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Last Updated :2024/07/25

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Master

Affiliation (Master)

  • Research Faculty of Agriculture　Fundamental AgriScience Research　Forest Science

Affiliation (Master)

  • Research Faculty of Agriculture　Fundamental AgriScience Research　Forest Science

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Profile and Settings

Degree

  • Dr (Agr.)（Nagoya Univ.）

Profile and Settings

  • Name (Japanese)

    Arakawa

  • Name (Kana)

    Keita

  • Name

    200901069220147094

Alternate Names

Achievement

Research Interests

  • 環境適応   寒冷環境   樹木生理学   

Research Areas

  • Environmental science/Agricultural science / Landscape science
  • Environmental science/Agricultural science / Environmental agriculture
  • Life sciences / Wood science

Published Papers

  • 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) 1340-7902 2023
  • Y. Jitsuyama, Y. Kita, K. Arakawa, T. Suzuki
    VITIS 61 (4) 125 - 132 0042-7500 2022 
    Hokkaido in northern Japan is a typical subpolar region and wine production using traditional cultivars can be done recently. However, there is an issue that the grapevines have short life spans in snowy central Hokkaido. This study investigates the effect of snow it-self and its interaction with vineyard slope and grape-vine stiffness on physical stress. In comparison of the flatness of the vineyard, the distortion of the basal trunk in the slope zone was greater than in the flat zones in the snowy mid-season. In comparison between cultivars, the distortion of the basal trunk in the flexible 'Kerner' was greater than in rigid 'Gewurztraminer', even in the flat zones. The magnitude of the distortion coincides with the mortality of grapevines. Although the chang-es in distortion showed a transition in just two winter durations, these results suggested a possibility that the snow cover might be linked to one of the physical dam-ages of grapevines in vineyards in heavy snowy regions.
  • Reiko Horiuchi, Keita Arakawa, Jun Kasuga, Takashi Suzuki, Yutaka Jitsuyama
    Cryobiology 101 44 - 51 0011-2240 2021/08 [Refereed]
  • Cryo-plate法を用いた交雑ポプラ培養体の超低温保存技術の開発
    田中大介, 佐久間義範, 安井雅範, 川村浩平, 荒川圭太
    低温生物工学会誌 67 (1) 47 - 51 2021/04 [Refereed][Not invited]
  • 古賀 泰雅, 鉄穴口 晃, 鈴木 伸吾, 重冨 顕吾, 荒川 圭太
    Cryobiology and Cryotechnology 低温生物工学会 67 (2) 141 - 145 1340-7902 2021 [Refereed]
  • Jun Kasuga, Yusuke Tsumura, Daisuke Kondoh, Yukata Jitsuyama, Reiko Horiuchi, Keita Arakawa
    Journal of Plant Physiology 253 153248 - 153248 0176-1617 2020/08 [Refereed][Not invited]
  • Seizo Fujikawa, Chikako Kuwabara, Jun Kasuga, Keita Arakawa
    Advances in Experimental Medicine and Biology 1081 289 - 320 0065-2598 2018 [Refereed][Not invited]
     
    © Springer Nature Singapore Pte Ltd. 2018. Studies on supercooling-promoting substances (SCPSs) are reviewed introducing name of chemicals, experimental conditions and the supercooling capability (SCC) in all, so far recognized, reported SCPSs and results of our original study are presented in order to totally show the functional properties of SCPSs which are known in the present state. Many kinds of substances have been identified as SCPSs that promote supercooling of aqueous solutions in a non-colligative manner by reducing the ice nucleation capability (INC) of ice nucleators (INs). The SCC as revealed by reduction of freezing temperature (°C) by SCPSs differs greatly depending on the INs. While no single SCPS that affects homogeneous ice nucleation to reduce ice nucleation point has been found, many SCPSs have been found to reduce freezing temperatures by heterogeneous ice nucleation with a large fluctuation of SCC depending on the kind of heterogeneous IN. Not only SCPSs increase the degree of SCC (°C), but also some SCPSs have additional SCC to stabilize a supercooling state for a long term to stabilize supercooling against strong mechanical disturbance and to reduce sublimation of ice crystals. The mechanisms underlying the diverse functions of SCPSs remain to be determined in future studies.
  • Keita Arakawa, Jun Kasuga, Naoki Takata
    Advances in Experimental Medicine and Biology 1081 129 - 147 0065-2598 2018 [Refereed][Not invited]
     
    © Springer Nature Singapore Pte Ltd. 2018. Boreal trees possess very high freezing resistance, which is induced by short-day length and low temperatures, in order to survive severe subzero temperatures in winter. During autumn, cooperation of photoreceptors and circadian clock system perceiving photoperiod shortening results in growth cessation, dormancy development, and first induction of freezing resistance. The freezing resistance is further enhanced by subsequent low temperature during seasonal cold acclimation with concomitant changes in various morphological and physiological features including accumulation of sugars and late embryogenesis abundant proteins. The mechanism of adaptation to freezing temperatures differs depending on the type of tissue in boreal trees. For example, bark, cambium, and leaf cells tolerate freezing-induced dehydration by extracellular freezing, whereas xylem parenchyma cells avoid intracellular freezing by deep supercooling. In addition, dormant buds in some trees respond by extraorgan freezing. Boreal trees have evolved overwintering mechanisms such as dormancy and high freezing resistance in order to survive freezing temperatures in winter.
  • Shingo Suzuki, Satoshi Fukuda, Yukiharu Fukushi, Keita Arakawa
    Bioscience, biotechnology, and biochemistry 81 (11) 2090 - 2097 0916-8451 2017/11 [Refereed][Not invited]
     
    Previous studies have shown that some polyphenols have anti-ice nucleation activity (anti-INA) against ice-nucleating bacteria that contribute to frost damage. In the present study, leaf disk freezing assay, a test of in vitro application to plant leaves, was performed for the screening of anti-INA, which inhibits the ice nucleation activity of an ice-nucleating bacterium Erwinia ananas in water droplets on the leaf surfaces. The application of polyphenols with anti-INA, kaempferol 7-O-β-glucoside and (-)-epigallocatechin gallate, to the leaf disk freezing assay by cooling at -4--6 °C for 3 h, revealed that both the compounds showed anti-INAs against E. ananas in water droplets on the leaf surfaces. Further, this assay also revealed that the extracts of five plant leaves showed high anti-INA against E. ananas in water droplets on leaf surfaces, indicating that they are the candidate resources to protect crops from frost damage.
  • 荒川 圭太
    冷凍 = Refrigeration 日本冷凍空調学会 92 (1078) 547 - 553 0034-3714 2017/08 [Not refereed][Not invited]
  • Takaaki Inada, Toshie Koyama, Hiroyuki Tomita, Takuya Fuse, Chikako Kuwabara, Keita Arakawa, Seizo Fujikawa
    JOURNAL OF PHYSICAL CHEMISTRY B 121 (27) 6580 - 6587 1520-6106 2017/07 [Refereed][Not invited]
     
    Various water-soluble substances are known as anti-ice nucleating agents (anti-INAs), which inhibit heterogeneous ice nucleation initiated by ice nucleating agents (INAs). Among them, several surfactants are reportedly effective as anti-INAs especially against silver iodide (AgI), which is a typical inorganic INA that induces heterogeneous ice nucleation at relatively high temperatures. In this study, the anti-ice nucleating activities of seven surfactants were examined in emulsified surfactant solutions containing AgI particles. Among previously reported anti-INAs (e.g., antifreeze proteins (AFPs), polyphenol compounds and synthetic polymers), a cationic surfactant used in this study, hexadecyltrimethylammonium bromide (C16TAB), showed the highest anti-ice nucleating activity against AgI. Based on the unique concentration-dependent dispersibility of AgI particles in C16TAB solution, anti-ice nucleating activity of C16TAB must be caused by the adsorption of C16TAB molecules on AgI surfaces either as a monolayer or a bilayer depending on the C16TAB concentration.
  • IZUHARA Nobuhiro, KUWABARA Chikako, SUZUKI Shingo, ODAKA Yuko, FUJIKAWA Seizo, ARAKAWA Keita
    Cryobiology and Cryotechnology 低温生物工学会 63 (2) 155 - 160 1340-7902 2017 [Refereed][Not invited]
     
    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.
  • SUZUKI Shingo, FUKUDA Satoshi, FUKUSHI Yukiharu, ARAKAWA Keita
    Cryobiology and Cryotechnology 低温生物工学会 63 (1) 33 - 36 1340-7902 2017 [Refereed][Not invited]
     
    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.
  • KAWAMURA Kohei, KAMI Daisuke, SUZUKI Shingo, TANAKA Daisuke, FUJIKAWA Seizo, ARAKAWA Keita
    Cryobiology and Cryotechnology 低温生物工学会 62 (2) 123 - 126 1340-7902 2016 [Refereed][Not invited]
     
    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%.
  • KUWABARA Chikako, SUZUKI Shingo, FUJIKAWA Seizo, ARAKAWA Keita
    Cryobiology and Cryotechnology 低温生物工学会 61 (2) 131 - 134 1340-7902 2015 [Refereed][Not invited]
     
    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.
  • Toshie Koyama, Takaaki Inada, Chikako Kuwabara, Keita Arakawa, Seizo Fujikawa
    CRYOBIOLOGY 69 (2) 223 - 228 0011-2240 2014/10 [Refereed][Not invited]
     
