Constitutive chemical defense mechanism of inner bark of Sorbus commixta against Trametes versicolor
Masato Koyama, Kengo Shigetomi, Yutaka Tamai, Keita Arakawa, Yuzou Sano
Tree Physiology, 45, 2, Oxford University Press (OUP), 13 Jan. 2025, [Peer-reviewed]
English, Scientific journal, Abstract

Tree bark is a crucial tissue that defends tree stems from invasions by microorganisms. However, our understanding of the constitutive chemical defense mechanisms of the tree barks remains limited. Our group recently discovered that the inner bark of Sorbus commixta Hedl. exhibited potent inhibitory effects on the growth of the white-rot fungus, Trametes versicolor (Linnaeus) Lloyd. It was hypothesized that this growth suppression was due to hydrogen cyanide (HCN) originating from cyanogenic glycosides such as amygdalin and prunasin, which are secondary metabolites in the inner bark of S. commixta. To test this hypothesis, we first quantified the amygdalin content in the inner bark of S. commixta and evaluated the antifungal activity (AFA) of HCN against T. versicolor by placing paper discs on potato dextrose agar with T. versicolor. Subsequently, we identified HCN in the inner bark of S. commixta using a picric acid paper. In the AFA tests, the amygdalin + β-glucosidase solution, the potassium cyanide (KCN) + benzaldehyde solution and the KCN solution exhibited elevated AFA against T. versicolor. Conversely, the amygdalin solution, the β-glucosidase solution and the benzaldehyde solution did not exhibit significant AFA. These findings demonstrate that HCN acts as an antifungal agent against T. versicolor. Furthermore, HCN was detected in the freeze-dried inner bark pieces and the trunks of living S. commixta. This suggests that the disruption of the cells in the inner bark of S. commixta caused the cyanogenic glycosides and β-glucosidase to interact, which resulted in the hydrolysis of cyanogenic glycosides and generation of HCN. From the perspective of constitutive chemical defense mechanisms, HCN plays a crucial role in the high AFA of the inner bark of S. commixta against T. versicolor, creating an unfavorable environment for the growth of white-rot fungus after injury breaches the periderm.

An Attempt to Reduce Freezing Injury by Anti-Ice Nucleation Activity (Supercooling-promoting Activity) of the Extracts of Plants
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, 1, 6, 2023, [Peer-reviewed], [Lead author, Corresponding author]
Japanese, Scientific journal

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
English, Scientific journal

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, Elsevier BV, Aug. 2021, [Peer-reviewed]
English, Scientific journal

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, Apr. 2021, [Peer-reviewed], [Last author, Corresponding author]
Japanese, Scientific journal

Ice Nucleation Substances Extracted from Bark of Cercidiphyllum japonicum
古賀 泰雅, 鉄穴口 晃, 鈴木 伸吾, 重冨 顕吾, 荒川 圭太
Cryobiology and Cryotechnology, 67, 2, 141, 145, Japanese Society of Cryobiology and Cryotechnology, 2021, [Peer-reviewed]
Japanese

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, Elsevier BV, Aug. 2020, [Peer-reviewed]
English, Scientific journal

Supercooling-promoting (Anti-ice nucleation) substances
Seizo Fujikawa, Chikako Kuwabara, Jun Kasuga, Keita Arakawa
Advances in Experimental Medicine and Biology, 1081, 289, 320, 2018, [Peer-reviewed]
English, In book

Mechanism of overwintering in trees
Keita Arakawa, Jun Kasuga, Naoki Takata
Advances in Experimental Medicine and Biology, 1081, 129, 147, 2018, [Peer-reviewed], [Lead author]
English, In book

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, Nov. 2017, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
English, Scientific journal

Winter Hardiness of Boreal Woody Plants and Supercooling-promoting Substances
荒川 圭太
冷凍 = Refrigeration, 92, 1078, 547, 553, 日本冷凍空調学会, Aug. 2017, [Corresponding author]
Japanese

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

Inhibition of Ice Nucleation Activity of Recombinant inaA Proteins by Polyphenols
IZUHARA Nobuhiro, KUWABARA Chikako, SUZUKI Shingo, ODAKA Yuko, FUJIKAWA Seizo, ARAKAWA Keita
Cryobiology and Cryotechnology, 63, 2, 155, 160, Japanese Society of Cryobiology and Cryotechnology, 2017, [Peer-reviewed], [Last author, Corresponding author]
Japanese, 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.

Comparison of Supercooling-facilitating Activities in Various Plant Extracts
SUZUKI Shingo, FUKUDA Satoshi, FUKUSHI Yukiharu, ARAKAWA Keita
Cryobiology and Cryotechnology, 63, 1, 33, 36, Japanese Society of Cryobiology and Cryotechnology, 2017, [Peer-reviewed], [Last author, Corresponding author]
Japanese, 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.

Cryopreservation of Hybrid Aspen by Vitrification
KAWAMURA Kohei, KAMI Daisuke, SUZUKI Shingo, TANAKA Daisuke, FUJIKAWA Seizo, ARAKAWA Keita
Cryobiology and Cryotechnology, 62, 2, 123, 126, Japanese Society of Cryobiology and Cryotechnology, 2016, [Peer-reviewed], [Last author, Corresponding author]
Japanese, 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%.

Ice Nucleation Activity of Escherichia coli Cells Expressing Recombinant inaA Proteins
KUWABARA Chikako, SUZUKI Shingo, FUJIKAWA Seizo, ARAKAWA Keita
Cryobiology and Cryotechnology, 61, 2, 131, 134, Japanese Society of Cryobiology and Cryotechnology, 2015, [Peer-reviewed], [Last author, Corresponding author]
Japanese, 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, Oct. 2014, [Peer-reviewed]
English, Scientific journal

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

Analysis of supercooling activities of surfactants
Chikako Kuwabara, Ryuji Terauchi, Hiroshi Tochigi, Hisao Takaoka, Keita Arakawa, Seizo Fujikawa
CRYOBIOLOGY, 69, 1, 10, 16, Aug. 2014, [Peer-reviewed]
English, Scientific journal

Compositional Changes in Soluble Proteins of Tree Buds During Endodormancy Release
SUZUKI Shingo, TAKAHASHI Daisuke, KUWABARA Chikako, UEMURA Matsuo, ARAKAWA Keita
Cryobiology and Cryotechnology, 60, 1, 59, 63, Japanese Society of Cryobiology and Cryotechnology, 2014, [Peer-reviewed], [Last author, Corresponding author]
Japanese, 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, Aug. 2013, [Peer-reviewed]
English, Scientific journal

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, May 2013, [Peer-reviewed]
English, Scientific journal

Freezing behavior of cells in evergreen needle leaves of fir (Abies sachalinensis)
ENDOH Keita, FUJIKAWA Seizo, ARAKAWA Keita
Cryobiology and Cryotechnology, 58, 2, 125, 134, Japanese Society for Cryobiology and Cryotechnology, Oct. 2012, [Peer-reviewed]
English, Scientific journal, 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, Jun. 2012, [Peer-reviewed]
English, Scientific journal

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, Apr. 2012, [Peer-reviewed], [Last author, Corresponding author]
English, Scientific journal

Evaluation on efficacy of supercooling-facilitating substances by various measurements
KUWABARA Chikako, WANG Donghui, KASUGA Jun, MORIWAKA Genta, ARAKAWA Keita, KOYAMA Toshie, INADA Takaaki, FUJIKAWA Seizo
Cryobiology and Cryotechnology, 58, 1, 99, 103, Japanese Society of Cryobiology and Cryotechnology, 2012, [Peer-reviewed]
Japanese, 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.

