 [査読無し][通常論文]
  
• 抗氷核(過冷却促進)ポリフェノールによる水溶液の凍結抑制効果について

  荒川 圭太 グリーンテクノ情報 14 (1) 13 -17 2018年06月 [査読無し][通常論文]
  
• 北海道で栽培される醸造用ブドウ樹体組織の耐凍性について

  堀内玲子, 実山豊, 荒川圭太, 鈴木卓 園芸学研究 別冊 17 (2) 2018年
• 交雑ポプラのER局在性GFP導入株におけるERの構造解析と超低温保存法の確立

  川村浩平, 桜井健至, 嘉見大助, 田中大介, 遠藤圭太, 鈴木伸吾, 山岸祐介, 荒川圭太 日本木材学会大会研究発表要旨集(完全版)(CD-ROM) 67th 2017年
• 交雑ポプラ培養細胞を用いたin vitro管状要素誘導に合成オーキシンNAAおよび2,4-Dが及ぼす影響

  山岸祐介, 鎌田裕, 工藤佳世, 半智史, 船田良, 荒川圭太, 佐野雄三 日本木材学会大会研究発表要旨集(完全版)(CD-ROM) 67th 2017年
• 樹木冬芽における有鱗芽と裸芽の凍結適応機構

  岡田 香織, 遠藤 圭太, 荒川 圭太 日本木材学会北海道支部講演集 45 48 -51 2013年11月 [査読無し][通常論文]
  
• カラマツ木部における冬季誘導性のデハイドリンの機能評価

  坂本友陽, 安達正博, 岡田香織, 鈴木伸吾, 宇梶慎子, 荒川圭太, 高田直樹 日本木材学会大会研究発表要旨集(完全版)(CD-ROM) 63rd 2013年
• 樹木冬芽の越冬過程における可溶性タンパク質の組成変化

  鈴木伸吾, 遠藤圭太, 岡田香織, 荒川圭太, 高橋大輔, 上村松生 日本木材学会北海道支部講演集(Web) (44) WEB ONLY P-2 2012年11月 [査読無し][通常論文]
  
• 冬季にカラマツ木部に蓄積するデハイドリンタンパク質の機能評価

  坂本友陽, 岡田香織, 鈴木伸吾, 宇梶慎子, 荒川圭太 日本木材学会北海道支部講演集(Web) (44) WEB ONLY P-6 2012年11月 [査読無し][通常論文]
  
• Supercooling-promoting (anti-ice nucleating) flavonoids and tannins (vol 63, pg 307, 2010)

  Chikako Kuwabara, Jun Kasuga, Donghui Wang, Yukiharu Fukushi, Keita Arakawa, Seizo Fujikawa CRYOBIOLOGY 64 (3) 308 -308 2012年06月 [査読無し][通常論文]
  
• J024031 氷核活性を抑制するポリフェノール化合物の効果

  稲田 孝明, 小山 寿恵, 桑原 慎子, 荒川 圭太, 藤川 清三 年次大会 2012 (0) _J024031 -1-_J024031-3 2012年 [査読無し][通常論文]
   

  Antifreeze proteins (AFPs) and several synthetic polymers are known as anti-ice nuclei (anti-IN), which inactivate ice nuclei (IN) in water and thus inhibit heterogeneous ice nucleation catalyzed by IN. Recently, it has been found that several types of polyphenol compounds also inhibit heterogeneous ice nucleation. In this study, ice nucleation catalyzed by silver iodide (Agl) particles, which are typical inorganic IN, in emulsified solutions of five different polyphenol compounds (a-oligoglucosyl quercetin 3-O-β-D-glucopyranoside (Q3(Glc)n), epigallocatechin gallate (EGCG), tea catechin, tannic acid, and oligonol) was investigated experimentally. Results showed that the ice nucleating activity of Agl was inactivated by all the polyphenols used here. For Q3(Glc)n and EGCG solutions, in the majority of the emulsified droplets, ice nucleation occurred within a narrow temperature range between -17 and -20℃. On the contrary, for tea catechin, tannic acid, and oligonol solutions, ice nucleation occurred within relatively wide temperature range between -17 and -38 ℃. The highest ice nucleation temperatures in the emulsified droplets of the polyphenol solutions were considerably lower than those of AFP and synthetic polymer solutions. Especially, the highest ice nucleation temperature for oligonol solution was -19.4 ℃. Such anti-ice nucleating activities of the polyphenols available at relatively high temperatures would be promising for practical applications in various fields.
• カツラ木部由来の過冷却促進物質とその構造類似物質における過冷却活性の比較

  森若元太, 宇梶槙子, 藤川清三, 荒川圭太 日本木材学会北海道支部講演集(Web) (43) WEB ONLY O-3 2011年11月 [査読無し][通常論文]
  
• ハイブリッドアスペンの凍結抵抗性に関する研究

  板羽貴史, 砂留光利, 上出奈央, 佐野雄三, 藤川清三, 荒川圭太 日本木材学会北海道支部講演集(Web) (42) WEB ONLY O-2 2010年11月 [査読無し][通常論文]
  
• カラマツ(Larix kaempferi)木部柔細胞の過冷却能に関与する冬季誘導性タンパク質(LkDRP1及びLkDRP2)に関する研究

  能美彩香, 森本和成, 藤川清三, 荒川圭太 日本木材学会北海道支部講演集(Web) (42) WEB ONLY O-1 2010年11月 [査読無し][通常論文]
  
• 異なる凍結挙動を示すカツラおよびシラカンバ冬芽の組織細胞

  遠藤圭太, 荒川圭太, 藤川清三 日本木材学会北海道支部講演集(Web) (42) WEB ONLY O-3 2010年11月 [査読無し][通常論文]
  
• 低温走査型電子顕微鏡(Cryo‐SEM)による冬芽の凍結挙動観察

  遠藤圭太, 荒川圭太, 藤川清三 日本顕微鏡学会北海道支部学術講演会講演要旨集 2010 2 2010年 [査読無し][通常論文]
  
• トドマツ針葉の耐凍性および凍結挙動

  遠藤圭太, 荒川圭太, 藤川清三 日本木材学会北海道支部講演集 (41) 7 -10 2009年10月20日 [査読無し][通常論文]
  
• カラマツ木部柔細胞の深過冷却能に関与する可溶性蛋白質の解析

  森本 和成, 森 仁志, 春日 純, 藤川 清三, 荒川 圭太 日本植物生理学会年会およびシンポジウム 講演要旨集 2009 (0) 512 -512 2009年 [査読無し][通常論文]
   

  寒冷地に生育する樹木の木部柔細胞は深過冷却によって氷点下温度に適応している。このような木部柔細胞では、季節的な低温馴化によって過冷却能力が向上することで冬季の厳しい氷点下温度に対する抵抗性（凍結抵抗性）が著しく高まる。これまでの研究により、木部柔細胞の深過冷却機構では細胞壁構造や細胞内に蓄積する糖類、氷核形成阻害物質などが関与することが示唆されているが、主たる細胞成分の一つである可溶性蛋白質が過冷却能に対してどのような役割を果たしているのかに関する知見は少ない。そこで本研究では、北方樹木の木部柔細胞の過冷却能の変動によく対応して変動する蛋白質を特定することを目的とした。野外に生育するカラマツ（Larix kaempferi）の2ないし5年生の枝を用い、季節的な低温馴化による過冷却能の上昇ならびに人為的な脱馴化処理による過冷却能の減少の双方に対応して、蓄積量が増減した可溶性蛋白質を二次元電気泳動法で検出した。そして、変動した16個の候補蛋白質をトリプシン消化後、LC-MALDI MS/MS解析に供試してde novo sequencingした。予測されたアミノ酸配列をもとにデータベースを検索した結果、機能未知な樹木由来の蛋白質と類似したものがいくつか見いだされた。現在、引き続きこれらの蛋白質の同定を試みている。
• カラマツ冬芽(長枝芽)の凍結挙動