    Freeze-avoiding organisms survive sub-zero temperatures without freezing in several ways, such as removal of ice nucleating agents (INAs), production of polyols, and dehydration. Another way is production of anti-ice nucleating agents (anti-INAs), such as has been reported for several antifreeze proteins (AFPs) and polyphenols, that inhibit ice nucleation by inactivating INAs. In this study, the anti-ice nucleating activity of five polyphenol compounds, including flavonoid and tannin compounds of both biological and synthetic origin, against silver iodide (AgI) was examined by measuring the ice nucleation temperature in emulsified polyphenol solutions containing AgI particles. The emulsified solutions eliminated the influence of contamination by unidentified INAs, thus enabling examination of the anti-ice nucleating activity of the polyphenols against AgI alone. Results showed that all five polyphenol compounds used here have anti-ice nucleating activities that are unique compared with other known anti-INAs, such as fish AFPs (type land III) and synthetic polymers (poly(vinyl alcohol), poly(vinylpyrrolidone) and poly(ethylene glycol)). All five polyphenols completely inactivated the ice nucleating activity of AgI even at relatively low temperatures, and the first ice nucleation event was observed at temperatures between -14.1 and 19.4 degrees C, compared with between -8.6 and 11.8 degrees C for the fish AFPs and three synthetic polymers. These anti-ice nucleating activities of the polyphenols at such low temperatures are promising properties for practical applications where freezing should be prevented. (C) 2014 Elsevier Inc. All rights reserved.
  • Keita Endoh, Chikako Kuwabara, Keita Arakawa, Seizo Fujikawa
    ENVIRONMENTAL AND EXPERIMENTAL BOTANY 106 52 - 59 0098-8472 2014/10 [Refereed][Not invited]
     
    Dormant buds of larch (Larix kaempferi) exhibit extraorgan freezing, in which extracellular ice crystals are formed in localized areas far from the primordia, as an adaptive response to freezing temperatures. In order to better understand why dormant buds have evolved such a complicated mechanism, the freezing behavior of isolated primordia from dormant buds of larch were examined using cryo-scanning electron microscopy (cryo-SEM) and by freeze-fracture replica electron microscopy (FR-EM). When isolated primordia were slowly frozen (5 degrees C/day), extracellular ice crystals developed within primordia. Thus, isolated primordia serves as a useful model to examine the freezing response of primordia in which extracellular freezing occurs. Slow freezing (5 degrees C/day) of isolated primordia resulted in a much lower survival rate (25% at -30 degrees C) than in intact buds (100% at -30 degrees C). In whole buds, exhibiting extraorgan freezing, cryo-SEM observations indicated that slow freezing to -30 degrees C resulted in deep supercooling with a slight shrinkage of all primordial cells, whereas in isolated primordia, slow freezing to -30 degrees C resulted in intracellular freezing of about half of the cells with other cells exhibiting extracellular freezing with significant shrinkage and deformation. FR-EM of whole buds subjected to slow freezing revealed the formation of intramembrane particle (IMP)-free areas with long rectangular shapes in the plasma membrane of many primordial cells at temperatures lower than -15 degrees C and in a small number of cells in isolated primordia. In isolated primordia, the number of cells exhibiting intracellular freezing, which in most cases did not reveal membrane fracture faces, corresponded to the numbers of cells exhibiting IMP-free areas in whole buds. We speculate that the formation of IMP-free areas in plasma membranes may induce intracellular freezing when extracellular ice crystals have developed within primordial tissues. FR-EM also revealed the formation of fracture-jump lesions only in the plasma membranes of primordial cells of slowly-frozen, isolated primordia. We propose that such fracture-jump lesions are produced not only by osmotic dehydration but also by the deformation of cells due to the presence of extracellular ice crystals within primordial tissues in isolated primordia. The results indicate that the plasma membrane of primordial cells in ecodormant buds of larch are subject to a phase transition that may give rise to intracellular freezing, as well as increased sensitivity to dehydration, when extracellular ice crystals form within primordial tissues. We propose that extraorgan freezing represents a mechanism that prevents the formation of extracellular ice crystals within primordial tissues, thus lowering the risk of intracellular freezing and dehydrative stress. (C) 2014 Elsevier B.V. All rights reserved.
  • Chikako Kuwabara, Ryuji Terauchi, Hiroshi Tochigi, Hisao Takaoka, Keita Arakawa, Seizo Fujikawa
    CRYOBIOLOGY 69 (1) 10 - 16 0011-2240 2014/08 [Refereed][Not invited]
     
    Supercooling-promoting activities (SCAs) of 25 kinds of surfactants including non-ionic, anionic, cationic and amphoteric types were examined in solutions (buffered Milli-Q water, BMQW) containing the ice nucleation bacterium (INB) Erwinia ananas, silver iodide (AgI) or BMQW alone, which unintentionally contained unidentified ice nucleators, by a droplet freezing assay. Most of the surfactants exhibited SCA in solutions containing AgI but not in solutions containing the INB E. ananas or BMQW alone. SCAs of many surfactants in solutions containing AgI were very high compared with those of previously reported supercooling-promoting substances. Cationic surfactants, hexadecyltrimethylammonium bromide (C16TAB) and hexadecyltrimethylammonium chloride (C16TAC), at concentrations of 0.01% (w/v) exhibited SCA of 11.8 degrees C, which is the highest SCA so far reported. These surfactants also showed high SCAs at very low concentrations in solutions containing AgI. C16TAB exhibited SCA of 5.7 degrees C at a concentration of 0.0005% (w/v). (C) 2014 Elsevier Inc. All rights reserved.
  • SUZUKI Shingo, TAKAHASHI Daisuke, KUWABARA Chikako, UEMURA Matsuo, ARAKAWA Keita
    Cryobiology and Cryotechnology 低温生物工学会 60 (1) 59 - 63 1340-7902 2014 [Refereed][Not invited]
     
    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.
  • Chikako Kuwabara, Donghui Wang, Keita Endoh, Yukiharu Fukushi, Keita Arakawa, Seizo Fujikawa
    CRYOBIOLOGY 67 (1) 40 - 49 0011-2240 2013/08 [Refereed][Not invited]
     
    Based on the discovery of novel supercooling-promoting hydrolyzable gallotannins from deep supercooling xylem parenchyma cells (XPCs) in Katsura tree (see Wang et al. (2012) [38]), supercooling capability of a wide variety of tannin-related polyphenols (TRPs) was examined in order to find more effective supercooling-promoting substances for their applications. The TRPs examined were single compounds including six kinds of hydrolyzable tannins, 11 kinds of catechin derivatives, two kinds of structural analogs of catechin and six kinds of phenolcarboxylic acid derivatives, 11 kinds of polyphenol mixtures and five kinds of crude plant tannin extracts. The effects of these TRPs on freezing were examined by droplet freezing assays using various solutions containing different kinds of identified ice nucleators such as the ice nucleation bacterium (INB) Erwinia ananas, the INB Xanthomonas campestris, silver iodide and phloroglucinol as well as a solution containing only unintentionally included unidentified airborne ice nucleators. Among the 41 kinds of TRPs examined, all of the hydrolyzable tannins, catechin derivatives, polyphenol mixtures and crude plant tannin extracts as well as a few structural analogs of catechin and phenolcarboxylic acid derivatives exhibited supercooling-promoting activity (SCA) with significant differences (p > 0.05) from at least one of the solutions containing different kinds of ice nucleators. It should be noted that there were no TRPs exhibiting ice nucleation-enhancing activity (INA) in all solutions containing identified ice nucleators, whereas there were many TRPs exhibiting INA with significant differences in solutions containing unidentified ice nucleators alone. An emulsion freezing assay confirmed that these TRPs did not essentially affect homogeneous ice nucleation temperatures. It is thought that not only SCA but also INA in the TRPs are produced by interactions with heterogeneous ice nucleators, not by direct interaction with water molecules. In the present study, several TRPs that might be useful for applications due to their high SCA in many solutions were identified. (C) 2013 Elsevier Inc. All rights reserved.
  • Jun Kasuga, Keita Endoh, Megumi Yoshiba, Ippei Taido, Keita Arakawa, Matsuo Uemura, Seizo Fujikawa
    PHYSIOLOGIA PLANTARUM 148 (1) 25 - 35 0031-9317 2013/05 [Refereed][Not invited]
     
    The supercooling capability of xylem parenchyma cells (XPCs) in boreal hardwood species differs depending not only on species, but also season. In this study, the roles of cell walls and intracellular contents in supercooling capability of XPCs were examined in three boreal hardwood species, Japanese beech, katsura tree and mulberry, whose supercooling capability differs largely depending on species and season. XPCs in these species harvested in winter and summer were treated by rapid freezing and thawing (RFT samples) or by RFT with further washing (RFTW samples) to remove intracellular contents from XPCs in order to examine the roles of cell walls in supercooling. RFT samples were also treated with glucose solution (RFTG samples) to examine roles of intracellular contents in supercooling. The supercooling capabilities of these samples were examined by differential thermal analysis after ultrastructural observation of XPCs by a cryo-scanning electron microscope to confirm effects of the above treatments. XPCs in RFTW samples showed a large reduction in supercooling capability to similar temperatures regardless of species or season. On the other hand, XPCs in RFTG samples showed a large increase in supercooling capability to similar temperatures regardless of species or season. These results indicate that although cell walls have an important role in maintenance of supercooling, change in supercooling capability of XPCs is induced by change in intracellular contents, but not by change in cell wall properties.
  • ENDOH Keita, FUJIKAWA Seizo, ARAKAWA Keita
    Cryobiology and Cryotechnology 低温生物工学会 58 (2) 125 - 134 1340-7902 2012/10 [Refereed][Not invited]
     