Freezing Behaviors of Cells in Dormant Buds of Katsura (Cercidiphyllum japonicum) Tree
ENDOH Keita, OKADA Kaori, SUZUKI Shingo, FUJIKAWA Seizo, ARAKAWA Keita
Cryobiology and Cryotechnology, 58, 2, 179, 184, Japanese Society of Cryobiology and Cryotechnology, 2012, [Peer-reviewed]
Japanese, 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, Jan. 2012, [Peer-reviewed]
English, Scientific journal

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

Characterization of Supercooling-Facilitating (Anti-Ice Nucleation) Hydrolyzable Gallotannins in Supercooling Xylem Parenchyma Cells in Katsura Tree (Cercidiphyllum japonicum)
WANG Donghui, KASUGA Jun, KUWABARA Chikako, FUKUSHI Yukiharu, FUJIKAWA Seizo, ARAKAWA Keita
Cryobiology and Cryotechnology, 57, 2, 147, 151, Japanese Society of Cryobiology and Cryotechnology, 2011, [Peer-reviewed], [Last author, Corresponding author]
English, 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, Jun. 2010, [Peer-reviewed]
English, Scientific journal

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

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

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

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

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, 15 Jul. 2009
In book

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, 15 Jul. 2009
In book

Freezing Resistance of Trees : -Unfrozen Water-
FUJIKAWA Seizo, KASUGA Jun, ARAKAWA Keita
Cryobiology and Cryotechnology, 55, 1, 37, 41, Japanese Society of Cryobiology and Cryotechnology, 2009
Japanese, 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, Dec. 2008, [Peer-reviewed]
English, Scientific journal

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

The Effects of Acid Substances under The Freezing Process on The Survival of Plant Cells
INADA Hidetoshi, FUJIKAWA Seizo, ARAKAWA Keita
Transactions of the Japan Society of Refrigerating and Air Conditioning Engineers, 25, 2, 233, 238, Japan Society of Refrigerating and Air Conditioning Engineers, 2008, [Peer-reviewed], [Last author]
Japanese, 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, Jan. 2008, [Peer-reviewed]
English, Scientific journal

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, [Peer-reviewed]
English, Scientific journal

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, Dec. 2007, [Peer-reviewed]
English, Scientific journal

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, Oct. 2007, [Peer-reviewed]
English, Scientific journal

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, Mar. 2007, [Peer-reviewed]
English, Scientific journal

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, [Peer-reviewed]
English, Scientific journal

Effects of Light Condition after Simulated Acid Snow Stress on Leaves of Winter Wheat
INADA Hidetoshi, FUJIKAWA Seizo, SAITO Hideyuki, ARAKAWA Keita
Environmental sciences : an international journal of environmental physiology and toxicology, 14, Supplement, 53, 71, 2007, [Peer-reviewed], [Last author, Corresponding author]
English, Scientific journal

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, Heron Publishing, 2007, [Peer-reviewed]
English, Scientific journal

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, Sep. 2006, [Peer-reviewed]
English, Scientific journal

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, Apr. 2006, [Peer-reviewed], [Domestic magazines]
English, Scientific journal, 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, Apr. 2006, [Peer-reviewed]
English, Scientific journal

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, 10 Jan. 2006
In book

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, 10 Jan. 2006
In book

Physiological responses of wintering plants to acid snow stress
INADA Hidetoshi, FUJIKAWA Seizo, ARAKAWA Keita
Cryobiology and Cryotechnology, 52, 2, 163, 168, Japanese Society of Cryobiology and Cryotechnology, 2006, [Peer-reviewed], [Last author, Corresponding author]
Japanese, 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, [Lead author]
English, Research society

Nonfreezing water. Searching for nonfreezing substance for water with supercooling/cryogenic property in tree xylem parenchymatous cell.
FUJIKAWA SEIZO, KASUGA JUN, ARAKAWA KEITA
化学と生物, 43, 5, 280, 282, 日本農芸化学会, 01 May 2005
Japanese

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, Feb. 2005, [Peer-reviewed]
English, Scientific journal

Accumulation of Anti-ice-nucleating Substances in Conifer Xylem
MIZUNO Kaoru, KASUGA Jun, ARAKAWA Keita, FUJIKAWA Seizo
Cryobiology and Cryotechnology, 51, 2, 111, 114, Japanese Society of Cryobiology and Cryotechnology, 2005, [Peer-reviewed]
Japanese, 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).

Influence of the Freeze-thaw Process under Acid Conditions on the Viability of Wintering Plants
INADA Hidetoshi, FUJIKAWA Seizo, ARAKAWA Keita
Cryobiology and Cryotechnology, 51, 2, 115, 119, Japanese Society of Cryobiology and Cryotechnology, 2005, [Peer-reviewed], [Last author, Corresponding author]
English, 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.

Study on Proteins Related to Deep Supercooling Ability of Xylem Tissues of Fagus crenata L.
TAKAHASHI Hirokazu, ARAKAWA Keita, FUJIKAWA Seizo
Cryobiology and Cryotechnology, 51, 2, 105, 109, Japanese Society of Cryobiology and Cryotechnology, 2005, [Peer-reviewed]
English, 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, Jan. 2005, [Peer-reviewed]
English, Scientific journal

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, Wiley, Sep. 2004, [Peer-reviewed]
English, Scientific journal

Deep Supercooling in Plant Cells
FUJIKAWA Seizo, ARAKAWA Keita
Cryobiology and Cryotechnology, 50, 1, 33, 36, Japanese Society of Cryobiology and Cryotechnology, 2004
Japanese, 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, Jan. 2004, [Peer-reviewed]
English, Scientific journal

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, Jan. 2004, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal

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, May 2003, [Peer-reviewed]
English, Scientific journal

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, Feb. 2003, [Peer-reviewed]
English, Scientific journal

Physiological Changes Associated with Abscisic Acid-Induced Freezing Tolerance in Physicomitrella patens
MINAMI Anzu, NAGAO Manabu, ARAKAWA Keita, FUJIKAWA Seizo, TAKEZAWA Daisuke
Cryobiology and Cryotechnology, 49, 2, 179, 183, Japanese Society of Cryobiology and Cryotechnology, 2003, [Peer-reviewed]
Japanese, 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.

Seasonal Changes in Accumulation of Soluble Sugars in Birch Xylem Tissues
KASUGA Jun, FUJIKAWA Seizo, ARAKAWA Keita
Cryobiology and Cryotechnology, 49, 2, 185, 189, Japanese Society of Cryobiology and Cryotechnology, 2003, [Peer-reviewed]
Japanese, 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.