  遠藤圭太, 荒川圭太, 藤川清三 日本木材学会北海道支部講演集 (40) 11 -14 2008年10月22日 [査読無し][通常論文]
  
• カラマツ木部柔細胞の深過冷却能に関与する可溶性蛋白質の検出

  森本 和成, 荒川 圭太, 藤川 清三 日本植物生理学会年会およびシンポジウム 講演要旨集 2008 (0) 874 -874 2008年 [査読無し][通常論文]
   

  寒冷地に生育する樹木は、季節的な低温馴化によって凍結抵抗性が著しく向上し、冬季の厳しい氷点下温度でも生存可能となる。このような樹木の凍結挙動は組織によって異なり、師部や形成層の柔細胞は細胞外凍結で、木部の柔細胞は深過冷却によって氷点下温度に適応している。なかでも深過冷却機構に関する研究例は限られており、元来、木部組織における厚い細胞壁の構造特性が深過冷却機構の主要因と考えられてきた。そのため、細胞内成分の関与については糖類の蓄積以外はほとんど検証されていなかった。最近、当研究室では、深過冷却機構に関与する因子として、氷核形成阻害活性を有する二次代謝産物や過冷却能の増加に伴って誘導される遺伝子群を特定するに至った。そこで本研究では、主たる細胞成分の一つである可溶性蛋白質が木部柔細胞の深過冷却能に関与する可能性について検証するため、カラマツ（Larix kaempferi）の2ないし5年生の枝を用い、季節的な低温馴化による過冷却能の上昇ならびに人為的な脱馴化処理による過冷却能の減少の双方に対応して蓄積量が増減した可溶性蛋白質を二次元電気泳動法にて検出した。次に、これらの蛋白質の単離を試みた後、N末端アミノ酸配列が解析できた蛋白質について相同性検索を行った結果、Late embryogenesis abundant proteins を含む数種の蛋白質が同定できた。現在、引き続きこれらの蛋白質の同定を試みている。
• カラマツ木部柔細胞の過冷却能力の変化に伴って変動する蛋白質の検索

  森本和成, 藤川清三, 荒川圭太 日本木材学会北海道支部講演集 (39) 11 -14 2007年10月22日 [査読無し][通常論文]
  
• 木部柔細胞の過冷却能に与える可溶性糖の効果の検証

  葭葉恵, 春日純, 荒川圭太, 藤川清三 日本木材学会北海道支部講演集 (39) 19 -22 2007年10月22日 [査読無し][通常論文]
  
• カツラ木部柔細胞の深過冷却機構に関与する過冷却促進成分の探索

  春日純, 葭葉恵, 橋床泰之, 荒川圭太, 藤川清三 日本木材学会北海道支部講演集 (39) 15 -18 2007年10月22日 [査読無し][通常論文]
  
• シラカンバ木部柔細胞の細胞内可溶性糖が深過冷却機構に果たす役割

  春日純, 荒川圭太, 藤川清三 日本顕微鏡学会北海道支部学術講演会プログラム・予稿集 2006 15 2007年02月 [査読無し][通常論文]
  
• ハイブリッドアスペン実生の低温馴化機構に関する研究

  砂留光利, 上出奈央, 高田直樹, 藤川清三, 荒川圭太 日本木材学会大会研究発表要旨集(完全版)(CD-ROM) 57th 2007年
• 酸性雪ストレス後の再生長初期過程における冬小麦緑葉の生理的変化

  稲田 秀俊, 藤川 清三, 荒川 圭太 日本植物生理学会年会およびシンポジウム 講演要旨集 2007 (0) 479 -479 2007年 [査読無し][通常論文]
   

  雪の酸性化は酸性雨と同様にこの10年間で着実に進行し、環境への影響が懸念されるが、その影響は定かではない。そこで我々は、酸性雪の積雪層下で植物が局所的に雪氷表面に濃縮された酸性物質に長期的に曝されたり、酸性度の強い融雪水に曝されたりすることを想定し、越冬性作物の冬小麦幼苗をpH 2からpH 4に調整した硫酸溶液中で凍結融解することで酸性雪ストレスを模し、酸性雪が越冬性植物に与える影響を調べることにした。これまでに、低温馴化した冬小麦緑葉を硫酸存在下で凍結融解（SAS処理）すると傷害が助長されることがわかった。そこで本研究では、低温馴化した冬小麦個体を-4℃のSAS処理（pH 2）に4時間施した後、融解してから10℃の12時間日長の条件下に移して再生長させ、個体への影響を調べた。冬小麦緑葉の葉齢の違いによりSAS処理に対する応答性に差異がみられたため、葉齢別に応答性を評価した。pH 2のSAS処理後に再生長させた冬小麦の第1葉（成熟葉）では、SAS処理直後に生じた可視傷害部位は広がらないが、著しく脱水されて生重量が低下することが分かった。一方、第2葉ではいずれも大きな影響は見られなかった。おそらく葉齢によって凍結抵抗性が異なるため、第1葉と第2葉でSAS処理後の再生長への影響にも違いが生じたものと考えられる。SAS処理直後に冬小麦に大きな傷害が観察されなくても、再生長過程において緑葉に影響が出ることが強く示唆された。
• カラマツ木部組織由来の冬季誘導性ガラクチノール合成酵素遺伝子に関する研究

  砂留光利, 高田直樹, 春日純, 藤川清三, 荒川圭太 日本木材学会大会研究発表要旨集(完全版)(CD-ROM) 56th 2006年
• カツラ木部組織に存在する過冷却促進成分の単離と同定

  春日純, 松本一, 荒川圭太, 橋床泰之, 藤川清三 日本木材学会大会研究発表要旨集(完全版)(CD-ROM) 56th B10-1130 2006年 [査読無し][通常論文]
  
• カラマツ木部粗抽出画分からの氷核形成阻害活性の検出

  水野薫, 春日純, 荒川圭太, 藤川清三 日本木材学会北海道支部講演集 (37) 13 -16 2005年10月21日 [査読無し][通常論文]
  
• 冬小麦PR-5タンパク質(WAS-3)の構造解析

  桑原 慎子, 近藤 英昌, 野呂 奈津子, 竹澤 大輔, 荒川 圭太, 津田 栄 日本植物生理学会年会およびシンポジウム 講演要旨集 2005 (0) 848 -848 2005年 [査読無し][通常論文]
   

  WAS-3は、病原菌関連タンパク質PR-5に属し、冬小麦の低温馴化によってアポプラストに蓄積する。私たちは、WAS-3の機能を明らかにするために、小麦培養細胞を用いた組み換えタンパク質発現系を作成し、組み換えWAS-3タンパク質（rWAS-3）がin vitroにおいて雪腐れ病菌やフザリウム菌の菌糸の成長を阻害する活性を持つことを明らかにしてきた。また、rWAS-3は雪腐れ病菌やフザリウム菌の菌糸の細胞壁画分に対し親和性を持ち、菌糸の成分であるβ-1,3-グルカンに結合することを示唆してきた。しかし、抗菌活性の詳細なメカニズムは、まだ明らかになっていない。そこで、本研究では、抗菌活性のメカニズムに関する知見を得るために、rWAS-3の結晶を作成し、結晶構造の解析を行った。ハンギングドロップ蒸気拡散法によって結晶化条件のスクリーニングを行った後、至適化した結果、0.8×0.5×0.01 mmの薄い板状結晶が得られた。構造解析は分子置換法を用いて行い、1.8オングストローム分解能の立体構造を決定した。その結果、rWAS-3は3個のドメインを構成しており、ドメインAとドメインBの間に「くぼみ」があることが明らかとなった。分子表面の静電ポテンシャルに基づきモデルを作成すると、ドメインAとドメインBの間のくぼみは、負に荷電した領域であることが示されたことから、この領域にβ-1,3-グルカンが結合する可能性が考えられた。
• ヒメツリガネゴケのアブシジン酸低感受性変異株における浸透圧及び凍結耐性の低下

  南 杏鶴, 長尾 学, 荒川 圭太, 藤川 清三, 竹澤 大輔 日本植物生理学会年会およびシンポジウム 講演要旨集 2005 (0) 222 -222 2005年 [査読無し][通常論文]
   