    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.
  • Chikako Kuwabara, Donghui Wang, Jun Kasuga, Yukiharu Fukushi, Keita Arakawa, Toshie Koyama, Takaaki Inada, Seizo Fujikawa
    CRYOBIOLOGY 64 (3) 279 - 285 0011-2240 2012/06 [Refereed][Not invited]
     
    In this study, we examined the effects on freezing of 26 kinds of flavonoid compounds, which were randomly selected as compounds with structures similar to those of flavonoid compounds existing in deep supercooling xylem parenchyma cells (XPCs) in trees, in solutions containing different kinds of ice nucleators, including the ice nucleation bacterium (INB) Erwinia ananas, INB Xanthomonas campestris, silver iodide, phloroglucinol and unidentified airborne impurities in buffered Milli-Q water (BMQW). Cumulative freezing spectra were obtained in each solution by cooling 2 mu L droplets at 0.2 degrees C/min by a droplet freezing assay. Freezing temperature of 50% droplets (FT50) was obtained from each spectra in a separate analysis with more than 20 droplets and mean FT50 were obtained from more than five separate analyses using more than 100 droplets in total in each flavonoid. Supercooling-promoting activities (SCA) or ice nucleation-enhancing activities (INA) of these flavonoids were determined by the difference in FT50 between control solutions without flavonoids and experimental solutions with flavonoids. In mean values, most of the compounds examined exhibited SCA in solutions containing the INB E. ananas, INB X. campestris, silver iodide, and phloroglucinol although the magnitudes of their activities were different depending on the ice nucleator. In solutions containing the INB E. ananas, 10 compounds exhibited SCAs with significant differences (p < 0.05) in the range of 1.4-4.2 degrees C. In solutions containing silver iodide, 23 compounds exhibited SCAs with significant differences in the range of 2.0-7.1 degrees C. In solutions containing phloroglucinol, six compounds exhibited SCAs with significant differences in the range of 2.4-3.5 degrees C. In solutions containing the INB X. campestris, only three compounds exhibited SCAs with significant differences in the range of 0.9-2.3 degrees C. In solutions containing unidentified airborne impurities (BMQW alone), on the other hand, many compounds exhibited INA rather than SCA. In mean values, only four compounds exhibited SCAs in the range of 2.4-3.2 degrees C (no compounds with significant difference at p < 0.05), whereas 21 compounds exhibited INAs in the range of 0.1-12.3 degrees C (eight compounds with significant difference). It was also shown by an emulsion freezing assay that most flavonoid glycosides examined did not affect homogeneous ice nucleation temperatures, except for a few compounds that become ice nucleators in BMQW alone. These results suggest that most flavonoid compounds affect freezing temperatures by interaction with unidentified ice nucleators in BMQW as examined by a droplet freezing assay. The results of our previous and present studies indicate that flavonoid compounds have very complex effects to regulate freezing of water. (C) 2012 Elsevier Inc. All rights reserved.
  • Donghui Wang, Jun Kasuga, Chikako Kuwabara, Keita Endoh, Yukiharu Fukushi, Seizo Fujikawa, Keita Arakawa
    PLANTA 235 (4) 747 - 759 0032-0935 2012/04 [Refereed][Not invited]
     
    Xylem parenchyma cells (XPCs) in trees adapt to subzero temperatures by deep supercooling. Our previous study indicated the possibility of the presence of diverse kinds of supercooling-facilitating (SCF; anti-ice nucleation) substances in XPCs of katsura tree (Cercidiphyllum japonicum), all of which might have an important role in deep supercooling of XPCs. In the previous study, a few kinds of SCF flavonol glycosides were identified. Thus, in the present study, we tried to identify other kinds of SCF substances in XPCs of katsura tree. SCF substances were purified from xylem extracts by silica gel column chromatography and Sephadex LH-20 column chromatography. Then, four SCF substances isolated were identified by UV, mass and nuclear magnetic resonance analyses. The results showed that the four kinds of hydrolyzable gallotannins, 2,2',5-tri-O-galloyl-alpha,beta-d-hamamelose (trigalloyl Ham or kurigalin), 1,2,6-tri-O-galloyl-beta-d-glucopyranoside (trigalloyl Glc), 1,2,3,6-tetra-O-galloyl-beta-d-glucopyranoside (tetragalloyl Glc) and 1,2,3,4,6-penta-O-galloyl-beta-d-glucopyranoside (pentagalloyl Glc), in XPCs exhibited supercooling capabilities in the range of 1.5-4.5A degrees C, at a concentration of 1 mg mL(-1). These SCF substances, including flavonol glycosides and hydrolyzable gallotannins, may contribute to the supercooling in XPCs of katsura tree.
  • KUWABARA Chikako, WANG Donghui, KASUGA Jun, MORIWAKA Genta, ARAKAWA Keita, KOYAMA Toshie, INADA Takaaki, FUJIKAWA Seizo
    Cryobiology and Cryotechnology 低温生物工学会 58 (1) 99 - 103 1340-7902 2012 [Refereed][Not invited]
     
    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.
  • ENDOH Keita, OKADA Kaori, SUZUKI Shingo, FUJIKAWA Seizo, ARAKAWA Keita
    Cryobiology and Cryotechnology 低温生物工学会 58 (2) 179 - 184 1340-7902 2012 [Refereed][Not invited]
     
    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.
  • Salma Begum Bhyan, Anzu Minami, Yasuko Kaneko, Shingo Suzuki, Keita Arakawa, Yoichi Sakata, Daisuke Takezawa
    JOURNAL OF PLANT PHYSIOLOGY 169 (2) 137 - 145 0176-1617 2012/01 [Refereed][Not invited]
     
    Overwintering plants develop tolerance to freezing stress through a cold acclimation process by which the cells provoke internal protective mechanisms against freezing. The stress hormone abscisic acid (ABA) is known to increase freezing tolerance of plant cells, but its role in cold acclimation has not been determined. In this study, we used ABA-insensitive lines of the moss Physcomitrella patens to determine whether cold acclimation in bryophytes involves an ABA-dependent process. Two ABA-insensitive lines, both impaired in ABA signaling without showing ABA-induced stress tolerance, were subjected to cold acclimation, and changes in freezing tolerance and accumulation of soluble sugars and proteins were compared to the wild type. The wild-type cells acquired freezing tolerance in response to cold acclimation treatment, but very little increase in freezing tolerance was observed in the ABA-insensitive lines. Analysis of low-molecular-weight soluble sugars indicated that the ABA-insensitive lines accumulated sucrose, a major compatible solute in bryophytes, to levels comparable with those of the wild type during cold acclimation. However, accumulation of the trisaccharide theanderose and of specific LEA-like boiling-soluble proteins was very limited in the ABA-insensitive lines. Furthermore, analysis of cold-induced expression of genes encoding LEA-like proteins revealed that the ABA-insensitive lines accumulate only small amounts of these transcripts during cold acclimation. Our results indicate that cold acclimation of bryophytes requires an ABA-dependent signaling process. The results also suggest that cold-induced sugar accumulation, depending on the sugar species, can either be dependent or independent of the ABA-signaling pathway. (C) 2011 Elsevier GmbH. All rights reserved.
  • Chikako Kuwabara, Jun Kasuga, Donghui Wang, Yukiharu Fukushi, Keita Arakawa, Toshie Koyama, Takaaki Inada, Seizo Fujikawa
    CRYOBIOLOGY 63 (3) 157 - 163 0011-2240 2011/12 [Refereed][Not invited]
     
    Deep supercooling xylem parenchyma cells (XPCs) in Katsura tree contain flavonol glycosides with high supercooling-facilitating capability in solutions containing the ice nucleation bacterium (INB) Erwinia ananas, which is thought to have an important role in deep supercooling of XPCs. The present study, in order to further clarify the roles of these flavonol glycosides in deep supercooling of XPCs, the effects of these supercooling-facilitating (anti-ice nucleating) flavonol glycosides, kaempferol 3-O-beta-D-glucopyranoside (K3Glc), kaempferol 7-O-beta-D-glucopyranoside (K7Glc) and quercetin 3-O-beta-D-glucopyranoside (Q3Glc), in buffered Milli-Q water (BMQW) containing different kinds of ice nucleators, including INB Xanthomonas campestris, silver iodide and phloroglucinol, were examined by a droplet freezing assay. The results showed that all of the flavonol glycosides promoted supercooling in all solutions containing different kinds of ice nucleators, although the magnitudes of supercooling capability of each flavonol glycoside changed in solutions containing different kinds of ice nucleators. On the other hand, these flavonol glycosides exhibited complicated nucleating reactions in BMQW, which did not contain identified ice nucleators but contained only unidentified airborne impurities. Q3Glc exhibited both supercooling-facilitating and ice nucleating capabilities depending on the concentrations in such water. Both K3Glc and K7Glc exhibited only ice nucleation capability in such water. It was also shown by an emulsion freezing assay in BMQW that K3Glc and Q3Glc had no effect on homogeneous ice nucleation temperature, whereas K7Glc increased ice nucleation temperature. The results indicated that each flavonol glycoside affected ice nucleation by very complicated and varied reactions. More studies are necessary to determine the exact roles of these flavonol glycosides in deep supercooling of XPCs in which unidentified heterogeneous ice nucleators may exist. (C) 2011 Elsevier Inc. All rights reserved.
  • WANG Donghui, KASUGA Jun, KUWABARA Chikako, FUKUSHI Yukiharu, FUJIKAWA Seizo, ARAKAWA Keita
    Cryobiology and Cryotechnology 低温生物工学会 57 (2) 147 - 151 1340-7902 2011 [Refereed][Not invited]
     