Identification of Gene Expressed in Xylem Ray Parenchyma Cells of Larix kaempferi That Adapt to Cold Environment by Deep Supercooling
TAKATA Naoki, MINAMI Anzu, ARAKAWA Keita, TAKEZAWA Daisuke, FUJIKAWA Seizo
Cryobiology and Cryotechnology, 49, 2, 191, 194, Japanese Society of Cryobiology and Cryotechnology, 2003, [Peer-reviewed]
Japanese, 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.

The Relation between Small Diameter Capillaries in Cell Wall and Deep-Supercooling in Xylem Ray Parenchyma Cells of Woody Plants
TANAKA Satoko, NAGAO Manabu, FUNADA Ryo, FUJIKAWA Seizo, ARAKAWA Keita
Cryobiology and Cryotechnology, 49, 2, 209, 213, Japanese Society of Cryobiology and Cryotechnology, 2003, [Peer-reviewed]
Japanese, 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.

Decrease in Level of Plasma Membrane Protein after Freezing Treatment of Tubers of Jerusalem Artichoke
ARAKAWA Keita, INADA Hidetoshi
Cryobiology and Cryotechnology, 49, 2, 203, 207, Japanese Society of Cryobiology and Cryotechnology, 2003, [Peer-reviewed], [Lead author, Corresponding author]
Japanese, 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.

Accumulation of Extracellular Proteins in Birch Xylem during Seasonal Cold Acclimation
ARAKAWA Keita, KASUGA Jun, TAKASHIMA Hiroshi, FUJIKAWA Seizo
Cryobiology and Cryotechnology, 49, 2, 195, 201, Japanese Society of Cryobiology and Cryotechnology, 2003, [Peer-reviewed], [Lead author]
English, 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, Sep. 2002
English, Scientific journal

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, May 2002, [Peer-reviewed]
English, Scientific journal

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, Aug. 2001, [Peer-reviewed]
English, Scientific journal

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, May 2001, [Peer-reviewed], [Corresponding author]
English, Scientific journal

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, Mar. 2000, [Peer-reviewed]
English, Scientific journal

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
English, Symposium

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, Jun. 1999, [Peer-reviewed]
English, Scientific journal

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, Jun. 1999, [Peer-reviewed]
English, Scientific journal

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, Jun. 1999, [Peer-reviewed]
English, Scientific journal

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, Feb. 1999, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Changes of Cell Wall in Xylem Ray Parenchyma Cells of Birch by Cold Acclimation
KUBOTA Masatoshi, ARAKAWA Keita, KURODA Katsushi, FUJIKAWA Seizo
Cryobiology and Cryotechnology, 45, 2, 124, 127, Japanese Society of Cryobiology and Cryotechnology, 1999, [Peer-reviewed]
Japanese, 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, Jun. 1997, [Peer-reviewed]
English, Scientific journal

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, [Peer-reviewed]
English, International conference proceedings

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

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

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, Wiley, Jan. 1994, [Peer-reviewed]
English, Scientific journal

BETAINE ALDEHYDE DEHYDROGENASE IN THE GRAMINEAE - LEVELS IN LEAVES OF BOTH BETAINE-ACCUMULATING AND NONACCUMULATING CEREAL PLANTS
ISHITANI, M, ARAKAWA, K, MIZUNO, K, KISHITANI, S, TAKABE, T
PLANT AND CELL PHYSIOLOGY, 34, 3, 493, 495, Japanese Society of Plant Physiologists, Apr. 1993, [Peer-reviewed]
English, Scientific journal, 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, Oct. 1992, [Peer-reviewed], [Lead author]
English, Scientific journal

LEVELS OF BETAINE AND BETAINE ALDEHYDE DEHYDROGENASE-ACTIVITY IN THE GREEN LEAVES, AND ETIOLATED LEAVES AND ROOTS OF BARLEY
ARAKAWA, K, KATAYAMA, M, TAKABE, T
PLANT AND CELL PHYSIOLOGY, 31, 6, 797, 803, Sep. 1990, [Peer-reviewed], [Lead author]
English, Scientific journal

Mechanisms of Salinity Tolerance in Plants.
ISHITANI Manabu, ARAKAWA Keita, TAKEBE Tetsuko
Chemical Regulation of Plants, 25, 149, 162, The Japanese Society for Chemical Regulation of Plants, 1990
Japanese, Scientific journal

Betaine accumulation and osmoregulation in higher plants and cyanobacteria　　　　　　　　　　　　　　　
Takabe T, Arakawa K
Plant water relations and growth under stress, 151, 158, 1989, [Last author]
English, In book

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, American Society of Plant Biologists (ASPB), 01 Dec. 1988, [Peer-reviewed]
English, Scientific journal

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, Oxford University Press, 1987, [Peer-reviewed], [Lead author]
English, Scientific journal

Freezing Resistance of Overwintering Plants
Keita Arakawa, Shingo Suzuki, Low Temperature Science, 83, 201, 210, Mar. 2025, [Peer-reviewed], [Lead author, Corresponding author]
Japanese, Introduction scientific journal, 49194462

Regional variation in spring root growth of Abies sachalinensis seedlings associated with provenance snow environments　　　　　　　　　　　　　　　
Tetsuto Sugai, Keita Arakawa, Wataru Ishizuka, International Society of Root Research 12th International Symposium, Jun. 2024
English, Summary international conference

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

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

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

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

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

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, Dec. 2019
北海道大学北方生物圏フィールド科学センター森林圏ステーション, Japanese

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, Oct. 2018
日本木材加工技術協会, Japanese

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, Jun. 2018
グリーンテクノバンク, Japanese

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

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

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

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, Nov. 2013

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

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, Nov. 2012
Japanese, Summary national conference

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, Nov. 2012
Japanese

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, Jun. 2012
English, Others

J024031 Inhibition of Ice Nucleation by Polyphenol Compounds
INADA Takaaki, KOYAMA Toshie, KUWABARA Chikako, ARAKAWA Keita, FUJIKAWA Seizo, The Proceedings of Mechanical Engineering Congress, Japan, 2012, 0, _J024031, 1-_J024031-3, 2012
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., The Japan Society of Mechanical Engineers, Japanese

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, Nov. 2011
Japanese

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, Nov. 2010
Japanese

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, Nov. 2010
Japanese

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, Nov. 2010
Japanese

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
Japanese

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, 20 Oct. 2009
Japanese

Study on the Proteins that Involve in Freezing Resistance of Xylem of Larch
Morimoto Kazunari, Mori Hitoshi, Ksuga Jun, Fujikawa Seizo, Arakawa Keita, Plant and Cell Physiology Supplement, 2009, 0, 512, 512, 2009
Deep supercooling (DSC) ability of the parenchyma cells of xylem (XPCs) is essential for freezing resistance of boreal woody plants in winter. DSC ability is induced during seasonal cold acclimation in XRCs with various physiological changes. In XRCs of larch (Larix kaempferi), changes in gene expression and sugar accumulation have been characterized in association with changes in DSC ability. However, roles of proteins on DSC ability are still unclear. Therefore, we examined to identify proteins that are related to the DSC ability. In parallel with the decrease in DSC ability of larch twigs of winter by artificial deacclimation, decrease in levels of sixteen winter-induced proteins were detected by two-dimensional electrophoresis. These proteins were excised from gel spots, digested with trypsin, and analyzed LC-MALDI MS/MS. Database search based on de novo sequences from MS/MS spectra revealed that some candidate proteins for increasing DSC ability were similar to several proteins., The Japanese Society of Plant Physiologists