  アブシジン酸（ABA）によるシグナル伝達は、植物の低温や浸透圧ストレスに対する応答や耐性の獲得に関与している。ヒメツリガネゴケ（Physcomitrella patens）原糸体細胞ではABA処理によってストレス応答性遺伝子の発現誘導や可溶性糖の蓄積などがおこり、凍結耐性（耐凍性）が向上する。また、高張のマンニトール処理では内生ABA量が増加し、生理学的変化を伴った耐凍性の上昇がみられる。
  ABAによる様々な生理的変化のなかで耐凍性を促進させる要因を探るため、ヒメツリガネゴケABA低感受性変異株AR1～7を紫外線照射処理によって単離した。すべてのAR変異株は、野生株が生育阻害をおこすABA濃度下でも生育することができ、ABA処理後の耐凍性は野生株よりも低い値を示した。特にABAによる耐凍性の上昇程度が低いAR4株では、ABA誘導性遺伝子の発現量やLEA様タンパク質、可溶性糖の蓄積において著しい減少がみられた。この変異株では、マンニトールによる浸透圧ストレスに対して強い感受性を示した。以上の結果は、ABA依存的な浸透圧シグナル伝達経路によって、凍結や高浸透圧溶液による脱水ストレス耐性が調節されていることを示唆している。
• クワ皮層柔細胞に冬季特異的に蓄積する蛋白質WAP27の長期凍結下での凍害保護活性

  長尾 学, 宇梶 徳史, 竹澤 大輔, 荒川 圭太, 藤川 清三 日本植物生理学会年会およびシンポジウム 講演要旨集 2005 (0) 221 -221 2005年 [査読無し][通常論文]
   

  ヤマグワ（Morus bombycis）の皮層柔細胞の耐凍性は夏季に。比べて冬季に大幅に上昇することが知られている。これまでにヤマグワの皮層柔細胞内のERにGroup 3 LEA蛋白質である分子量27-kDaのWAP27蛋白質 が冬季特異的に蓄積することがわかっている。
  植物は野外では一定範囲の凍結温度下に長く置かれる。特に氷点に近い凍結温度下では低温や脱水のみでなく、未凍結溶液中の塩類濃縮に長期間さらされる。そこでWAP27の耐凍性における機能を知るために一定の凍結温度で長期間保持した凍結感受性蛋白質、乳酸脱水素酵素（LDH）に対する組み替えWAP27（rWAP27）凍害保護活性を調べた。rWAP27を添加し-2、-4、-10、-20℃で長期間保持したLDHはすべての凍結温度で80％以上の高い活性を維持した。それに対し牛血清アルブミン（BSA）は、-20℃ではrWAP27と同等の保護活性を維持したが、-2、-4℃では凍結時間に依存して活性が低下する傾向が見られた。卵白アルブミン（Ovalbumin）ではすべての凍結温度で活性が経時的に低下した。今回の結果からrWAP27は長期凍結下で凍結感受性蛋白質に対し、他の凍結保護物質より高い保護活性を持つことが明らかになった。
• 酸性条件下における越冬性植物の凍結傷害機構

  稲田 秀俊, 伊藤 利章, 長尾 学, 藤川 清三, 荒川 圭太 日本植物生理学会年会およびシンポジウム 講演要旨集 2005 (0) 381 -381 2005年 [査読無し][通常論文]
   

  我々は、酸性雪が越冬性植物に及ぼす影響を調べるため、冬小麦(Triticum aestivum L. cv. Chihokukomugi)緑葉の組織切片を用いて酸性条件下で耐凍性試験を行い、酸性雪ストレスによる越冬性植物の傷害発生機構の解析を進めてきた。これまでに冬小麦の緑葉を硫酸溶液(pH 2.0)の存在下で細胞外凍結させると融解後の生存率が著しく低下することを明らかにした。本研究では、気温の寒暖差の大きい初冬や初春に酸性融雪水中で植物が凍結融解される影響を見積もるため、冬小麦緑葉の切片を純水または硫酸溶液の存在下で平衡凍結融解を繰り返し行った。すると、硫酸溶液共存下では凍結融解を繰り返す度毎に緑葉の生存率は徐々に低下し、純水で処理したものに比べてより生存率が低下した。次に、冬小麦緑葉組織の凍結融解過程のどの段階で酸性化すると生存率が低下するかを調べるために、硫酸溶液を添加する時期を植氷前、融解時、融解後にそれぞれ設定して耐凍性試験を行い、緑葉組織の生存率への影響について評価した。その結果、細胞外凍結した冬小麦の緑葉組織では、融解時に酸性物質が存在することが傷害発生を助長させる要因となりうることが予想された。
• シロイヌナズナ由来の転写制御因子CBF1を導入したハイブリッドアスペンの耐凍性及び組織構造に関する研究

  古俣寛隆, 伊藤利章, 佐野雄三, 荒川圭太, 藤川清三 日本木材学会北海道支部講演集 (36) 27 -30 2004年10月28日 [査読無し][通常論文]
  
• FE‐SEMによる急速凍結固定した樹木組織柔細胞の微細構造の観察

  山根健一, 伊藤利章, 佐野雄三, 荒川圭太, 藤川清三 日本木材学会北海道支部講演集 (36) 23 -26 2004年10月28日 [査読無し][通常論文]
  
• 深過冷却するシラカンバ木部柔細胞に蓄積する冬季誘導性蛋白質の機能解析

  高島寛, 荒川圭太, 藤川清三 日本木材学会北海道支部講演集 (36) 31 -34 2004年10月28日 [査読無し][通常論文]
  
• 越冬性植物の耐凍性機構

  荒川圭太 北海道芝草研究会報 2004年 [査読無し][通常論文]
  
• 越冬性植物の酸性凍結処理に対する応答

  稲田 秀俊, 荒川 圭太 日本植物生理学会年会およびシンポジウム 講演要旨集 2004 (0) 359 -359 2004年 [査読無し][通常論文]
   

  大気中の酸性物質を含む酸性雨が植物の生育や代謝に影響を与えることはすでによく知られている。しかし、酸性雪が積雪地域の越冬性植物に与える影響についてはほとんど調べられていない。そこで本研究では、酸性雪が越冬性植物の生存にどのような影響を及ぼすのかを簡易的に評価するために、組織切片を用いて酸性条件下での耐凍性試験を試みた。試料には低温馴化させた冬小麦（Triticum aestivum L. cv. Chihokukomugi）緑葉の切片を用い、硫酸でpHを調整した酸性溶液を添加し平衡凍結法による耐凍性試験を行った。凍結融解後の試料の生存率を測定すると、pH 4.0や3.0の酸性凍結処理は対照区の純水(pH 5.6)での凍結処理とほぼ同程度であった。一方、pH 2.0の凍結処理では対照区よりも生存率が著しく低下していた。この酸性凍結の場合、植氷した試料を直ちに4℃に移してゆっくり融解させても生存率はほとんど低下しなかった。同様の試料を緩速冷却によって平衡凍結すると、凍結処理温度の低下に伴って生存率が低下し純水凍結との差が徐々に大きくなった。-12℃での純水凍結処理の生存率は約40%であったのに対して酸性凍結処理では約10%であった。また、pH 2.0の酸性溶液を添加した試料を-12℃まで過冷却させると、生存率はとほんど低下せず約90%であった。これらの結果から、冬小麦の緑葉を硫酸共存下で凍結融解すると著しく凍結傷害を被ることが明らかになった。
• コケ植物の耐凍性獲得過程で発現する遺伝子の同定

  南 杏鶴, 長尾 学, 荒川 圭太, 藤川 清三, 竹澤 大輔 日本植物生理学会年会およびシンポジウム 講演要旨集 2004 (0) 803 -803 2004年 [査読無し][通常論文]
   