    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.
  • 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 0931-1890 2010/06 [Refereed][Not invited]
     
    A dye injection method was used to elucidate the xylem water-conducting pathways of 34 broadleaved evergreen trees growing in southern Japan: two semi-ring-porous, 26 diffuse-porous, five radial-porous and one non-vessel species. The large earlywood vessels in semi-ring-porous species have a water transport function in only the outermost annual ring, as in deciduous ring-porous species. On the other hand, the small vessels in semi-ring-porous species maintain the water transport function in many outer annual rings. For the other xylem-type species, the many vessels in many outer annual rings have a water transport function. In diffuse-porous species, we categorized the water-conducting pattern within the annual rings into two types: d1 type, where water travels through vessels in the whole region; and d2 type, where water travels mainly through the earlywood vessels. The pattern in radial-porous species is similar to that in the d1 type; the pattern in non-vessels species is similar to that in the d2 type. The vessel diameter in radial-porous species is similar to that of the earlywood vessels of semi-ring-porous species. These results suggest that the conduit diameter size is only one of many factors determining the water-conducting pathways of broadleaved evergreen species.
  • Norifumi Ukaji, Chikako Kuwabara, Yuri Kanno, Mitsunori Seo, Daisuke Takezawa, Keita Arakawa, Seizo Fujikawa
    TREE PHYSIOLOGY 30 (4) 502 - 513 0829-318X 2010/04 [Refereed][Not invited]
     
    With seasonal changes, several proteins accumulate in the endoplasmic reticulum (ER)-enriched fraction in the bark of mulberry tree (Morus bombycis Koidz.). Results of partial amino acid sequence analysis in our previous study suggested that one of these proteins is the ER-localized small heat shock protein (sHSP), designated 20-kD winter-accumulating protein (WAP20). In the present study, molecular and biochemical properties of WAP20 were investigated in detail. The deduced amino acid sequence of the cDNA has the predicted signal sequence to the ER, retention signal to the ER and two consensus regions conserved in sHSPs. Recombinant WAP20 expressed in Escherichia coli also showed typical biochemical features of sHSPs, including the formation of a high-molecular-mass complex between 200 and 300 kD under native conditions, promotion of the renaturation of chemically denaturated citrate synthase and prevention of heat stress-induced aggregation of the enzyme. Transcript levels of WAP20 in the bark tissue were seasonally changed, showing high expression levels from mid-October to mid-December, and the transcript levels were additionally increased and decreased by cold treatment and warm treatment, respectively. WAP20 transcripts were detected abundantly in bark tissue rather than xylem and winter bud tissues during seasonal cold acclimation. The bark tissue specificity of WAP20 accumulation was also observed by exogenous application of phytohormone abscisic acid (ABA) in de-acclimated twigs, whereas WAP20 transcripts were increased in all of these tissues by heat shock treatment at 37 degrees C in summer twigs. The results suggest that ABA may be involved in the expression of the WAP20 gene in bark tissue of the mulberry tree during seasonal cold acclimation.
  • Jun Kasuga, Yukiharu Fukushi, Chikako Kuwabara, Donghui Wang, Atsushi Nishioka, Emiko Fujikawa, Keita Arakawa, Seizo Fujikawa
    CRYOBIOLOGY 60 (2) 240 - 243 0011-2240 2010/04 [Refereed][Not invited]
     
    Deep Supercooling Xylem parenchyma cells (XPCs) of katsura tree (Cercidiphyllum japonicum) contain four kinds of flavonol glycosides with high supercooling-facilitating (anti-ice nucleation) activities These flavonol glycosides have very similar structures, but their supercooling-facilitating activities are very different In this study, we analyzed the supercooling-facilitating activities of 12 kinds of flavonol glycosides in order to determine the chemical structures that might affect supercooling-facilitating activity All of the flavonol glycosides tested showed supercooling-facilitating activity, although the magnitudes of activity differed among the compounds It was clear that the combination of the position of attachment of the glycosyl moiety, the kind of attached glycosyl moiety and the structure of aglycone determined the magnitude of anti-ice nucleation activity However, there is still some ambiguity preventing the exact identification of features that affect the magnitude of supercooling-facilitating activity. (C) 2009 Elsevier Inc. All rights reserved
  • 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 0918-9440 2009/11 [Refereed][Not invited]
     
    Plants have evolved intricate mechanisms to respond and adapt to a wide variety of biotic and abiotic stresses in their environment. The Arabidopsis DEAR1 (DREB and EAR motif protein 1; At3g50260) gene encodes a protein containing significant homology to the DREB1/CBF (dehydration-responsive element binding protein 1/C-repeat binding factor) domain and the EAR (ethylene response factor-associated amphiphilic repression) motif. We show here that DEAR1 mRNA accumulates in response to both pathogen infection and cold treatment. Transgenic Arabidopsis overexpressing DEAR1 (DEAR1ox) showed a dwarf phenotype and lesion-like cell death, together with constitutive expression of PR genes and accumulation of salicylic acid. DEAR1ox also showed more limited P. syringae pathogen growth compared to wild-type, consistent with an activated defense phenotype. In addition, transient expression experiments revealed that the DEAR1 protein represses DRE/CRT (dehydration-responsive element/C-repeat)-dependent transcription, which is regulated by low temperature. Furthermore, the induction of DREB1/CBF family genes by cold treatment was suppressed in DEAR1ox, leading to a reduction in freezing tolerance. These results suggest that DEAR1 has an upstream regulatory role in mediating crosstalk between signaling pathways for biotic and abiotic stress responses.
  • Keita Endoh, Jun Kasuga, Keita Arakawa, Toshiaki Ito, Seizo Fujikawa
    CRYOBIOLOGY 59 (2) 214 - 222 0011-2240 2009/10 [Refereed][Not invited]
     
    The freezing behavior of dormant buds in larch, especially at the cellular level, was examined by a Cryo-SEM. The dormant buds exhibited typical extraorgan freezing. Extracellular ice crystals accumulated only in basal areas of scales and beneath crown tissues, areas in which only these living cells had thick walls unlike other tissue cells. By slow cooling (5 degrees C/day) of dormant buds to -50 degrees C, all living cells in bud tissues exhibited distinct shrinkage without intracellular ice formation detectable by Cryo-SEM. However, the recrystallization experiment of these slowly cooled tissue cells. which was done by further freezing of slowly cooled buds with LN and then rewarming to -20 degrees C, confirmed that some of the cells in the leaf primordia, shoot primordia and apical meristem, areas in which cells had thin walls and in which no extracellular ice accumulated, lost freezable water with slow cooling to -30 degrees C, indicating ability of these cells to adapt by extracellular freezing, whereas other cells in these tissues retained freezable water with slow cooling even to -50 degrees C, indicating adaptation of these cells by deep supercooling. On the other hand, all cells in crown tissues and in basal areas of scales, areas in which cells had thick walls and in which large masses of ice accumulated, had the ability to adapt by extracellular freezing. It is thought that the presence of two types of cells exhibiting different freezing adaptation abilities within a bud tissue is quite unique and may reflect sophisticated freezing adaptation mechanisms in dormant buds. (C) 2009 Elsevier Inc. All rights reserved.
  • Seizo Fujikawa, Jun Kasuga, Naoki Takata, Keita Arakawa
    Plant Cold Hardiness: From the Laboratory to the Field 29 - 42 2009/07/15 [Not refereed][Not invited]
  • Keita Arakawa, Hidetoshi Inada, Seizo Fujikawa
    Plant Cold Hardiness: From the Laboratory to the Field 173 - 182 2009/07/15 [Not refereed][Not invited]
  • FUJIKAWA Seizo, KASUGA Jun, ARAKAWA Keita
    Cryobiology and Cryotechnology 低温生物工学会 55 (1) 37 - 41 1340-7902 2009 [Not refereed][Not invited]
     
    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.
  • Daisuke Kami, Jun Kasuga, Keita Arakawa, Seizo Fujikawa
    CRYOBIOLOGY 57 (3) 242 - 245 0011-2240 2008/12 [Refereed][Not invited]
     
    The effect of kaempferol-7-O-glucoside (KF7G), one of the supercooling-facilitating flavonol glycosides which was originally found in deep supercooling xylem parenchyma cells of the katsura tree and was found to exhibit the highest level of supercooling-facilitating activity among reported substances, was examined for successful cryopreservation by vitrification procedures, with the aim of determining the possibility of using diluted vitrification solution (VS) to reduce cryoprotectant toxicity and also to inhibit nucleation at practical cooling and rewarming by the effect of supplemental KF7G. Examination was performed using shoot apices of cranberry and plant vitrification solution 2 (PVS2) with dilution. Vitrification procedures using the original concentration (100%) of PVS2 caused serious injury during treatment with PVS2 and resulted in no regrowth after cooling and rewarming (cryopreservation). Dilution of the concentration of PVS2 to 75% or 50% (with the same proportions of constituents) significantly reduced injury by PVS2 treatment, but regrowth was poor after cryopreservation. It is thought that dilution of PVS2 reduced injury by cryoprotectant toxicity, but such dilution caused nucleation during cooling and/or rewarming, resulting in poor survival. On the other hand, addition of 0.5 mg/ml (0.05% w/v) KF7G to the diluted PVS2 resulted in significantly (p < 0.05) higher regrowth rates after cryopreservation. It is thought that addition of supercooling-facilitating KF7G induced vitrification even in diluted PVS2 probably due to inhibition of ice nucleation during cooling and rewarming and consequently resulted in higher regrowth. The results of the present study indicate the possibility that concentrations of routinely used VSs can be reduced by adding supercooling-facilitating KF7G, by which more successful cryopreservation might be achieved for a wide variety of biological materials. (C) 2008 Elsevier Inc. All rights reserved.
  • Jun Kasuga, Yasuyuki Hashidoko, Atsushi Nishioka, Megumi Yoshiba, Keita Arakawa, Seizo Fujikawa
    PLANT CELL AND ENVIRONMENT 31 (9) 1335 - 1348 0140-7791 2008/09 [Refereed][Not invited]
     