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, 22 Oct. 2008
Japanese

Detection of Soluble Proteins That Are Responsive to the Changes in Deep Supercooling Ability in Xylem Parenchyma Cells of Larch (Larix kaempferi)
Morimoto Kazunari, Arakawa Keita, Fujikawa Seizo, Plant and Cell Physiology Supplement, 2008, 0, 874, 874, 2008
Parenchyma cells of xylem (XPCs) in larch (Larix kaempferi) adapt to subzero temperatures by deep supercooling (DSC). Recently, secondary metabolites possessing anti-ice nucleation activities and genes expressed in association with increased DSC ability have been characterized. However, role of soluble proteins on DSC mechanism is still unknown. In this study, detection of soluble proteins that are responsive to the changes in DSC ability in XPCs of larch was examined. DSC ability of XPCs in larch twigs was kept at the maximum level (LT₅₀: <-50^oC) in winter. After the artificial deacclimation of larch twigs of winter, DSC ability was promptly decreased to -34^oC within a week. In parallel with this, levels of many winter-inducible proteins were also decreased. N-terminal amino acid sequencing of the proteins that are responsive to the changes in DSC ability revealed that some of them are homologous to the late embryogenesis abundant proteins., The Japanese Society of Plant Physiologists

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, 22 Oct. 2007
Japanese

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, 22 Oct. 2007
Japanese

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, 22 Oct. 2007
Japanese

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, Feb. 2007
Japanese

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

Response Of Winter Wheat After Simulated Acid Snow Stress During A Re-growth Period
Inada Hidetoshi, Fujikawa Seizo, Arakawa Keita, Plant and Cell Physiology Supplement, 2007, 0, 479, 479, 2007
In the last decade, precipitation of snow containing acid pollutants (acid snow) has been observed. Wintering plants under acid snow layer are subjected to the exposure to the acid pollutants locally concentrated on snow crystal surface and the immersion or freeze-thaw in strong acid meltwater. In this study, we examined the response of winter wheat that were re-grown under light/dark condition after treatment of simulated acid snow (SAS) stress. When wheat seedlings were re-grown for 48 hours after SAS treatment with sulfuric acid solution (pH 2), fresh weight of mature leaves markedly decreased within 24 hours, but that of younger leaves hardly decreased. During the re-growth period, relative water content of mature leaves gradually decrease to 75%, although that of younger leaves decreased to 90% within 12 hours and was kept at this level. Damage of mature leaves by SAS treatment was expanded under the re-growth condition., The Japanese Society of Plant Physiologists

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

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
Japanese

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, 21 Oct. 2005
Japanese

Crystal structure of PR-5 protein from winter wheat
Kuwabara Chikako, Kondo Hidemasa, Noro Natsuko, Takezawa Daisuke, Arakawa Keita, Tsuda Sakae, Plant and Cell Physiology Supplement, 2005, 0, 848, 848, 2005
WAS-3 protein, a member of PR-5 family, accumulates in an apoplastic space of winter wheat in response to cold acclimation. We previously revealed that recombinant WAS-3 protein (rWAS-3) produced using high efficiency wheat expression system inhibited hyphal growth of pink snow mold and Fusarium oxysporum. Furthermore, rWAS-3 bound to fungal cell wall itself and β-1,3-glucan that was main components of fungal cell walls. In order to clarify the biochemical interaction of WAS-3 and β-1,3-glucan, we determined the crystal structure of rWAS-3 using X-ray crystallographic analysis. The thin plate-shaped crystals of rWAS-3 were obtained by hanging-drop vapor diffusion method. The structure of rWAS-3 was determined at 1.8 angstrom resolution by molecular replacement method. The electrostatic potential analysis revealed that rWAS-3 had an acidic cleft region. Therefore, we concluded that this region might be β-1,3-glucan binding site of rWAS-3., The Japanese Society of Plant Physiologists

Reduced tolerance to freezing and osmotic stress in Physcomitrella ABA-insensitive mutants
Minami Anzu, Nagao Manabu, Arakawa Keita, Fujikawa Seizo, Takezawa Daisuke, Plant and Cell Physiology Supplement, 2005, 0, 222, 222, 2005
Abscisic acid (ABA) is known to play an important role in responses to environmental stresses. We are investigating mechanisms of ABA-induced freezing tolerance in the moss Physcomitrella patens. ABA induced physiological changes such as expression of various genes, accumulation of boiling soluble proteins and increase in soluble sugar contents.
We isolated mutants with reduced ABA sensitivity by UV mutagenesis. These mutants grew normally in a medium containing ABA, whereas the wild type showed inhibited growth. Freezing and osmotic stress tolerance in these mutants was lower than that of wild type, but the extents of tolerance varied among the mutants. In these mutants, levels of expression of stress-related genes, a few boiling-soluble proteins and soluble sugars after ABA treatment were reduced as compared with those of wild-type. The degrees of freezing tolerance were correlated with levels of proteins and sugars in ABA-insensitive mutants., The Japanese Society of Plant Physiologists

Cryoprotective activity of winter- accumulating WAP27 protein from mulberry cortical parenchyma cells
Nagao Manabu, Ukaji Norifumi, Takezawa Daisuke, Arakawa Keita, Fujikawa Seizo, Plant and Cell Physiology Supplement, 2005, 0, 221, 221, 2005
Freezing tolerance of cortical parenchyma cells of mulberry (Morus bombycis) drastically increases in winter. 27-kDa group 3 late embryogenesis protein (WAP27) accumulated in ER of the cells in winter.
In natural conditions, plants are subjected to prolonged freezing that at high subzero temperature results in prolonged exposure of cells to concentrated solutions. We therefore examined cryoprotection of recombinant WAP27 (rWAP27) on lactate dehydrogenase (LDH) activity against prolonged freezing. The rWAP27 preserved above 80% of LDH activity during prolonged freezing at -2, -4, -10, -20^oC. The cryoprotective activity of bovine serum albumin was nearly equal to that of rWAP27 during prolonged freezing at -20^oC, but decreased with freezing time at -2 and -4^oC. The cryoprotective activity of ovalbumin was also decreased with freezing time at -2 to -20^oC. The rWAP27 has a high cryoprotective activity to freeze-labile proteins against prolonged freezing., The Japanese Society of Plant Physiologists