  コケ植物は熱帯から極地まで幅広く生育し、その中には極端な乾燥や凍結に対して耐性を持つ種が少なからず存在する。蘚類ヒメツリガネゴケ（Physcomitrella patens）の原糸体は、通常の培養条件では耐凍性を持たないが、アブシジン酸（ABA）処理によって高い耐凍性を獲得し、-80℃での凍結保存も可能になる。このことから、ABAが細胞の凍結脱水ストレス耐性に重要な役割を持つと考えられた。私たちは、ABA処理したヒメツリガネゴケ原糸体細胞において葉緑体や液胞の小型化、細胞内浸透濃度の増加および熱安定性タンパク質や可溶性糖の蓄積がおこり、凍結傷害の主要因である細胞膜の損傷が緩和されることを明らかにした。ABAで誘導される耐凍性上昇はシクロヘキシミド処理で著しく抑制されることから、この過程には核コードタンパク質の合成が必須であることが示された。
  ヒメツリガネゴケの耐凍性上昇に関わる遺伝子を同定するため、フィルターアレイを用いたディファレンシャルスクリーニングによりABA処理で増加する60以上の遺伝子を単離した。その結果、これらの中にはLEAや水チャネルなどのように高等植物のストレス応答性遺伝子と相同性のあるものが多数含まれる一方、高等植物には保存されていない葉緑体タンパク質LI818や、UVI-1など原核生物遺伝子のホモログが新たに同定された。
• 深過冷却で寒冷環境に適応するカラマツ木部柔細胞において冬季特異的に増加する遺伝子とその組織的発現様式

  高田直樹, 藤川清三, 荒川圭太, 南杏鶴, 竹沢大輔 日本木材学会北海道支部講演集 (35) 1 -4 2003年10月15日 [査読無し][通常論文]
  
• オゾン感受性を示すパラコート耐性突然変異体 rcd1 の反応機構の解析

  藤部 貴宏, 佐治 光, 荒川 圭太, 竹内 裕一, 山本 興太朗 日本植物生理学会年会およびシンポジウム 講演要旨集 2003 (0) 347 -347 2003年 [査読無し][通常論文]
   

  植物は様々なストレスを受けることにより活性酸素を発生させることが知られている。これらのストレスに対抗するため、植物はストレスを受けると活性酸素除去酵素群の発現を誘導する。rcd1-1（Overmyer ら、2000） と同じ遺伝子座上に変異が起こっているパラコート耐性突然変異体 rcd1-2 は活性酸素が関与すると考えられる紫外線の照射や塩ストレスに対して耐性を示したが（日本植物学会第66回大会、2002）、同じく活性酸素発生を伴うオゾンの曝露に対しては感受性を示した。その損傷は活性酸素による一次的な反応ではなく、プログラム細胞死的な反応であった。rcd1 突然変異は劣性なので、rcd1-2 変異体はストレス反応系の負の調節因子の機能が欠損していて常にストレス反応を起こしてしまい、構成的にストレス防御系が働いて活性酸素ストレスに対して耐性を示すと考えられる。また、オゾンの曝露はその作用機構が病原菌の感染に似ているため、rcd1 のオゾン感受性は感染部位の拡大を防ぐためのプログラム細胞死が誤作動することによって起こっていると考えられる。本研究では rcd1-2 変異体の紫外線やオゾン曝露等に対するストレス応答、および原因遺伝子のクローニングとそのシグナル経路について報告する。
• PR-10/Bet v 1ファミリーは細胞質と核に局在する

  宇梶 徳史, 竹澤 大輔, 荒川 圭太, 藤川 清三 日本植物生理学会年会およびシンポジウム 講演要旨集 2003 (0) 308 -308 2003年 [査読無し][通常論文]
   

  冬季に-80℃以下の高い凍結耐性を獲得するクワ(Morus bombycis Koidz.)皮層柔細胞では、秋から冬にかけての季節的低温馴化に伴い、可溶性画分に18kDの分子サイズを有するタンパク質（WAP18）の蓄積が見出される。精製したWAP18は、in vitroに於いて凍結・融解による乳酸脱水素酵素活性低下を防止したことから、WAP18はクワ皮層柔細胞が獲得する冬季の凍結耐性獲得に、何らかの形で貢献するものと推測される。cDNAクローニングにより、WAP18はPR-10/Bet v 1ファミリーと高い相同性を有することが示された。ノーザン解析を行ったところ、WAP18遺伝子は、4℃の低温処理のみならず、wounding、エテフォン、およびサリチル酸による誘導が見られた。
  一方、PR-10/Bet v 1ファミリーはその推定されるアミノ酸配列から、細胞質に局在すると推測されているが、正確な細胞内局在は示されていない。そこで、WAP18のクワ皮層柔細胞における細胞内局在を、免疫電子顕微鏡法を用いて観察したところ、細胞質と核に金コロイド標識が見いだされた。このことから、PR-10/Bet v 1ファミリーは細胞質と核に局在することが示唆された。
• シラカンバ木部組織における冬季誘導性の細胞外蛋白質の解析

  荒川 圭太, 春日 純, 藤川 清三 日本植物生理学会年会およびシンポジウム 講演要旨集 2003 (0) 307 -307 2003年 [査読無し][通常論文]
   

  寒冷地域に分布する落葉広葉樹のシラカンバ(Betula platyphylla L.)は、秋から冬にかけて季節的な低温馴化過程を経て耐寒性が著しく上昇する。シラカンバの木部放射柔細胞は深過冷却によって凍結抵抗性を高めるが、この抵抗性の誘導には細胞内部だけでなく細胞外部の要因も関連する可能性が考えられる。そこで本研究では、木部組織の凍結抵抗性と関連性のある細胞外因子を見い出すため、低温馴化過程で生じる木部組織での細胞外蛋白質の組成変化を調べ、冬季誘導性蛋白質の同定を試みた。
  野外で採集したシラカンバの枝から皮相組織を取り除いて得られた木部組織を材料に用い、短時間の酸性溶液処理により細胞外蛋白質を抽出した。夏と冬に採集した組織からそれぞれ調製した細胞外蛋白質画分の組成をSDS-PAGEにより比較したところ、季節的な低温馴化によって30 kDa付近の複数の蛋白質バンドが顕著に増加していることが明らかになった。これらの冬季誘導性の細胞外蛋白質(WCWPs)を単離して、N末端アミノ酸配列の分析をおこなったところ、互いにアミノ酸配列が類似しており、生体防御に関連するPR蛋白質と相同性を示すことが判明した。また、WCWPsに対する抗体を用いてイムノブロット解析をおこなった結果、これらは互いに冬季誘導性のアイソフォーム蛋白質であることが示唆された。
• ヒメツリガネゴケの耐凍性に関わるABA・低温誘導性プロテインキナーゼの機能解析

  南 杏鶴, 長尾 学, 荒川 圭太, 藤川 清三, 竹澤 大輔 日本植物生理学会年会およびシンポジウム 講演要旨集 2003 (0) 459 -459 2003年 [査読無し][通常論文]
   

  植物が低温馴化により耐凍性を獲得する過程では、タンパク質のリン酸化や脱リン酸化が関わっていると考えられている。我々は、蘚類ヒメツリガネゴケ（Physcomitrella patens）原糸体が、ABAや低温、高浸透圧処理によって凍結耐性を上昇することを以前に示した。ABA・低温処理によってmRNAの顕著な発現誘導が起こるPARK(P. patens ABA-responsive protein kinase)遺伝子は、セリン・スレオニンプロテインキナーゼをコードしていた。PARKはシロイロナズナのS-ドメインレセプター様プロテインキナーゼとキナーゼドメインにおいて56.4％の相同性があるが、細胞外ドメインを持たず、N末端は膜貫通ドメインと思われる疎水領域から始まっていた。GST融合タンパク質を用いたPARKのリン酸化活性の解析では、ミエリン塩基性タンパク質やヒストンIIISの基質をリン酸化し、PARK自身も自己リン酸化することが分かった。また、GFP融合遺伝子による解析では、PARKの細胞膜周辺への局在が示された。PARK遺伝子破壊株の耐凍性を調べた結果、ABAや低温処理した破壊株では、野生株より低い凍結耐性を示した。これらの結果から、PARKが細胞膜結合型プロテインキナーゼとして、ヒメツリガネゴケのABAや低温による凍結耐性上昇のシグナル伝達の制御に関与している可能性が示唆された。
• Molecular chaperone activity of ER-localized small heat shock proteins that accumulated in cortical parenchyma cells of mulberry tree during winter in vitro