    Xylem parenchyma cells (XPCs) of boreal hardwood species adapt to sub-freezing temperatures by deep supercooling to maintain a liquid state of intracellular water near -40 degrees C. Our previous study found that crude xylem extracts from such tree species exhibited anti-ice nucleation activity to promote supercooling of water. In the present study, thus, we attempted to identify the causative substances of supercooling. Crude xylem extracts from katsura tree (Cercidiphyllum japonicum), of which XPCs exhibited deep supercooling to -40 degrees C, were prepared by methanol extraction. The crude extracts were purified by liquid-liquid extraction and then by silica gel column chromatography. Although all the fractions obtained after each purification step exhibited some levels of anti-ice nucleation activity, only the most active fraction was retained to proceed to the subsequent level of purification. High-performance liquid chromatography (HPLC) analysis of a fraction with the highest level of activity revealed four peaks with high levels of anti-ice nucleation activity in the range of 2.8-9.0 degrees C. Ultraviolet (UV), mass and nuclear magnetic resonance (NMR) spectra revealed that these four peaks corresponded to quercetin-3-O-beta-glucoside (Q3G), kaempferol-7-O-beta-glucoside (K7G), 8-methoxykaempferol-3-O-beta-glucoside (8MK3G) and kaempferol-3-O-beta-glucoside (K3G). Microscopic observations confirmed the presence of flavonoids in cytoplasms of XPCs. These results suggest that diverse kinds of anti-ice nucleation substances, including flavonol glycosides, may have important roles in deep supercooling of XPCs.
  • INADA Hidetoshi, FUJIKAWA Seizo, ARAKAWA Keita
    Transactions of the Japan Society of Refrigerating and Air Conditioning Engineers 公益社団法人 日本冷凍空調学会 25 (2) 233 - 238 1344-4905 2008 [Refereed][Not invited]
     
    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.
  • M. Nagao, K. Arakawa, D. Takezawa, S. Fujikawa
    PLANTA 227 (2) 477 - 489 0032-0935 2008/01 [Refereed][Not invited]
     
    In nature, intact plant cells are subjected to freezing and can remain frozen for prolonged periods. We assayed the survival of Arabidopsis thaliana leaf cells following freezing and found that short- and long-term exposures produced different types of cellular injury. To identify the cause of these injuries, we examined the ultrastructure of the cell plasma membranes. Our results demonstrate that ultrastructural changes in the plasma membrane due to short-term freezing are associated with interbilayer events, including close apposition of the membranes. In both acclimated and non-acclimated leaf cells, these interbilayer events resulted in "fracture-jump lesions" in the plasma membrane. On the other hand, long-term freezing was associated with the development of extensive protein-free areas caused by the aggregation of intramembrane proteins with consequent vesiculation of the affected membrane regions; this effect was clearly different from the ultrastructural changes induced by interbilayer events. We also found that prolonged exposure of non-acclimated leaf cells to a concentrated electrolyte solution produced effects that were similar to those caused by long-term freezing, suggesting that the ultrastructural changes observed in the plasma membrane following long-term freezing are produced by exposure of the leaf cells to a concentrated electrolyte solution. This study illustrates multiple causes of freezing-induced injury in plant cells and may provide useful information regarding the functional role of the diverse changes that occur during cold acclimation.
  • Toshihiro Umebayashi, Yasuhiro Utsumi, Shinya Koga, Susumu Inoue, Seizo Fujikawa, Keita Arakawa, Junji Matsumura, Kazuyuki Oda
    IAWA JOURNAL 29 (3) 247 - 263 0928-1541 2008 [Refereed][Not invited]
     
    The interspecific variation of dye ascent in the stems of 44 broadleaved deciduous species growing in Japan was studied using freeze-dried samples after dye injection. The dye ascending pattern differed both within and between ring-porous and diffuse-porous species. In large earlywood vessels of all ring-porous species, the dye ascended only in the outermost annual ring, and the inner annual rings had lost their water transport function. The dye ascending pattern within the inner annual rings in the ring-porous species was categorized into three types: i) the dye ascended both in the many latewood vessels throughout the latewood and small earlywood vessels; ii) the dye ascended in the many vessels throughout the latewood; and iii) the dye ascended mainly in the late latewood vessels. In diffuse-porous species, the dye ascending pattern within the annual rings also was categorized into three types: i) the dye ascended throughout the annual rings; ii) the dye ascended mainly in the earlywood vessels, and iii) the dye ascended mainly in the latewood vessels. Xylem water distribution was also examined by cryo-SEM in three ring-porous and three diffuse-porous species that had different dye ascending patterns. The water distribution pattern within annual rings was correlated with the dye ascending pattern except for one diffuseporous species (Salix gracilistyla). In this case, water was distributed in the whole region of the annual rings although dye was mainly distributed in the earlywood. These results showed that the functional area of water transport within annual rings differed among ring-porous species and diffuse-porous species.
  • Jun Kasuga, Kaoru Mizuno, Keita Arakawa, Seizo Fujikawa
    CRYOBIOLOGY 55 (3) 305 - 314 0011-2240 2007/12 [Refereed][Not invited]
     
    Boreal hardwood species, including Japanese white birch (Betula platyphylla Sukat. var. japonica Hara), Japanese chestnut (Castanea crenata Sieb. et Zucc.), katsura tree (Cercidiphyllum japonicum Sieb. et Zucc.), Siebold's beech (Fagus crenata Blume), mulberry (Morus bombycis Koidz.), and Japanese rowan (Sorbus commixta Hedl.), had xylem parenchyma cells (XPCs) that adapt to subfreezing temperatures by deep supercooling. Crude extracts from xylem in all these trees were found to have anti-ice nucleation activity that promoted supercooling capability of water as measured by a droplet freezing assay. The magnitude of increase in supercooling capability of water droplets in the presence of ice-nucleation bacteria, Erwinia ananas, was higher in the ranges from 0.1 to 1.7 degrees C on addition of crude xylem extracts than freezing temperature of water droplets on addition of glucose in the same concentration (100 mosmol/kg). Crude xylem extracts from C japonicum provided the highest supercooling capability of water droplets. Our additional examination showed that crude xylem extracts from C. japonicum exhibited anti-ice nucleation activity toward water droplets containing a variety of heterogeneous ice nucleators, including ice-nucleation bacteria, not only E ananas but also Pseudomonas syringae (NBRC3310) or Xanthomonas campestris, silver iodide or airborne impurities. However, crude xylem extracts from C. japonicum did not affect homogeneous ice nucleation temperature as analyzed by emulsified micro-water droplets. The possible role of such anti-ice nucleation activity in crude xylem extracts in deep supercooling of XPCs is discussed. (C) 2007 Elsevier Inc. All rights reserved.
  • Naoki Takata, Jun Kasuga, Daisuke Takezawa, Keita Arakawa, Seizo Fujikawa
    JOURNAL OF EXPERIMENTAL BOTANY 58 (13) 3731 - 3742 0022-0957 2007/10 [Refereed][Not invited]
     
    Xylem parenchyma cells (XPCs) in larch adapt to subfreezing temperatures by deep supercooling, while cortical parenchyma cells (CPCs) undergo extracellular freezing. The temperature limits of supercooling in XPCs changed seasonally from -30 degrees C during summer to -60 degrees C during winter as measured by freezing resistance. Artificial deacclimation of larch twigs collected in winter reduced the supercooling capability from -60 degrees C to -30 degrees C. As an approach to clarify the mechanisms underlying the change in supercooling capability of larch XPCs, genes expressed in association with increased supercooling capability were examined. By differential screening and differential display analysis, 30 genes were found to be expressed in association with increased supercooling capability in XPCs. These 30 genes were categorized into several groups according to their functions: signal transduction factors, metabolic enzymes, late embryogenesis abundant proteins, heat shock proteins, protein synthesis and chromatin constructed proteins, defence response proteins, membrane transporters, metal-binding proteins, and functionally unknown proteins. All of these genes were expressed most abundantly during winter, and their expression was reduced or disappeared during summer. The expression of all of the genes was significantly reduced or disappeared with deacclimation of winter twigs. Interestingly, all but one of the genes were expressed more abundantly in the xylem than in the cortex. Eleven of the 30 genes were thought to be novel cold-induced genes. The results suggest that change in the supercooling capability of XPCs is associated with expression of genes, including genes whose functions have not been identified, and also indicate that gene products that have been thought to play a role in dehydration tolerance by extracellular freezing also have a function by deep supercooling.
  • Jun Kasuga, Naoki Takata, Kenichi Yamane, Katsushi Kuroda, Keita Arakawa, Seizo Fujikawa
    CRYOLETTERS 28 (2) 77 - 81 0143-2044 2007/03 [Refereed][Not invited]
     