Study on the Mechanism of Freezing Injury under Acid Condition in Wintering Plant
Inada Hidetoshi, Ito Toshiaki, Nagao Manabu, Fujikawa Seizo, Arakawa keita, Plant and Cell Physiology Supplement, 2005, 0, 381, 381, 2005
We have already reported that the acidification to pH 2.0 with sulfuric acid solution in the freeze-thaw process promoted the freezing injury of leaves of cold-acclimated winter wheat (Triticum aestivum L. cv. Chihokukomugi). In this study, we examined a modified freezing test to estimate the influence of freeze-thaw cycles in the acid meltwater in early winter or early spring on the viability of wintering plants. When freeze-thaw cycles were repeated four times, survival rates of leaves treated with sulfuric acid solution gradually decreased as freeze-thaw was repeated. Further, we estimated the influence of acid condition in the freezing, thawing or post-thawing process on the viability of leaves of winter wheat. Survival rates of leaves decreased by existence of sulfuric acid solution in the thawing process. These results suggested that acid condition in the thawing process might become a factor to promote freezing injury of winter wheat., The Japanese Society of Plant Physiologists

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, 28 Oct. 2004
Japanese

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, 28 Oct. 2004
Japanese

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, 28 Oct. 2004
Japanese

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
Japanese, Lecture materials

Response of Wintering Plants to Acid Freeze-thawing
Inada Hidetoshi, Arakawa Keita, Plant and Cell Physiology Supplement, 2004, 0, 359, 359, 2004
It has been reported that the acid rain influenced the growth and metabolism of plants. However, the influence of acid snow on wintering plant has been hardly reported. In this study, the equilibrium-freezing test under acid condition (acid freezing test) was done to study the response of wintering plant to acid freezing stress, which may be caused by acid snow. When leaf segments of cold acclimated winter wheat (Triticum aestivum L. cv. Chihokukomugi) were applied to the acid freezing test, the survival rate after the acid freeze-thaw (pH 2.0) was markedly decreased as compared with that after the freeze-thaw in pure water (pH 5.6), a control experiment. On the other hand, survival rate after supercooling in the acid solution (pH 2.0) was hardly lowered. These results suggested that the acidic condition in a freeze-thaw process of winter wheat promoted the freezing injury., The Japanese Society of Plant Physiologists

Analysis of genes involved in acquisition of freezing tolerance in mosses
Minami Anzu, Nagao Manabu, Arakawa Keita, Fujikawa Seizo, Takezawa Daisuke, Plant and Cell Physiology Supplement, 2004, 0, 803, 803, 2004
Bryophytes live throughout the world from the tropics to Antarctica. Many mosses can grow in extreme environment such as lands with dry and cold seasons. The moss Physcomitrella patens acquires high freezing tolerance (FT) upon treatment with abscisic acid (ABA) and survives after cryopreservation. We demonstrated that ABA induced morphological changes in cellular organelles and accumulation of boiling-stable proteins and soluble sugars. The increase in FT was strongly inhibited by cycloheximide suggesting critical roles of synthesis of nuclear encoded protein in ABA-induced FT.
To identify genes involved in development of FT of P. patens, we carried out differential screening. Results of analysis of more than 60 isolated clones indicated that a large proportion of the ABA-induced genes had similarity to higher plant stress-induced genes such as those encoding LEA and aquaporin, whereas several other genes encoded proteins not conserved in higher plants such as LI818 and UVI-1., The Japanese Society of Plant Physiologists

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, 15 Oct. 2003
Japanese

Molecular genetic characterization of rcd1 mutant which is resistant to paraquat but sensitive to ozone
Fujibe Takahiro, Saji Hikaru, Arakawa Keita, Takeuchi Yuichi, Yamamoto Kotaro T., Plant and Cell Physiology Supplement, 2003, 0, 347, 347, 2003
Various stress treatments can cause excess active oxygen species (AOS) production. Since generation of AOS is involved in stress-induced injury, enhancement of AOS scavenging activities would confer resistance to stresses to plants. rcd1-2, which is allelic to rcd1-1 (Overmyer et al., 2000), is resistant to paraquat, UV-B and salt stresses because of the enhanced activities of the AOS scavenging enzymes in chloroplasts. On the other hand, rcd1 is more sensitive to ozone fumigation than wild type, and displays programmed cell death after ozone treatment. rcd1 appears to be a mutant that shows stress responses constitutively. Consequently, it accumulates AOS scavenging enzymes without any stresses, and displays programmed cell death even after removal of ozone. RCD1 gene should negatively regulate wide range of stress-related down-stream genes in an unstressed condition., The Japanese Society of Plant Physiologists

PR-10/Bet v 1 protein family localized in cytosol and nucleus
Ukaji Norifumi, Takezawa Daisuke, Arakawa Keita, Fujikawa Seizo, Plant and Cell Physiology Supplement, 2003, 0, 308, 308, 2003
During seasonal cold acclimation, 18 kD proteins, designated WAP18, were accumulated in the soluble protein fractions in mulberry (Morus bombycis Koidz.) tree. The purified WAP18 showed cryoprotective activity against freeze-labile lactate-dehydrogenase, LDH, suggesting that WAP18 has roles to contribute freezing tolerance in cortical parenchyma cells of mulberry tree in winter. The cDNA cloning revealed that WAP18 has high sequence similarity to pathogenesis-related (PR)-10/Bet v 1 protein family. Northern blot analysis showed that transcript levels of WAP18 increased not only by cold treatment at 4^oC but also by wounding, ethephon and salicylic acid.
It has been predicted that PR-10/Bet v 1 proteins localize in the cytosol based on the absence of apparent signal sequence. Immuno-electronmicroscpe analysis showed that gold particles against anti-WAP18 antibodies localized not only in cytosol but also in nucleus in mulberry tree. This result indicates that PR-10/Betv1 proteins localize in cytosol and nucleus., The Japanese Society of Plant Physiologists

Study Of The Extracellular Proteins Induced During Seasonal Cold Acclimation In Birch Xylem Tissue
Arakawa Keita, Kasuga Jun, Fujikawa Seizo, Plant and Cell Physiology Supplement, 2003, 0, 307, 307, 2003
Freezing resistance of birch (Betula platyphylla L.) xylem tissue is induced during seasonal cold acclimation with the development of supercooling ability. Both intracellular and extracellular components may contribute to the supercooling ability of winter xylem tissue. In this study, identification of winter-induced extracellular proteins (WCWPs) was examined to study the physiological roles of WCWPs in the freezing resistance mechanism of birch xylem tissue.
Extracellular proteins were eluted with acidic solution from xylem tissues of birch twigs harvested in the field. SDS-PAGE analysis showed that the extracellular protein composition changed drastically after seasonal cold acclimation. Levels of proteins with molecular masses of about 30 kDa, namely WCWPs, increased markedly in winter. The N-terminal amino acid sequencing revealed that some of WCWPs are similar to each other and are homologous to a pathogenesis-related protein. Immunoblot analysis also showed the similarity between the WCWPs., The Japanese Society of Plant Physiologists