  N Ukaji, D Takezawa, K Arakawa, S Fujikawa PLANT AND CELL PHYSIOLOGY 43 S116 -S116 2002年 [査読無し][通常論文]
  
• ABA-induced freezing tolerance in moss Physcomitrella patens and physiological changes

  M Nagao, A Minami, K Arakawa, S Fujikawa, D Takezawa PLANT AND CELL PHYSIOLOGY 43 S168 -S168 2002年 [査読無し][通常論文]
  
• ABA-INDUCED FREEZING TOLERANCE IN Physcomitrella patens AND GENE EXPRESSION :

  NAGAO Manabu, MINAMI Anzu, TAKEZAWA Daisuke, ARAKAWA Keita, FUJIKAWA Seizo Plant and cell physiology 42 s121 2001年
• ANTIFUNGAL EFFECT OF WAS-3a PROTEIN TO SNOW MOLD IN WINTER WHEAT :

  KUWABARA Chikako, TAKEZAWA Daisuke, Fujikawa Seizo, ARAKAWA Keita Plant and cell physiology 42 s142 2001年
• Accumulation of 18kD Intracellular Pathogenesis-Related Proteins in Cortical Parenchyma Cells of Mulberry Tree during Seasonal Cold Acclimation :

  Ukaji Norifumi, Takezawa Daisuke, Arakawa Keita, Fujikawa Seizo Plant and cell physiology 42 s143 2001年
• POSSIBLE FUNCTIONS OF EXTRACELLULAR PROTEINS IN WINTER HARDINESS OF PLANTS :

  ARAKAWA Keita, TAKEZAWA Daisuke, KUWABARA Chikako, KURODA Katsushi, YOSHIDA Shizuo, FUJIKAWA Seizo Plant and cell physiology 42 s12 2001年
• The Function of the ABA-induced Thaumatin-like Protein in Winter Wheat Cells :

  KUWABARA Chikako, ARAKAWA Keita, TAKEZAWA Daisuke, FUJIKAWA Seizo Plant and cell physiology 41 s50 2000年
• Gene expression of WAP20 and WAP27 :

  UKAJI Norifumi, TAKEZAWA Daisuke, ARAKAWA Keita, FUJIKAWA Seizo Plant and cell physiology 41 s48 2000年
• DIVERSITY OF PROTEINS EXPRESSED IN OVERWINTERING PLANTS UNDER THE LOW TEMPERATURE ENVIRONMENT :

  TAKEZAWA Daisuke, ARAKAWA Keita, YOSHIDA Shizuo, FUJIKAWA Seizo Plant and cell physiology 41 s10 2000年
• WAS-3, the ABA-induced Thaumatin-like Protein, in Suspension-cultured Cells of Winter Wheat

  KUWABARA Chikako, ARAKAWA Keita, TAKEZAWA Daisuke, FUJIKAWA Seizo Plant and cell physiology 40 s75 -s75 1999年03月
• Characterization of WAP27 ; the proteins which accumulation in the ER during winter of mulbery (Morus bombycis Koidz) cortical cells

  UKAJI Norifumi, TAKEZAWA Daisuke, ARAKAWA Keita, FUJIKAWA Seizo Plant and cell physiology 40 s104 -s104 1999年03月
• DECREASE IN LEVELS OF PLASMA MEMBRANE PROTEINS FROM JERUSALEM ARTICHOKE TUBERS FROZEN AT SUBLETHAL TEMPERATURE

  ARAKAWA Keita, HANAZAKI Mitsuru, YOSHIDA Shizuo Plant and cell physiology 40 s104 -s104 1999年03月
• Molecular Cloning of ABA-induced Secretory Protein (WAS-2) in Winter Wheat Cultured Cells

  KUWABARA Chikako, TAKEZAWA Daisuke, ARAKAWA Keita, YOSHIDA Shizuo Plant and cell physiology 39 S92 -S92 1998年05月
• Increase of ER Protein During Cold Accumulation in Mulberry Twig

  UKAJI Norifumi, TAKEZAWA Daisuke, ARAKAWA Keita, FUJIKAWA Seizo, YOSHIDA Shizuo Plant and cell physiology 39 S139 -S139 1998年05月
• ABA-INDUCED ACCUMULATION OF A 19-kDa PLASMA MEMBRANE POLYPEPTIDE IN SUSPENSION CULTURED-CELLS OF WINTER WHEAT AND ITS RELATIONSHIP TO THE INDUCTION OF FREEZING TOLERANCE

  KOIKE Michiya, TAKEZAWA Daisuke, ARAKAWA Keita, YOSHIDA Shizuo Plant and cell physiology 38 s97 1997年03月
• CHANGES IN PROTEIN COMPOSITION DURING COLD ACCLIMATION IN ORCHARD GRASS

  ARAKAWA Keita, YOSHIDA Shizuo Plant and cell physiology 38 s97 1997年03月
• BIOCHEMICAL ANALYSIS OF ABSCISIC ACID-INDUCED SECRETION OF PROTEINS IN SUSPENSION-CULTURED CELLS OF WINTER WHEAT

  KUWABARA Chikako, ARAKAWA Keita, YOSHIDA Shizuo Plant and cell physiology 38 s42 1997年03月
• 低温馴化過程におけるオーチャードグラスのタンパク質組成の変動

  荒川 圭太, 吉田 静夫 日本植物学会大会研究発表記録 = Proceedings of the annual meeting of the Botanical Society of Japan 60 114 -114 1996年10月 [査読無し][通常論文]
  
• キクイモ細胞の凍結傷害と細胞壁の役割

  村井 麻理, 荒川 圭太, 吉田 静夫 日本植物学会大会研究発表記録 = Proceedings of the annual meeting of the Botanical Society of Japan 60 261 -261 1996年10月 [査読無し][通常論文]
  
• ANALYSIS OF PHYSIOLOGICAL FACTORSGOVERNING THE DEVELOPMENT OF FREEZING TOLERANCE IN SALT GREEN(Brassica campestris L.)

  MURAI Mari, ARAKAWA Keita, YOSHIDA Shizuo Plant and cell physiology 36 S141 1995年03月
• INACTIVATION OF VACUOLAR H^+-ATPase IN CUCUMBER COTYLEDONS CAUSED BY CHILLING TREATMENT

  KAWAMURA Yukio, ARAKAWA Keita, YOSHIDA Shizuo Plant and cell physiology 36 S141 1995年03月
• PURIFICATION OF ABA-INDUCED 19-KD PLASMAMEMBRANE POLYPEPTIDE FROM CELL SUSPENSION CULTURES OF WINTERWHEAT

  KOIKE Michiya, ARAKAWA Keita, YOSHIDA Shizuo Plant and cell physiology 36 S127 1995年03月
• 厳しい冬を乗り越える植物 -植物の耐凍性について-

  荒川圭太 東北大学遺伝生態研究センター通信 4 -5 1995年 [査読無し][通常論文]
  
• BETAINE ALDEHYDE DEHYDROGENASE PROTEIN IS PRESENT IN LEAVES OF BOTH BETAINE ACCUMULATORS AND NONACCUMULATORS IN VARIOUS CEREAL PLANTS

  ARAKAWA, K, MIZUNO, K, TAKABE, T PHOTOSYNTHESIS RESEARCH 34 (1) 218 -218 1992年10月 [査読無し][通常論文]
  
• IMMUNOLOGICAL STUDIES OF BETAINE ALDEHYDE DEHYDROGENASE IN BARLEY

  ARAKAWA, K, MIZUNO, K, TAKABE, T PHOTOSYNTHESIS RESEARCH 34 (1) 217 -217 1992年10月 [査読無し][通常論文]
  