    In previous studies, xylem parenchyma cells (XPCs) in the boreal softwood species larch, which has thick and rigid walls similar to those of XPCs in boreal hardwood species, were shown to respond to subfreezing temperature by deep supercooling during summer but change their freezing behavior to extracellular freezing during winter (4). In this study, we reexamined freezing behavior of XPCs in larch by observation of deep etching of frozen samples as well as observation of re-warmed samples after freezing using a cryo-scanning electron microscope. The results showed that XPCs in larch adapts to subfreezing temperature by deep supercooling throughout all seasons. Such freezing behavior is the same as that of XPCs in boreal hardwood species.
  • Jun Kasuga, Keita Arakawa, Seizo Fujikawa
    NEW PHYTOLOGIST 174 (3) 569 - 579 0028-646X 2007 [Refereed][Not invited]
     
    Seasonal changes in the accumulation of soluble sugars in extracellular freezing cortical parenchyma cells and deep supercooling xylem parenchyma cells in Japanese white birch (Betula platyphylla var. japonica) were compared to identify the effects of soluble sugars on the mechanism of deep supercooling, which keeps the liquid state of water in cells under extremely low temperatures for long periods. Soluble sugars in both tissues were analyzed by high-performance liquid chromatography (HPLC), and the concentrations of sugars in cells were estimated by histological observation of occupancy rates of parenchyma cells in each tissue. Relative and equilibrium melting points of parenchyma cells were measured by differential thermal analysis and cryoscanning electron microscopy, respectively. In both xylem and cortical parenchyma cells, amounts of sucrose, raffinose and stachyose increased in winter, but amounts of fructose and glucose exhibited little change throughout the entire year. In addition, no sugars were found to be specific for either tissue. Combined results of HPLC analyses, histological observation and melting point analyses confirmed that the concentration of sugars was much higher in xylem cells than in cortical cells. It is thought that the higher concentration of soluble sugars in xylem cells may contribute to facilitation of deep supercooling in xylem cells by depressing the nucleation temperature.
  • INADA Hidetoshi, FUJIKAWA Seizo, SAITO Hideyuki, ARAKAWA Keita
    Environmental sciences : an international journal of environmental physiology and toxicology 14 (Supplement) 53 - 71 0915-955X 2007 [Refereed][Not invited]
  • Toshihiro Umebayashi, Yasuhiro Utsumi, Shinya Koga, Susumu Inoue, Yasuki Shiiba, Keita Arakawa, Junji Matsumura, Kazuyuki Oda
    Tree Physiology 27 (7) 993 - 999 0829-318X 2007 [Refereed][Not invited]
     
    To elucidate the water-conducting pathways in living trees by the dye injection method, suitable sample preparation procedures are needed. We evaluated quantitatively the properties and concentrations of three dyes (acid fuchsin, basic fuchsin and safranin) widely used for this purpose, and determined the optimal conditions required to avoid artifacts after dye injection into the sap stream of Pieris japonica D. Don. Among the dyes tested, an aqueous solution of acid fuchsin at a concentration of 0.1% or more was the most useful for delineating water movement. In non-transpiring stem segments, the vertical movement of acid fuchsin by capillarity and diffusion from the dye injection site was limited. However, acid fuchsin moved rapidly in the horizontal direction by capillarity and diffusion, and most xylem cells were stained within 2 h. A delay of more than 2 h between dye injection and examination of the tissues greatly reduces the precision of the method. Use of the dye injection method without appropriate, well-defined experimental procedures may give rise to misleading information about the functional water-conducting pathway in living trees. © 2007 Heron Publishing.
  • Jun Kasuga, Kaoru Mizuno, Natsuko Miyaji, Keita Arakawa, Seizo Fujikawa
    CRYOLETTERS 27 (5) 305 - 310 0143-2044 2006/09 [Refereed][Not invited]
     
    In order to find the possible role of intracellular contents in facilitating the supercooling capability of xylem parenchyma cells, changes in the temperature of supercooling levels were compared before and after the release of intracellular substances from beech xylem parenchyma cells by DTA. Various methods were employed to release intracellular substances from xylem parenchyma cells and all resulted in a reduction of supercooling ability. It was concluded that the reduction of supercooling ability primarily resulted from changes of intracellular conditions, including the release of intracellular contents or their mixing with extracellular solutions, rather than due to changes of cell wall structures. It is therefore suggested that any unidentified intracellular contents may function to facilitate supercooling capability in xylem parenchyma cells.
  • Hidetoshi Inada, Manabu Nagao, Seizo Fujikawa, Keita Arakawa
    Plant & cell physiology 47 (4) 504 - 12 0032-0781 2006/04 [Refereed][Not invited]
     
    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.
  • M Nagao, K Oku, A Minami, K Mizuno, M Sakurai, K Arakawa, S Fujikawa, D Takezawa
    PHYTOCHEMISTRY 67 (7) 702 - 709 0031-9422 2006/04 [Refereed][Not invited]
     
    Mosses are known to have the ability to develop high degrees of resistance to desiccation and freezing stress at cellular levels. However, underlying cellular mechanisms leading to the development of stress resistance in mosses are not understood. We previously showed that freezing tolerance in protonema cells of the moss Physcomitrella patens was rapidly increased by exogenous application of the stress hormone abscisic acid (ABA) [Minami, A., Nagao, M., Arakawa, K., Fujikawa, S., Takezawa, D., 2003a. Abscisic acid-induced freezing tolerance in the moss Physcomitrella patens is accompanied by increased expression of stress-related genes. J. Plant Physiol. 160, 475483]. Herein it is shown that protonema cells with acquired freezing tolerance specifically accumulate low-molecular-weight soluble sugars. Analysis of the most abundant trisaccharide revealed that the cells accumulated theanderose (G(6)-alpha-glucosyl sucrose) in close association with enhancement of freezing tolerance by ABA treatment. The accumulation of theanderose was inhibited by cycloheximide, an inhibitor of nuclear-encoded protein synthesis, coinciding with a remarkable decrease in freezing tolerance. Furthermore, theanderose accumulation was promoted by cold acclimation and treatment with hyperosmotic solutes, both of which had been shown to enhance cellular freezing tolerance. These results reveal a novel role for theanderose, whose biological function has been obscure, in high freezing tolerance in moss cells. (c) 2006 Elsevier Ltd. All rights reserved.
  • S. Fujikawa, N. Ukaji, M. Nagao, K. Yamane, D. Takezawa, K. Arakawa
    Cold Hardiness in Plants: Molecular Genetics, Cell Biology, and Physiology 181 - 202 2006/01/10 [Not refereed][Not invited]
  • A. Minami, M. Nagao, K. Arakawa, S. Fujikawa, D. Takezawa
    Cold Hardiness in Plants: Molecular Genetics, Cell Biology, and Physiology 138 - 152 2006/01/10 [Not refereed][Not invited]
  • INADA Hidetoshi, FUJIKAWA Seizo, ARAKAWA Keita
    Cryobiology and Cryotechnology 低温生物工学会 52 (2) 163 - 168 1340-7902 2006 [Refereed][Not invited]
     
    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 [Not refereed][Not invited]
  • FUJIKAWA SEIZO, KASUGA JUN, ARAKAWA KEITA
    化学と生物 日本農芸化学会 43 (5) 280 - 282 0453-073X 2005/05/01 [Not refereed][Not invited]
  • M Nagao, A Minami, K Arakawa, S Fujikawa, D Takezawa
    JOURNAL OF PLANT PHYSIOLOGY 162 (2) 169 - 180 0176-1617 2005/02 [Refereed][Not invited]
     
    Abscisic acid (ABA) has been postulated to play a role in the development of freezing tolerance during the cold acclimation process in higher plants, but its role in cold tolerance in lower land plants has not been elucidated. The moss Physcomitrella patens rapidly developed freezing tolerance when its protonemata were grown in a medium containing ABA, with dramatic changes in the LT50 value from -2 degrees C to over -10 degrees C. We examined physiological and morphological. alterations in protonema cells caused by ABA treatment to elucidate early cellular events responsible for rapid enhancement of freezing tolerance. Microscopic observations revealed that ABA treatment for 1 day resulted in a dramatic alteration in the appearance of intracellular organelles. ABA-treated cells had slender chloroplasts, with a reduced amount of starch grains, in comparison with those of non-treated cells. The ABA-treated cells also had several segmented vacuoles while many of non-treated cells had one central vacuole. When frozen to -4 degrees C, freezing injury-associated ultrastructural. changes such as formation of aparticulate domains and fracture-jump lesions were frequently observed in the plasma membrane of non-treated protonema cells but not in that of ABA-treated cells. The ABA treatment increased the osmotic concentration of the protonema cells, in correlation with accumulation of free soluble sugars. These results suggest that ABA-induced accumulation of soluble sugars, associated with morphological changes in organelles, mitigated freezing-induced structural damage in the plasma membrane, eventually leading to enhancement of freezing tolerance in the protonema cells. (c) 2004 Elsevier GmbH. All rights reserved.
  • MIZUNO Kaoru, KASUGA Jun, ARAKAWA Keita, FUJIKAWA Seizo
    Cryobiology and Cryotechnology 低温生物工学会 51 (2) 111 - 114 1340-7902 2005 [Refereed][Not invited]
     
    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).
  • INADA Hidetoshi, FUJIKAWA Seizo, ARAKAWA Keita
    Cryobiology and Cryotechnology 低温生物工学会 51 (2) 115 - 119 1340-7902 2005 [Refereed][Not invited]
     
    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.
  • TAKAHASHI Hirokazu, ARAKAWA Keita, FUJIKAWA Seizo
    Cryobiology and Cryotechnology 低温生物工学会 51 (2) 105 - 109 1340-7902 2005 [Refereed][Not invited]
     