Functional Analysis of ABA-and Cold-Responsive Protein Kinase in Physcomitrella patens
Minami Anzu, Nagao Manabu, Arakawa Keita, Fujikawa Seizo, Takezawa Daisuke, Plant and Cell Physiology Supplement, 2003, 0, 459, 459, 2003
Protein phosphorylation and dephosphorylation are implicated in the development of freezing tolerance during cold acclimation in plants. We previously showed that freezing tolarance of moss Physcomitrella patens protonema cells increased by treatment with ABA, cold. We isolated the PARK gene encoding protein Ser/Thr kinase induced by ABA and cold. The PARK kinase domain was similar to S-domain receptor like protein kinase, but it did not possess the extracellular domain.
The GST-PARK fusion protein phosphorylated histon IIIS and myelin basic protein and that the kinase itself underwent rapid autophosphorylation. When the PARK-GFP fusion gene was introduced into plant cells, the fluorescence signal was observed in the plasma membrane. Gene knock-out plants of PARK had reduced survival rates after freezing as compared to the wild type plants. These results indicate that PARK might regulate signal transduction processes of ABA- or cold-induced freezing tolerance., The Japanese Society of Plant Physiologists

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
English, Summary international conference

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
English, Summary international conference

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
Japanese Society of Plant Physiologists, English

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
Japanese Society of Plant Physiologists, English

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
Japanese Society of Plant Physiologists, English

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
Japanese Society of Plant Physiologists, English

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
Japanese Society of Plant Physiologists, English

Gene expression of WAP20 and WAP27 :
UKAJI Norifumi, TAKEZAWA Daisuke, ARAKAWA Keita, FUJIKAWA Seizo, Plant and cell physiology, 41, s48, 2000
Japanese Society of Plant Physiologists, English

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
Japanese Society of Plant Physiologists, English

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, Mar. 1999
English

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, Mar. 1999
English

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, Mar. 1999
English

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, May 1998
English

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, May 1998
English

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, Mar. 1997
English

CHANGES IN PROTEIN COMPOSITION DURING COLD ACCLIMATION IN ORCHARD GRASS
ARAKAWA Keita, YOSHIDA Shizuo, Plant and cell physiology, 38, s97, Mar. 1997
English

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, Mar. 1997
English

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, Oct. 1996
Japanese

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, Oct. 1996
Japanese

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, Mar. 1995
English

INACTIVATION OF VACUOLAR H^+-ATPase IN CUCUMBER COTYLEDONS CAUSED BY CHILLING TREATMENT
KAWAMURA Yukio, ARAKAWA Keita, YOSHIDA Shizuo, Plant and cell physiology, 36, S141, Mar. 1995
English

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, Mar. 1995
English

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, [Lead author, Corresponding author]
Japanese, Introduction other

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, Oct. 1992
English, Summary international conference

IMMUNOLOGICAL STUDIES OF BETAINE ALDEHYDE DEHYDROGENASE IN BARLEY　　　　　　　　　　　　　　　
ARAKAWA, K, MIZUNO, K, TAKABE, T, PHOTOSYNTHESIS RESEARCH, 34, 1, 217, 217, Oct. 1992
English, Summary international conference

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), 11.5 低温応答
共立出版, Nov. 2020, 9784320058125, xvi, 235p, 図版 [8] p, Japanese, [Contributor]

低温環境の科学事典
河村, 公隆, 樹木細胞の凍結挙動
朝倉書店, 29 Jul. 2016, 425416128X, 432, Japanese, [Contributor]

木質の構造
日本木材学会, 第３章３ 環境ストレスと形成層活動
文永堂出版, Apr. 2011, 9784830041204, xii, 313p, Japanese, [Contributor]

Function of the apoplast of trees in winter
Grants-in-Aid for Scientific Research
01 Apr. 2024 - 31 Mar. 2027
荒川 圭太, 鈴木 伸吾
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido University, 24K09012

Roles of Cell Walls of Trees at Subzero Temperatures
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
01 Apr. 2021 - 31 Mar. 2024
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。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido University, 21K05699

Control of freezing of water at subzero temperatures in woody plant cells
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
01 Apr. 2015 - 31 Mar. 2018
Arakawa Keita
It is well known that existence of the ice nucleation substances (ice nucleators) such as silver iodide and ice nucleation bacteria promote freezing of supercooled water. Because these substances catalyze freezing of water, ice nucleation activity (INA) increases freezing temperature of the solution. On the other hand, recent studies showed that some polyphenols repressed INA of ice nucleators and depressed freezing temperature of the solution containing ice nucleators. Existence of these polyphenols (anti-INA polyphenols) maintained supercooling state of the solution. For study on the anti-INA process by anti-INA polyphenols, His-inaA was highly purified from the soluble fraction prepared from the crude extracts of Escherichia coli cells overexpressing His-inaA and soluble form of His-inaA was characterized. INA was detected in the soluble form of His-inaA and repressed by addition of some anti-INA polyphenols.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 15H04615

New strategy for prevention of frost damage to plants
Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
01 Apr. 2015 - 31 Mar. 2017
FUKUSHI YUKIHARU
The crude extracts of plant leaves (65 species) were examined in this research revealed that they showed various levels of anti-ice nucleation activity (supercooling activity). In particular, the leaf extracts of Picea glehnii, Brassica juncea (red mustard), Sasa senanensis, Salix bakko, Laurus nobilis, Citrus sudachi, Perilla frutescens, Camellia sinensis, and Taraxacum officinale showed relatively high anti-ice nucleation activity by droplet freezing assays. Anti-ice nucleation effects were clearly shown on leaves by using our new leaf-disc freezing assay system. Application of plant extracts in a tea plantation is in consultation.
Japan Society for the Promotion of Science, Grant-in-Aid for Challenging Exploratory Research, Hokkaido University, 15K14667

Study on overwintering mechanism of dormant buds of trees for sustainable production of woody biomass
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
2011 - 2013
ARAKAWA Keita
In this study, extraorgan-freezing and endodormancy release of larch dormant buds were studied for understanding overwintering mechanism of dormant buds of trees.
Cryo-scanning electron microscopic study revealed that primordial cells were partially dehydrated at subzero temperatures and water remaining in primordial cells was kept in the deep supercooling state. In this tissue, accumulation of several carbohydrates such as pinitol and sucrose were detected as major components. However, it is difficult to explain that high freezing tolerance by deep supercooling capability of primordial tissue is determined by accumulation of major carbohydrates alone. Possible contribution of minor components such as supercooling-facilitating substances might be studied in larch dormant buds. In addition, proteomic analysis by using LC-MS/MS showed that only minor changes in soluble protein composition occurred in larch dormant buds within three weeks during endodormancy release.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido University, Principal investigator, Competitive research funding, 23580453

Production of transgenic trees that are tolerate to environmental stresses for supply of forest tree resources in the future
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
2008 - 2010
ARAKAWA Keita
In this study, winter-induced galactinol synthase gene in xylem of Larix kaempferi driven by cauliflower mosaic virus 35S promoter were introduced into hybrid aspen (Populus tremula×P. alba) to examine the enhancement of freezing stress tolerance. In transgenic hybrid aspen, levels of raffinose in green leaf, bark and xylem tissues were increased as compared with those of wild type, but freezing tolerance was not increased in these tissues. However, freezing tolerance of leaves after cold acclimation was only increased slightly with increase in levels of raffinose content.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido University, Principal investigator, Competitive research funding, 20580360