書籍等出版物

• 木本植物の生理生態

  小池, 孝良, 北尾, 光俊, 市栄, 智明, 渡辺, 誠(農学) (担当:分担執筆範囲:11.5 低温応答)
  共立出版 2020年11月 (ISBN: 9784320058125) xvi, 235p, 図版 [8] p
• 低温環境の科学事典

  河村, 公隆 (担当:分担執筆範囲:樹木細胞の凍結挙動)
  朝倉書店 2016年07月 (ISBN: 425416128X) 432
• 木質の構造

  日本木材学会 (担当:分担執筆範囲:第３章３ 環境ストレスと形成層活動)
  文永堂出版 2011年04月 (ISBN: 9784830041204) xii, 313p

所属学協会

• 日本木材学会北海道支部   日本植物学会   日本農芸化学会   低温生物工学会   日本木材学会   日本植物生理学会   

共同研究・競争的資金等の研究課題

• 氷点下における樹木細胞壁の役割

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  研究期間 : 2021年04月 -2024年03月 

  代表者 : 荒川 圭太, 重冨 顕吾, 鈴木 伸吾

   

  多くの植物植物は、氷点下温度にさらされると細胞外の水から凍結が始まり、それにともなって細胞は脱水を生じる。このような挙動は細胞外凍結と呼ばれ、樹木では緑葉や樹皮の細胞などが細胞外凍結する。そこでカツラの樹皮から細胞壁画分を調製して調べると、水を凍結へと導く氷核形成を促す活性（氷核活性）が検出された。そこで本研究では、カツラ樹皮から検出された氷核活性の原因物質を特定し、細胞外凍結における役割について調べることを目的とした。初年度は、カツラ樹皮由来の氷核活性成分の特定とその性質について調べることを主な目標とした。 氷核活性成分の調製には、冬季のカツラ樹皮から抽出した細胞壁画分を用い、既知の氷核活性物質と比較しながら活性成分の分画を進めた。細胞壁から水で抽出できた活性画分を用い、熱処理や酸処理、アルカリ処理などの活性への影響について調べたところ、既知の氷核物質であるシュウ酸カルシウムや氷核タンパク質とは性質は異なることが示唆された。これらの処理に加え、限外濾過を利用した簡易的なサイズ分画も併用し、活性成分の部分精製を進めて性質を調べるとともに、スケールアップして活性画分を回収して構造解析にも用いた。 これまでの実験によって知り得た活性成分の性質を鑑み、二次元NMR解析などで構造解析を進めたところ、活性成分は多糖類であることが示唆され、その主たる構成糖などの知見が得られた。氷核活性成分の機能解析については、活性の濃度依存性や季節変動の有無、抗氷核ポリフェノール類による活性阻害の有無について検証などを進めている。また、既知の氷核物質との活性比較を試みたが、それぞれの研究によって活性測定条件が異なることもあり、構造特性や活性の比較を論ずるには慎重な判断を要することを改めて認識した。２年目も研究計画に沿って引き続き氷核活性成分の構造や活性の特性に注目して実験を進めていく予定である。
• 樹木の越冬に学ぶ水の凍結制御機構

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2015年04月 -2018年03月 

  代表者 : 荒川 圭太, 福士 幸治

   

  抗氷核ポリフェノールが有する氷核形成阻害活性（過冷却促進活性または抗氷核活性）の作用機構を解析するため、氷核細菌Erwinia ananasの氷核タンパク質inaAを活性評価系に利用することにした。inaAにヒスチジンタグを付した組換えタンパク質（His-inaA）を大腸菌で発現させ、その菌体の粗抽出液からHis-inaAをアフィニティ精製したところ、可溶性His-inaA画分でも氷核活性が検出された。さらに、この可溶性His-inaA画分に抗氷核ポリフェノールを添加すると氷核活性が抑制された。
• 植物の凍霜害防止のための新戦略

  日本学術振興会：科学研究費助成事業 挑戦的萌芽研究

  研究期間 : 2015年04月 -2017年03月 

  代表者 : 福士 幸治, 荒川 圭太, 実山 豊

   

  65種類の植物葉の粗抽出を従来のドロップレット凍結法で試験した結果、植物葉抽出物に氷核形成阻害物質(過冷却促進活性)が広く認められ、特に、アカエゾマツ、レッドマスタード、クマイザサ、バッコヤナギ、ゲッケイジュ、スダチ、アカジソ、チャノキ、セイヨウタンポポ葉の活性が強かった。特に活性が強かった植物葉粗抽出物を、新たに開発したリーフディスク凍結試験法に供試した結果、植物葉上でも凍結を押さえる作用が認められた。従って、本法を用いて未利用の植物葉から新規な過冷却促進物質が期待される。現在、植物葉抽出物を用いた茶園の防霜に関する共同研究について協議中である。
• 木質バイオマス生産を支える樹木冬芽の越冬機構の解明

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  研究期間 : 2011年 -2013年 

  代表者 : 荒川 圭太

   

  冬芽の越冬機構である凍結適応機構と休眠について調べた。カラマツ冬芽の凍結挙動を低温走査型電子顕微鏡（cryo-SEM）等で調べると、原基の細胞は部分脱水するものの、細胞内に残存する水は深過冷却していることが示唆された。また、冬芽ではピニトールやスクロースなどが主要な炭水化物として蓄積していたが、それだけでは過冷却能を説明できないものと考えられた。また、カラマツ冬芽の自発休眠が解除されて強制休眠へ移行する過程での可溶性タンパク質組成の変化をLC-MS/MS法で調べると、自発休眠から強制休眠に移行する比較的短期間ではタンパク質組成は大きく変化しないことが示唆された。
• 森林資源の供給と有効利用を目指した環境ストレス耐性樹種の創製

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  研究期間 : 2008年 -2010年 

  代表者 : 荒川 圭太

   

  本研究では、形質転換可能なモデル樹種の交雑ポプラ(Populus tremula×P.alba)に、ニホンカラマツ(Larix kaempferi)木部由来の冬季誘導性ガラクチノール合成遺伝子を導入し、ラフィノース合成による凍結ストレス耐性の向上を試みた。当該遺伝子発現によって緑葉、師部、木部の各組織でラフィノース含量が有意に増加したが、師部や木部の凍結抵抗性には明確な差は見られなかったものの、低温馴化した緑葉で凍結ストレス耐性に有意な差が見られた。
• 樹木木部柔細胞の過冷却促進物質の網羅的構造同定と大量獲得法の検討

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2008年 -2010年 

  代表者 : 藤川 清三, 福士 幸治, 荒川 圭太

   

  寒冷環境において-40℃まで凍らない樹木木部柔細胞から、過冷却を促進する物質(氷核形成阻害物質)の探索を行い、数種類のフラボノール配糖体および数種類の加水分解型タンニンを同定した。この知見に基づき、類似の化学構造を持つ数十種類のフラボノイド配糖体、および数十種類の加水分解型・縮合型タンニンが過冷却活性を持つことを解明した。これらのうち、大量に獲得できる化合物を用い、過冷却促進新素材としての応用を検討した。
• 産業利用を目的とした遺伝子組換えポプラの野外実験

  日本学術振興会：科学研究費助成事業 基盤研究(A)

  研究期間 : 2007年 -2010年 

  代表者 : 林 隆久, 谷口 亨, 馬場 啓一, 海田 るみ, 太田 誠一, 磯貝 明, 舘野 正樹, 吉田 正人, 菊池 彰, 西窪 伸之, 荒川 圭太, 加来 友美, 朴 龍又, 栗田 学, 坪村 美代子, 渡邊 和男, 間野 絵梨子

   