    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.
  • A Minami, M Nagao, K Ikegami, T Koshiba, K Arakawa, S Fujikawa, D Takezawa
    PLANTA 220 (3) 414 - 423 0032-0935 2005/01 [Refereed][Not invited]
     
    Bryophyte species growing in areas in which temperatures fall below zero in winter are likely to have tolerance to freezing stress. It is well established in higher plants that freezing tolerance is acquired by exposure to non-freezing low temperatures, accompanied by expression of various genes and increases in levels of the stress hormone abscisic acid (ABA). However, little is known about the physiological changes induced by cold acclimation in non-vascular plants such as bryophytes. We examined the effects of low temperatures on protonema cells of the moss Physcomitrella patens (Hedw.) Bruch & Schimp. The freezing tolerance of protonema cells was clearly increased by incubation at low temperatures ranging from 10degreesC to 0degreesC, with maximum tolerance achieved by incubation at 0degreesC for several days. The enhancement of freezing tolerance by low temperatures occurred in both light and dark conditions and was accompanied by accumulation of several transcripts for late-embryogenesis-abundant (LEA) proteins and boiling-soluble proteins. By de-acclimation, low-temperature-induced expression of these transcripts and proteins, as well as the freezing tolerance, was reduced. Interestingly, endogenous levels of ABA in tissues or that secreted into the culture medium were not specifically increased by low-temperature treatment. Furthermore, removal of ABA from the medium by addition of activated charcoal did not affect low-temperature-induced freezing tolerance of the protonema cells. Our results provide evidence that bryophytes have an ABA-independent cold-signaling pathway leading to expression of stress-related genes and resultant acquisition of freezing tolerance.
  • N. UKAJI, C. KUWABARA, D. TAKEZAWA, K. ARAKAWA, S. FUJIKAWA
    Plant, Cell and Environment 27 (9) 1112 - 1121 0140-7791 2004/09 [Refereed][Not invited]
  • FUJIKAWA Seizo, ARAKAWA Keita
    Cryobiology and Cryotechnology 低温生物工学会 50 (1) 33 - 36 1340-7902 2004 [Not refereed][Not invited]
     
    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.
  • T Fujibe, H Saji, K Arakawa, N Yabe, Y Takeuchi, KT Yamamoto
    PLANT PHYSIOLOGY 134 (1) 275 - 285 0032-0889 2004/01 [Refereed][Not invited]
     
    To better understand the role of active oxygen species (AOS) in acquired resistance to increased levels of ultraviolet (UV)-B irradiation in plants, we isolated an Arabidopsis mutant that is resistant to methyl viologen, and its sensitivity to UV-B was investigated. A complementation test revealed that the obtained mutant was allelic to the ozone-sensitive radical-induced. cell death1-1 (rcd1-1). Therefore, this mutant was named rcd1-2. rcd1-2 was recessive and nearly 4-fold more resistant to methyl viologen than wild type. It exhibited a higher tolerance to short-term UV-B supplementation treatments than the wild type: UV-B-induced formation of cyclobutane pyrimidine dimers was reduced by one-half after 24 h of exposure; the decrease in quantum yield of photosystem II was also diminished by 40% after 12 h of treatment. Furthermore, rcd1-2 was tolerant to freezing. Steady-state mRNA levels of plastidic Cu/Zn superoxide dismutase and stromal ascorbate peroxidase were higher in rcd1-2 than in wild type, and the mRNA level of the latter enzyme was enhanced by UV-13 exposure more effectively in rcd1-2. UV-B-absorbing compounds were more accumulated in rcd1-2 than in wild type after UV-B exposure for 24 h. These findings suggest that rcd1-2 methyl viologen resistance is due to the enhanced activities of the AOS-scavenging enzymes in chloroplasts and that the acquired tolerance to the short-term UV-B exposure results from a higher accumulation of sunscreen pigments. rcd1 appears to be a mutant that constitutively shows stress responses, leading to accumulation of more pigments and AOS-scavenging enzymes without any stresses.
  • K Arakawa, M Hanazaki, S Yoshida
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 68 (1) 175 - 182 0916-8451 2004/01 [Refereed][Not invited]
     
    When plasma membranes were prepared from tubers of Helianthus tuberosus L. (Jerusalem artichoke) frozen at a sublethal temperature (-10degreesC), the levels of some plasma membrane proteins, named frost-susceptible proteins (FSPs), decreased [Uemura, M., et al., Plant Physiol., 80, 187-195 (1986)]. The aim of this study was to characterize the response of FSP120, which is named FSP-3 in a previous report, to freezing treatment by immunoblotting. Levels of FSP120 in the plasma membranes of tubers decreased after sublethal freezing, whereas no degraded products were detected in the microsomes or the soluble fraction. The amount of FSP120 in the crude extract of frozen tubers remained at a comparable level to that of the unfrozen tubers. These results suggest that FSP120 might be released from plasma membranes during freezing treatment of the tubers of Jerusalem artichoke.
  • A Minami, M Nagao, K Arakawa, S Fujikawa, D Takezawa
    JOURNAL OF PLANT PHYSIOLOGY 160 (5) 475 - 483 0176-1617 2003/05 [Refereed][Not invited]
     
    Abscisic acid (ABA)-induced genes are implicated in the development of freezing tolerance during cold acclimation in higher plants, but their roles in lower land plants have not been determined. We examined ABA- and cold-induced changes in freezing tolerance and gene expression in the moss Physcomitrella patens. Slow equilibrium freezing to -4degreesC of R patens protonemata grown under normal growth conditions killed more than 90% of the cells, indicating that the protonema cells are freezing-sensitive. ABA treatment for 24h dramatically increased the freezing tolerance of the protonemata, while cold treatment only slightly increased the freezing tolerance within the same period. We examined the expressions of fourteen Physcomitrella patens ABA-responsive genes (PPARs), isolated from ABA-treated protonemata. ABA treatment resulted in a remarkable increase in the expression of all the PPAR genes within 24 h. Several of the PPAR genes (PPAR 1 to 8, and 14) were also responsive to cold, but the response was much slower than that to ABA. Treatment with hyperosmotic concentrations of NaCl and mannitol increased freezing tolerance of protonemata and also increased the expression levels of eleven PPAR genes (PPAR2, 3, 5 to 8, and 10 to 14). These results suggest that ABA and environmental stresses positively affect the expression of common genes that participate in protection of protonema cells leading to the development of freezing tolerance.
  • K Kuroda, J Kasuga, K Arakawa, S Fujikawa
    PLANT PHYSIOLOGY 131 (2) 736 - 744 0032-0889 2003/02 [Refereed][Not invited]
     
    It has been accepted that xylem ray parenchyma cells (XRPCs) in hardwood species respond to subfreezing temperatures either by deep supercooling or by extracellular freezing. Present study by cryo-scanning electron microscopy examined the freezing responses of XRPCs in five boreal hardwoods: Salix sachalinensis Fr. Schmit, Populus sieboldii Miq., Betula platyphylla Sukat. var japonica Hara, Betula pubescens Ehrh., and red osier dogwood (Cornus sericea), in which XRPCs have been reported to respond by extracellular freezing. Cryo-scanning electron microscopy observations revealed that slow cooling of xylem to -80degreesC resulted in intracellular freezing in the majority of XRPCs in S. sachalinensis, an indication that these XRPCs had been deep supercooled. In contrast, in the majority of XRPCs in P. sieboldii, B. platyphylla, B. pubescens, and red osier dogwood, slow cooling to -80degreesC produced slight cytorrhysis without clear evidence of intracellular freezing, suggesting that these XRPCs might respond by extracellular freezing. In these XRPCs exhibited putative extracellular freezing; however, deep etching revealed the apparent formation of intracellular ice crystals in restricted local areas. To confirm the occurrence of intracellular freezing, we rewarmed these XRPCs after cooling and observed very large intracellular ice crystals as a result of the recrystallization. Thus, the XRPCs in all the boreal hardwoods that we examined responded by deep supercooling that was accompanied with incomplete desiccation. From these results, it seems possible that limitations to the deep-supercooling ability of XRPCs might be a limiting factor for adaptation of hardwoods to cold climates.
  • MINAMI Anzu, NAGAO Manabu, ARAKAWA Keita, FUJIKAWA Seizo, TAKEZAWA Daisuke
    Cryobiology and Cryotechnology 低温生物工学会 49 (2) 179 - 183 1340-7902 2003 [Refereed][Not invited]
     
    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.
  • KASUGA Jun, FUJIKAWA Seizo, ARAKAWA Keita
    Cryobiology and Cryotechnology 低温生物工学会 49 (2) 185 - 189 1340-7902 2003 [Refereed][Not invited]
     
    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.
  • TAKATA Naoki, MINAMI Anzu, ARAKAWA Keita, TAKEZAWA Daisuke, FUJIKAWA Seizo
    Cryobiology and Cryotechnology 低温生物工学会 49 (2) 191 - 194 1340-7902 2003 [Refereed][Not invited]
     
    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.
  • TANAKA Satoko, NAGAO Manabu, FUNADA Ryo, FUJIKAWA Seizo, ARAKAWA Keita
    Cryobiology and Cryotechnology 低温生物工学会 49 (2) 209 - 213 1340-7902 2003 [Refereed][Not invited]
     
    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.
  • ARAKAWA Keita, INADA Hidetoshi
    Cryobiology and Cryotechnology 低温生物工学会 49 (2) 203 - 207 1340-7902 2003 [Refereed][Not invited]
     