Comprehensive analysis to determine chemical structures in supercoolingpromoting substances from xylem parenchyma cells of trees and considerations to obtain much volume of supercooling-promoting substances for their applications
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
2008 - 2010
FUJIKAWA Seizo, FUKUSHI Yukiharu, ARAKAWA Keita
From xylem parenchyma cells in trees that can supercool to -40C during winter, we identified a several kinds of flavonol glycosides and hydrolysable tannins. Based on such results, we could also identified more than 50 kinds of flavonol and tannin-related compounds that promoted supercooling of water. We also tried to use these supercooling-promoting substances as novel materials that inhibit freezing of water.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, Coinvestigator not use grants, Competitive research funding, 20380099

Field trial of transgenic poplars for industrial use
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
2007 - 2010
HAYASHI Takahisa
The transgenic expression of Aspergillus xyloglucanase cDNA(AaXEG2) with 35S promoter in the leaves of open-field grown poplars was studied. This level was slightly decreased in the poplars grown in fertile soil for four years. Xyloglucan content was greatly decreased in the walls of the tissues in the transgenic lines, although the degradation levels of xyloglucan were slightly lower for the poplars grown in fertile soil than for those grown in non-fertile soil. The leaves exhibited a smaller surface area with more rounded teeth than did those of the wild type plants, as if they were of the sun leaf variety that was grown in the incubation room and subsequently greenhoused. Large numbers of proteins were markedly downregulated in the leaves of the transgenic plants via proteomic analysis. Significant reduction occurred not only on the moduli of elasticity and rupture in both their live stems and dried trunks compared with those in the wild type.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (A), 19208016

Study on identification and application of supercooling substances from xylem parenchyma cells of trees
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
2005 - 2007
FUJIKAWA Seizo, ARAKAWA Keita
Xylem parenchyma cells of trees have an unique adaptation mechanism to subzero temperatures which is called as deep supercooling. In order to clarify mechanism of deep supercooling, supercooling capability of xylem crude extracts from several trees that contained deep supercooling xylem parenchyma cells, was measured by droplet freezing assay. The results showed that crude xylem extracts from these trees had anti-ice nucleation activity, which promotes supercooling of water droplets. Among tree species examined, xylem crude extracts from Katsura-tree showed highest anti-ice nucleation activity. Therefore, the causative substances for the anti-ice nucleation activity were analyzed by using Katsura-tree. As a result of purification by liquid-liquid extraction, silica gelcolumn chromatography and HPLC, existence of diverse kinds of numerous anti-ice nucleation substances in xylem of Katsura-tree was suggested. As a part of them, we could identify 4 kinds of flavonol glycosides with high anti-ice nucleation activity. These flavonol glycosides were quercetin-3-0-glucoside, kaempferol-7-0-glucoside, 8-methoxykaempferol-3-0-glucoside and kaempferol-3-0-glucoside with anti-ice nucleation activity of 2.8C, 9.0C, 3.4C and 4.0C, respectively. Among already-known anti-ice nucleation substances, anti-ice nucleation activity of kaempferol-7-0-glucoside was highest. This is first discovery that flavonol glucosides have anti ice nucleation activity. The accumulation of flavonoids in cytoplasm of XPCs was also comfirmed by fluorescence microscopy. These results strongly indicated that anti-ice nucleation substances had important role to supercooling of XPCs.
We confirmed that these anti-ice nucleation flavonoids could facilitate supercooling of 250cc water at-7.5C for 1 week, and applied to a abroad patent as "supercooling facilitating substances". Based upon such supercooling capability, these substances were applied for cold preservation of animal organs with successful I result for long time preservation of the function at supercooling state. We also used these substances to reduce concentration of vitrification solutions (VS2) for successful cryopreservation of biological tissues. Due to lowering concentration of VS2, successful preservation was achieved. We are currently considering application of these substances to wide variety of technologies that use "unfrozen water".
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, Coinvestigator not use grants, Competitive research funding, 17380101

Study on the responses of wintering plants to stresses caused by acid snow
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
2004 - 2005
ARAKAWA Keita
Acid precipitation (acid rain or acid mist) is an environmental problem that has affected ecosystems and agriculture. Acid snow might be also 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 (Triticum aestivum L.). 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 (pH 2.0) decreased markedly. Leaf samples after supercooling to -4 and -8℃ 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℃ for 7 days 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℃ for 7 days with sulfuric acid (pH 2.0). The results suggest that an acid condition in the process of extracellular freezing and/or thawing promotes freezing injury of wheat leaves, because of the combined effects of acid substances concentrated in snow crystals and freeze-thaws in an acid solution concentrated by freezing. For further characterization of the responses of wintering plants to acid-snow stress, studies on the mechanism of injury caused by acid-snow stress are in progres.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido University, Principal investigator, Competitive research funding, 16580270

Molecular study on usage of unused available resources from northern trees
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
2002 - 2004
FUJIKAWA Seizo, FUNADA Ryo, SANO Yuzou, ARAKAWA Keita, TAKEZAWA Daisuke
We studied freezing adaptation mechanisms of northern trees including the cortical and xylem parenchyma cells at molecular level.
Cortical parenchyma cells of the northern trees adapt to subfreezing temperatures by extracellular freezing. These cells can adapt freezing even to liquid nitrogen temperature by obtaining high tolerance to freezing-induced dehydration. We identified that group 3 LEA proteins were specifically accumulated in ER of the cortical parenchyma cells of mulberry during winter. We named this protein as WAP27 and examined the function both in planta and in vitro. Transgenic Arabidopsis plants introduced WAP27 obtained higher freezing tolerance after cold acclimation as compared with wild type plants. Furthermore, purified WAP27 had cryoprotective activity to freezing sensitive proteins, such as LDH.
Xylem parenchyma cells of trees, on the other hand, adapt to subfreezing temperatures by deep supercooling. The xylem parenchyma cells from northern trees can supercool -60C. In order to examine mechanisms that water keep supercooling in xylem parenchyma cells, we analyzed gene expression, accumulation of proteins and soluble sugars. With comprehensive gene analysis about xylem of Larch, we could find several genes that were never reported as cold-induced genes in plants. We confirmed by northern blot analysis hat these genes were expressed in parallel with increased supercooling ability. On protein analysis using beech, we could identified a few tens protein-spots that were specifically accumulated in parallel with increased supercooling ability. Among these proteins, we purified chitinase-like proteins, which are known as one of antifreezing proteins in plants, and antifreeze activity was examined with negative result. We also analyzed soluble proteins in xylem of birch. However, the accumulation was same with the cortical parenchyma cells that adapt to subfreezing temperature by extracellular freezing. Finally, we obtained crude extracts from xylem (parenchyma cells) and the effect to supercooling of water was examined. We could find that the crude extracts from xylem that exhibited high supercooling in the parenchyma cells provided high ability of supercooling to water. Now, we are continuing identifications of substances within crude extracts that may provide high supercooling.
Our results showed that winter-induced genes and gene products from northern trees that exhibit extraordinary high freezing resistance may be useful resources for developing crops with high freezing resistance as well as for the use of them for freezing regulate substances in wide range of industries.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, Coinvestigator not use grants, Competitive research funding, 14360093