  キシログルカンを分解するキシログルカナーゼ(AaXEG2)を構成発現させたポプラを野外で4年間植栽した。発現レベルの変化、木部・葉・根の形態を解析した。発現のレベルは貧栄養土壌で高くなり、富貧栄養土壌で低くなった。葉は、陽葉の表現型を保った。プロテオミクスにより、全ての部位でストレスに関連する遺伝子の発現が増加したが、木部ではあて材に関するタンパク質の発現が認められた。木部の強度は、野生株と比べて減少し、枝を振動させると、振幅は長く続いた。
• 樹木木部柔細胞からの過冷却促進物質の同定とその応用に関する研究

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2005年 -2007年 

  代表者 : 藤川 清三, 荒川 圭太

   

  樹木の木部柔細胞は-40℃以下の低温まで長期間にわたり細胞内の水を液体状態で保つ、深過冷却という特殊な氷点下温度への適応機構を示す。この深過冷却のメカニズムを明らかにするために、深過冷却する木部柔細胞を持つ数種の樹木の木部からメタノール抽出により木部粗抽出物を得て過冷却活性を測定した。その結果、いずれも高い過冷却活性が得られた。抽出物が最も高い過冷却活性を示したカツラを用いて、氷核形成阻害物質の同定を行った。この結果、カツラ木部には非常に多種類の氷核形成阻害物質が存在することが明らかになった。液一液分離、シリカゲルカラムクロマトグラフィー、HPLCによる段階的な精製の結果、最終的に最も活性の高いシリカゲルカラムクロマトグラフィー画分から4つのピークを得て、化合物をNMRなどにより同定した結果、これらはケルセチンー3-0一グルコシド、ケンフェロールー7-0一グルコシド、8一メトキシケンフェロールー3-0一グルコシド、ケンフェロールー3-0一グルコシドであり、それぞれ2.8℃,9.0℃,3.4℃,4.0℃の高い過冷却活性を示した。これらのうち、ケンフェロールー7-0一グルコシドの示す9.0℃の過冷却活性はこれまでに知られている物質中で最大の活性であった。これらフラボノール配糖体の木部柔細胞内の局在を明らかにすると共に、引き続きこれら物質の木部柔細胞への蓄積の季節性および組織特異性を明らかにした。 併せて、樹木からの粗抽出物、および精製したフラボノール配糖体にはバルクの水を過冷却させる効果を持つことを明らかにし、特許出願した。これに基づいて、動物からの摘出臓器の過冷却保存への応用、清涼飲料への添加の効果、および凍結制御剤としての使用を検討した。いずれも良好な結果が得られ現在、その応用に関する幾つかの周辺特許権の獲得を目指している。
• 酸性雪による越冬性植物の傷害発生機構の解明と耐性付与のための分子基盤の構築

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  研究期間 : 2004年 -2005年 

  代表者 : 荒川 圭太

   

  化石燃料の消費によって大気中に放出された硫黄酸化物(SOx)や窒素酸化物(NOx)による酸性雨・酸性霧は地球規模の環境問題である。降雨などに混じる酸性物質(硫酸、硝酸など)が直接的もしくは間接的に植生や農作物の生産性・品質に少なからぬ影響を及ぼすことが懸念される。 降雨の酸性化と同様に雪氷の酸性化(酸性雪)も徐々に進行している。酸性雪は冬期間かつ寒冷地域での気象現象であるため、酸性雨に比べてその関心度は低い。酸性雪は地表面に留まる時間が長いため、酸性雪に覆われた植物は、氷点下温度と酸性物質による複合的環境ストレスを長期間にわたって被ることになる。そのため、酸性雪は融雪後の植物の生長に影響を及ぼす環境要因のひとつと考えられるが、酸性雪による植物への影響やその応答性について検証した例は見ない。そこで本研究では、酸性雪によるストレスを実験室レベルでシミュレーションし、酸性雪による越冬性植物の傷害発生機構を解析して酸性雪耐性付与への分子基盤の構築を目指した。 これまでに、越冬性作物である冬小麦を用い、酸性雪ストレスをシミュレーションする実験系を構築して酸性雪ストレスに対する冬小麦緑葉の応答性を調べてきた。その結果、酸性物質(硫酸)存在下で凍結融解処理することによって冬小麦緑葉の生存率が低下するのは、共存する硫酸が凍結濃縮されることによって生じるpH低下が主たる要因であることを明らかにした(Plant and Cell Physiology誌に掲載予定)。しかし、酸性雪ストレスによる越冬性作物の傷害発生の分子機構を詳細に解明するまでにはまだ至っていない。今後も当研究課題に関連した生理学的・生化学的解析を継続し、越冬性植物の分子育種による酸性雪耐性の付与や酸性雪耐性品種の選抜による生産性向上・植生回復などへ応用するための分子基盤の構築を試みる所存である。
• 北方樹木の未利用有効資源活用のための分子生物学的研究

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2002年 -2004年 

  代表者 : 藤川 清三, 船田 良, 佐野 雄三, 荒川 圭太

   

  北方樹木の寒冷環境適応機構を分子レベルで研究を行った。樹木構成細胞は組織により、冬季の樹幹内水分の凍結に対する適応機構が異なり、師部組織柔細胞は細胞外凍結による適応をするのに対して、木部組織柔細胞は深過冷却という凍結回避により適応する。 細胞外凍結では、凍結脱水に対する抵抗性を獲得することにより、北方樹木の師部柔細胞は液体窒素温度の凍結にも耐える。このような高い凍結耐性を持つ北方樹木、クワの皮層柔細胞内の小胞体(ER)にグループ3Late Embryogenesis Aboundant (LEA)タンパク質が,冬季特異的に蓄積することを明らかにした。このタンパク質をWAP27と命名し、その機能を解析した。WAP27を導入した形質転換シロイヌナズナは植物体レベルで凍結耐性が上昇する事、および単離・精製したWAP27は凍害防止剤として高い活性を持つことを明らかにした。 一方、北方樹木の木部柔細胞は、細胞内水分が液体状態のまま-60℃付近まで過冷却して氷点下温度に適応するという樹木特有の特徴的な適応機構を示す。この機構、すなわち水が安定的に-60℃付近まで凍らない機構を明らかにするため、冬季特異的に発現する遺伝子のフイルター・アレイおよびディファレンシャル・ディスプレイ法による解析、冬季特異的に蓄積するタンパク質の解析、冬季特異的に蓄積する可溶性糖質の解析を行った。樹木木部の網羅的な遺伝子解析は、世界初と思われる。カラマツを用いた遺伝子解析により植物においてはこれまでに報告されていない低温誘導性遺伝子を10数個明らかにした。これらの冬季特異的な遺伝子発現はノーザン・ブロット法により過冷却能力の変化と並行的に発現することを確認した。ブナを用いたタンパク質解析により、季節的および各種処理による過冷却能力の変化と並行的に発現する20数個のタンパク質スポットを確認した。これらのうち、既存の報告で、過冷却を促進する可能性のある不凍タンパク質と高い相同性を示すタンパク質を単離・精製して不凍活性を調べたが顕著な不凍活性は得られなかった。可溶性糖質については過冷却する木部特異的に蓄積する糖はシラカンバを用いた研究では得られなかった。しかし、木部から80%エタノールで抽出した粗画分は、これまでの報告には無い高い過冷却能力を持つことを明らかにし、引き続き物質の同定を行っている。 北方樹木由来のこれら物質は、高凍結抵抗性植物の作出、及び凍結制御剤として広範な産業的応用が期待できる。
• 樹木木部柔細胞における深過冷却機構の解析

  研究期間 : 2003年
• Study on the mechanism of deep supercooling in xylem cells of woody plants

  研究期間 : 2003年
• 遺伝子導入による越年性牧草の耐寒性、耐病性向上のための基礎研究

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2000年 -2002年 

  代表者 : 竹澤 大輔, 荒川 圭太, 小澤 修二

   