樹木木部柔細胞における深過冷却機構の解析　　　　　　　　　　　　　　　
2003
Competitive research funding

Study on the mechanism of deep supercooling in xylem cells of woody plants　　　　　　　　　　　　　　　
2003
Competitive research funding

BASIC RESARCH FOR IMPROVEMENT OF COLD AND DESEASE RESISTANCE OF PERRENIAL WEED PLANTS BY GENETIC ENGENEERING
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
2000 - 2002
TAKEZAWA Daisuke, ARAKAWA Keita, OZAWA Shuji
The goal of this research was to generate weeds that has increased tolerance to freezing and disease by genetic engineering. Experiments were focused on (1) isolation of genes that can confer plants with tolerance to freezing and disease, and (2) production of transgenic alfalfa plants by using the isolated genes.
As a candidate gene for production of transgenics, WAS-3 gene, which has similarity to antifreeze proteins and antifungal proteins was isolated from winter wheat that has high degree of winter-stress resistance. The WAS-3 protein produced in transgenic wheat cells efficiently blocked growth of hyphae of the snow mold, Microdochium nivale. In addition, transgenic plants expressing the WAS-3 protein exhibited a better resistance to Fusarium oxysporum than control non-transgenic plants. We also isolated genes encoding dehydrins and group III LEA from mulberry, which exhibits extremely high freezing tolerance in winter, and several ABA and cold-responsive genes from the moss Physcomitrella patens.
For production of transgenic alfalfa plants, we used Arabidopsis CBF3, a gene encoding a transcription factor that plays a critical role in the up-regulation of a number of cold-inducible genes. Agrobacterium tumefaciens carrying a construct for overexpression of the CBF3 gene was used for inoculation of leaf discs of alfalfa (Medicago sativa var. Regen-SY). Kanamycin-resistant calli were selected and shoots were regenerated by further cultivation. Nine transgenic plants were successfully regenerated. These transgenic plants showed normal growth indistinguishable from non-transgenics, suggesting they have less somatic mutations. These transgenic plants were being used for the freezing tests.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), HOKKAIDO UNIVERSITY, Coinvestigator not use grants, Competitive research funding, 12556044

越冬性植物の酸性雪ストレス耐性機構　　　　　　　　　　　　　　　
2002
Competitive research funding

Change of Cell Wall Properties in Woody Plant Cells by Low Temperature
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
1999 - 2001
FUJIKAWA Seizo, FUNADA Ryo, TAKEZAWA Daisuke, ARAKAWA Keita, SANO Yuzou
This study concerned to cold temperature adaptation of woody plant cells with the relation to the cell wall properties. This study provided a new evidence by cryo-scanning electron microscopy that xylem parenchyma cells of all hardwood species with thick and rigid cell walls, including boreal hardwood species, responded to subfreezing temperatures by deep supercooling. The mechanism of deep supercooling in xylem parenchyma cells has been studied in previous works and hypothesized to be due to the smaller size of micro-capillaries in the cell walls. However, this study confirmed that there was no distinct change in capillary size of the cell walls between cells exhibiting extracellular freezing arid deep supercooling. This study provided a new evidence that degree of supercooling is distinctly reduced by extraction of soluble substances in xylem cell protoplasts, suggesting the accumulation of antifreeze substances, which might drastically promote deep supercooling. We are currently analyzing candidates of antifreeze proteins and cloning their genes. In addition to these studies, this study also concerned to freezing adaptation mechanisms of cortical parenchyma cells of woody plants, which had comparatively thin soft cell walls and adapted to subfreezing temperature by extracellular freezing. All these results revealed specific physiological response of woody plant cells to environmental stresses as well as suggested the possibility of industrial application of stress-induced proteins from woody plant cells.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, Coinvestigator not use grants, Competitive research funding, 11460075

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
Grants-in-Aid for Scientific Research Grant-in-Aid for Encouragement of Young Scientists (A)
1995 - 1995
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。
Japan Society for the Promotion of Science, Grant-in-Aid for Encouragement of Young Scientists (A), Hokkaido University, Principal investigator, Competitive research funding, 07740608

An existence of cold stable vacuolar H^+-ATPase in Plants and its structural properties.
Grants-in-Aid for Scientific Research Grant-in-Aid for General Scientific Research (B)
1993 - 1994
YOSHIDA Shizuo, ARAKAWA Keita
The cold stability of vacuolar H^+-ATPase in vivo is markedly different between mung bean (Vigna radiata L.) and pea (Pisum sativum L.), the plants which are representatives of cold-sensitive and cold-tolerant species, respectively. Upon cold exposure of mung bean seedlings the H^+-ATPase activity declined significantly soon after the start of the cold treatment. In pea seedlings, by contrast, the H^+-ATPase remained highly active for long periods of the cold treatment. On cold incubation of isolated vacuolar membranes in vitro in the presence of MgATP and chaotropic anions such as C1^-, NO^-_2 and NO^-_3, the vacuolar H^+-ATPases exhibited also a marked difference in the cold-induced destabilization between these plants, especially in the sensitivity to the concentration of the anions. The enzyme from mung bean appeared to be more susceptible to the anions, particularly to NO^-_2, than the enzyme from pea, resulting in more severe cold-inactivation at the lower concentrations. The MgATP-dependent cold inactivation of the H^+-ATPase was closely associated with a release of the peripheral sectors from the membranes in the same way as in vacuolar-type H^+-ATPases from wide range of organisms. Thus, the cold stability of the enzyme complex both in vivo and in vitro is considered to be dependent on the intrinsic properties of the enzyme, suggesting the existence of "cold-labile" and "cold-stable" types in plants. SDS-PAGE and immunoblot analysis of the subunit components of the H^+-ATPase from both plants revealed that the molecular structures, especially the epitopes of the membrane sectors (16kD), differ each other, albeit that the molecular masses of other minor subunits are also slightly different. These findings in the present study are considered to provide a new insight into the mechanism to control the cold stability of vacuolar H^+-ATPase in plants.
Japan Society for the Promotion of Science, Grant-in-Aid for General Scientific Research (B), The Institute of Low Temperature Science Hokkaido University, Coinvestigator not use grants, Competitive research funding, 05454012

木本ならびに草本植物の寒冷適応機構の研究　　　　　　　　　　　　　　　
1992
Competitive research funding

Study on plant cold hardiness　　　　　　　　　　　　　　　
1992
Competitive research funding

排気浄化システム　　　　　　　　　　　　　　　
Patent right, 布施卓哉, 藤川清三, 荒川圭太, 宇梶慎子
特願2016-026154, 15 Feb. 2016
特許6655414
05 Feb. 2020, 株式会社デンソー; 国立大学法人北海道大学

不凍性液体　　　　　　　　　　　　　　　
Patent right, 藤川清三, 荒川圭太, 宇梶慎子, 福士幸治, 浦木康光, 寺内隆二, 高岡尚生, 栃木弘
特願2011-169821, 03 Aug. 2011
特許6319932, 13 Apr. 2018
国立大学法人北海道大学; コスモ石油ルブリカンツ株式会社

Supercooling promoting agents　　　　　　　　　　　　　　　
Patent right
WO2008/007684