  本研究では、遺伝子工学的手法によって牧草に凍結耐性や耐病性を付与することを目的とし、マメ科牧草アルファルファを用いて耐病性や耐凍性に関わる因子の探索を行うとともに、形質転換による改変を目的として研究を行ってきた。 導入する遺伝子の侯補として、高い耐寒性を持つ冬小麦からアンチフリーズタンパク質、抗菌タンパク質と相同性のあるタウマチン様タンパク質WAS-3を単離した。形質転換小麦細胞で産生させたWAS-3タンパク質は、雪腐れ菌Microdochium nivaleの菌糸成長を抑制した。WAS-3を過剰発現する形質転換植物は、Fusarium oxysporumの感染が野生株と比べて遅れる傾向が見られた。また、高い耐凍性を持つクワや、蘚類Physcomitrella patensから耐凍性関連遺伝子の探索を行った。クワからは季節的耐凍性上昇とともに蓄積するデハイドリンやLEAIII遺伝子を単離し、これら遺伝子を過剰発現する形質転換植物を作出した。 また、耐凍性を高めることが知られているシロイヌナズナ転写因子CBF3をアルファルファに導入し、選抜薬剤であるカナマイシンに耐性を示す耐性カルスを得、そこから9個体の植物体を再生させることに成功した。形質転換植物体は、CBF3発現植物でしばしばおこる矮化も観察されず、体細胞変異の少ない形質転換体であることが示された。形質転換体におけるCBF3遺伝子の発現をRT-PCRなどにより解析した。これら植物体の低温馴化前後の耐凍性の変化について、電解質溶出法により解析した。
• 越冬性植物の酸性雪ストレス耐性機構

  研究期間 : 2002年
• 低温による樹木構成細胞の細胞壁構造特性の変化

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 1999年 -2001年 

  代表者 : 藤川 清三, 船田 良, 竹澤 大輔, 荒川 圭太, 佐野 雄三

   

  平成11年度から開始し、本年度で終了する本課題研究では、樹木の寒冷地適応と細胞壁の構造特性について研究を行った。多年生植物で高木の樹木の木部柔細胞は強固な厚い・硬い細胞壁を持つ一方、皮層柔細胞は比較的薄い・柔らかい細胞壁を持つ。当研究では、樹木構成細胞中最も凍結抵抗性が低いために、その凍結抵抗性の限度が樹木の寒冷地への適応の限界要因となっている広葉樹の木部柔細胞が、北方樹木の細胞も含め、すべて深過冷却という凍結回避機構により寒冷環境に適応するという新規の知見を低温走査電子顕微鏡を用いた広汎な観察により明らかにした。また、深過冷却のメカニズムが細胞壁のマイクロ・キャピラリーのサイズに基づくという仮説の誤りを、原形質分離法を用いて、細胞外凍結する皮層柔細胞と深過冷却する木部柔細胞の細胞壁キャピラリーのサイズを測定して明らかにした。深過冷却の度合いが細胞(プロトプラスト)内物質の抽出により大きく減少する事実を明らかにし、木部柔細胞内に新規の不凍蛋白質様物質が蓄積する可能性を示した。細胞内蛋白質の季節的変化の解析を行い、深過冷却能力が最も高い冬季に特異的に蓄積する蛋白質において、これまでに知られていなかった高い過冷却能力を持つ不凍蛋白質の候補を見出し、遺伝子クローニングを続けている。これと共に、比較的薄い・柔らかい細胞壁を持つために、他の草本植物の組織細胞と同様に細胞外凍結により寒冷環境に適応する樹木の皮層柔細胞の凍結耐性獲得のメカニズムに関する研究をモデル植物などを用いて研究した。これらの結果は、植物の中でも樹木ならではの特徴的な環境適応機構を明らかにするとともに、未利用資源としての樹木のストレス誘導性蛋白質の利用の可能性を示唆する。
• 低温馴化過程で誘導される細胞膜タンパク質の細胞膜構造における機能特性に関する研究

  日本学術振興会：科学研究費助成事業 奨励研究(A)

  研究期間 : 1995年 -1995年 

  代表者 : 荒川 圭太

   

  冬コムギは低温馴化の過程で耐凍性が増大することに並行して細胞膜のタンパク質組成が変動する。そこで本研究では、耐凍性の増大と並行して増加する低分子の細胞膜タンパク質のひとつ(18kDa)に着目し、本タンパク質のcDNAクローニングを通じて細胞膜構造における生理的および機能的役割に関する基礎的知見を得ることを目的とした。そこで、まず最初にcDNAクローニングに必要な18-kDa細胞膜タンパク質のN末端ならびに部分的なアミノ酸配列を決定するとともに、目的タンパク質に対する特異抗体を調製することを試みた。 低温馴化した冬コムギ(Triticum aestivum L.cv.Norstar)苗条から細胞膜画分を調製して、18-kDa細胞膜タンパク質を単離するとともにN末端ならびにプロテアーゼ処理によって得られるペプチド断片のアミノ酸配列決定を試みた。しかし、HPLCによる精製において、目的タンパク質ならびにペプチド断片のカラムからの回収率が極端に低いこと等が原因で最終的な量的調製は非常に困難となり、当初の計画の遂行に支障をきたした。そのため、Norstar品種よりも生長量が大きく栽培し易い冬コムギの別品種(T.aestivum L.cv.Chihoku)に急遽材料を代えて耐凍性の検定や細胞膜タンパク質組成の解析をおこなった。その結果、本品種の耐凍性(LT_<50>)はNorstar品種(-18℃)よりやや低く(-15℃)、18-kDa細胞膜タンパク質に相当すると考えられるタンパク質に関しては、Norstar品種に比べて量的に低いながらも低温馴化によってその含量は増加することが判明した。そのため、本品種を用いて本研究計画の遂行が可能になるものと判断した。現在、当初の計画に基づいてcDNAクローニングをすすめるため、低温馴化した本品種から大量調製した細胞膜画分をもとにして、再度18-kDa細胞膜タンパク質の単離ならびに部分的アミノ酸配列の決定や特異抗体の調製などを試みている。
• 低温耐性型液胞膜H^+-ATPaseの存在とその分子構造特性に関する研究

  日本学術振興会：科学研究費助成事業 一般研究(B)

  研究期間 : 1993年 -1994年 

  代表者 : 吉田 静夫, 荒川 圭太, 前島 正義

   

  植物の液胞は、細胞生長、浸透圧調節、塩類および二次代謝産物などの輸送と貯蔵に重要な役割を果たしており、その原動力は液胞膜にあるプロトン輸送酵素(H^+-ATPase,H^+-PPase)によって生み出される。亜熱帯原産のヤエナリでは、液胞膜H^+-ATPaseの低温による早期失活が液胞内pHの上昇と細胞質pHの酸性化を引き起こすことから、本酵素は細胞内のイオン環境の恒常性維持にきわめて重要な働きを持つと考えられる。これと対照的に低温耐性なエンドウ(温帯植物)では、本酵素は低温に安定で長期間機能が維持される。6数種の豆科植物について検討したところ、本酵素のin vivo条件での低温安定性は植物の低温耐性と密接に関連していることが示された。分離された液胞膜小胞画分にMgATPと共に異なる濃度のchaotropic anionを加えて低温でインキュベートしたときの酵素失活につして比較検討したところ、低温耐性植物と低温感受性植物ではアニオンに対する感受性に著しい違いが認められた。とりわけ、亜硝酸イオンに対する感受性の違いは際だっていた。亜硝酸イオン感受性から本酵素は低温耐性型と低温感受性型に明確に区別される。精製酵素標品についてサブユニット構成を比較すると、68.57.16.13.12kDaの5種は全ての植物に共通であるが、30-4zkDaの4種類のサブユニットには分子サイズに違いが忍められた16kDa(Vo)k等電点は低温耐性型酵素により酸性側であり、免疫科学的な性質も著しく異なっていた。ヤエナリの16kDa抗体は低温感受性のそれを認識できるが低温耐性型のそれを全く認識できない。この事実は、16kDaの分子構造が酵素複合体の低温安定性を大きく左右する要素の一つであることを示唆しているものと思われ、現在、分子構造特性の解析を急いでいる。
• 木本ならびに草本植物の寒冷適応機構の研究

  研究期間 : 1992年
• Study on plant cold hardiness

  研究期間 : 1992年

産業財産権

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