    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.
  • ARAKAWA Keita, KASUGA Jun, TAKASHIMA Hiroshi, FUJIKAWA Seizo
    Cryobiology and Cryotechnology 低温生物工学会 49 (2) 195 - 201 1340-7902 2003 [Refereed][Not invited]
     
    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.
  • T Yamada, K Kuroda, Y Jitsuyama, D Takezawa, K Arakawa, S Fujikawa
    PLANTA 215 (5) 770 - 778 0032-0935 2002/09 [Not refereed][Not invited]
     
    In an effort to clarify the responses of a wide range of plant cells to freezing, we examined the responses to freezing of the cells of chilling-sensitive and chilling-resistant tropical and subtropical plants. Among the cells of the plants that we examined, those of African violet (Saintpaulia grotei Engl.) leaves were most chilling-sensitive, those of hypocotyls in mungbean [Vigna radiata (L.) R. Wilcz] seedlings were moderately chilling-sensitive, and those of orchid [Paphiopedilum insigne (Wallich ex Lindl.) Pfitz.] leaves were chilling-resistant, when all were chilled at -2 degreesC. By contrast, all these plant cells were freezing-sensitive and suffered extensive damage when they were frozen at -2 degreesC. Cryo-scanning electron microscopy (Cryo-SEM) confirmed that, upon chilling at -2 degreesC, both chilling-sensitive and chilling-resistant plant cells were supercooled. Upon freezing at -2 degreesC, by contrast, intracellular freezing occurred in Saintpaulia leaf cells, frost plasmolysis followed by intracellular freezing occurred in mungbean seedling cells, and extracellular freezing (cytorrhysis) occurred in orchid leaf cells. We postulate that chilling-related destabilization of membranes might result in the loss of the ability of the plasma membrane to act as a barrier against the propagation of extracellular ice in chilling-sensitive plant cells. We also examined the role of cell-walls in the response to freezing using cells in which the plasma membrane had been disrupted by repeated freezing and thawing. In chilling-sensitive Saintpaulia and mungbean cells, the cells with a disrupted plasma membrane responded to freezing at -2 degreesC by intracellular freezing. By contrast, in chilling-resistant orchid cells, as well as in other cells of chilling-resistant and freezing-resistant plant tissues, including leaves of orchard grass (Dactylis glomerata L.), leaves of Arabidopsis thaliana (L.) Heynh. and cortical tissues of mulberry (Morus bombycis Koids.). cells with a disrupted plasma membrane responded to freezing by extracellular freezing. Our results indicate that, in the chilling-sensitive plants cells that we examined. not only the plasma membrane but also the cell wall lacked the ability to serve as a barrier against the propagation of extracellular ice, whereas in the chilling-resistant plant cells that we examined, not only the plasma membrane but also the cell wall acted as a barrier against the propagation of extracellular ice. It appears. therefore, that not only the plasma membrane but also the cell wall greatly influences the freezing behavior of plant cells.
  • C Kuwabara, D Takezawa, T Shimada, T Hamada, S Fujikawa, K Arakawa
    PHYSIOLOGIA PLANTARUM 115 (1) 101 - 110 0031-9317 2002/05 [Refereed][Not invited]
     
    Cold acclimation of winter wheat (Triticum aestivum L.) seedlings induces accumulation in the apoplast of taTLPs that are similar to thaumatin-like proteins (TLPs), which are pathogenesis-related proteins. We characterized a cDNA of WAS-3a encoding the major isoform of taTLPs from winter wheat cells and showed that WAS-3a transcripts were markedly increased by treatment with ABA and by treatment with elicitors (chitosan, beta-glucan and cell wall fractions of Fusarium oxysporum and Microdochium nivale) in wheat cells. To analyse the function of WAS-3a, a highly efficient expression system using wheat cells was established, and a large amount of recombinant WAS-3a protein (rWAS-3a) was obtained with near homogeneity. Antifungal assays using various fungi grown on agar plates revealed that rWAS-3a inhibits hyphal growth of pink snow mould, Microdochium nivale, at a low temperature. The results suggest that cold-induced taTLPs that accumulate in the apoplast contribute to snow mould resistance of winter wheat.
  • N Ukaji, C Kuwabara, D Takezawa, K Arakawa, S Fujikawa
    PLANT PHYSIOLOGY 126 (4) 1588 - 1597 0032-0889 2001/08 [Refereed][Not invited]
     
    We have shown that two 27-kD proteins, designated as WAP27A and WAP27B, were abundantly accumulated in endoplasmic reticulum-enriched fractions isolated from cortical parenchyma cells of mulberry tree (Mortis bombycis Koidz.) during winter (N. Ukaji, C. Kuwabara, D. Takezawa, K. Arakawa, S. Yoshida, S. Fujikawa [1999] Plant Physiol 120: 480-489). In the present study, cDNA clones encoding WAP27A and WAP27B were isolated and characterized. The deduced amino acid sequences of WAP27A and WAP27B cDNAs had 12 repeats of an 11-mer amino acid motif that was the common feature of group 3 late-embryogenesis-abundant proteins. Under field conditions, transcripts of WAP27 genes were initially detected in mid-October, reached maximum level from mid-November to mid-December, and then gradually decreased. The transcript levels of WAP27 genes in cortical parenchyma cells harvested in October was drastically induced by cold treatment within a few days, whereas those in cortical parenchyma cells harvested in August were low even by cold treatment for 3 weeks. Immunocytochemical analysis by electron microscopy confirmed that WAP27 was localized specifically in vesicular-form ER and also localized in dehydration-induced multiplex lamellae-form ER. The role of WAP27 in the ER is discussed in relation to acquisition of freezing tolerance of cortical parenchyma cells in mulberry tree during winter.
  • Y Kawamura, K Arakawa, M Maeshima, S Yoshida
    EUROPEAN JOURNAL OF BIOCHEMISTRY 268 (10) 2801 - 2809 0014-2956 2001/05 [Refereed][Not invited]
     
    Vacuolar H+-ATPase (V-ATPase) consists of a catalytic head, a stalk part and a membrane domain. We indirectly investigated the interaction between the A subunit (catalytic head) and the E subunit (stalk part) using an ATP analogue, adenosine 5'-[beta,gamma -imino]triphosphate (AMP-PNP), which holds the enzyme in the substrate-binding state. AMP-PNP treatment caused a mobility shift of the E subunit with a faster migration in SDS/polyacrylamide gel electrophoresis without a reductant, while ATP treatment did not. A mobility shift of the E subunit has been detected in several plants. As polypeptides with intramolecular disulfide bonds migrate faster than those without disulfide bonds, the mobility shift may be due to the formation of an intramolecular disulfide bond by two cysteine residues conserved among several plant species. The mobility shift may be involved in the binding of AMP-PNP to the ATP-binding site, which exists in the A and B subunits, as it was inhibited by the addition of ATP. Pretreatment with 2'-3'-O-(4-benzoylbenzoyl)-ATP (Bz-ATP), which modifies the ATP-binding site of the B subunit under UV illumination, did not inhibit the mobility shift of the E subunit caused by AMP-PNP treatment. The response of V-ATPase following the AMP-PNP binding may cause a conformational change in the E subunit into a form that is susceptible to oxidation of cysteine residues. This is the first demonstration of interaction between the A and E subunits in the substrate-binding state of a plant V-ATPase.
  • Y Kawamura, K Arakawa, M Maeshima, S Yoshida
    JOURNAL OF BIOLOGICAL CHEMISTRY 275 (9) 6515 - 6522 0021-9258 2000/03 [Refereed][Not invited]
     
    Immunoblot analyses and partial amino acid sequencings revealed that both the 40- (E1) and 37-kDa (E2) subunits of V-ATPase in the pea epicotyl were E subunit isoforms. Similarly, both the 35- (D1) and 29-kDa (D2) subunits were D subunit isoforms, although the similarity of the amino acid sequences is still unknown. In immunoblot analyses, two or three E subunit isoforms with molecular masses ranging from 29 to 40 kDa were detected in other plants. Two isotypes of V-ATPase from the pea epicotyl were separated by ion exchange chromatography and had subunit compositions differing only in the ratio of E1 and E2. There was a difference in the V-max and K-m of ATP hydrolysis between the two isotypes. E1 was scarcely detected in crude membrane fractions from the leaf and cotyledon, while E2 was detected in fractions from all of the tissues examined. The compositions of D subunit isoforms in the leaf and epicotyl were different, and the vacuolar membrane in the leaf did not contain D2. The efficiency of H+ pumping activity in the vacuolar membrane of the leaf was higher than that of the epicotyl. The results suggest that the presence of the isoforms of D and E subunits is characteristic to plants and that the isoforms are closely related to the enzymatic properties.
  • Freezing adaptation mechanisms of living tissue cells in Betula platyphylla var. japonica Hara and their relation to cell wall properties.
    Fujikawa S, Kubota M, Kuroda K, Takezawa D, Arakawa K
    Tree Sap II 93 - 96 2000 [Not refereed][Not invited]
  • S Yoshida, K Hotsubo, Y Kawamura, M Murai, K Arakawa, D Takezawa
    JOURNAL OF PLANT RESEARCH 112 (1106) 225 - 236 0918-9440 1999/06 [Refereed][Not invited]
     
    In chilling-sensitive plants, the inactivation of the vacuolar H+-ATPase is one of the primary cellular events directly resulting from cold exposure. We demonstrate here that cold-induced inactivation of the proton translocating enzyme is closely linked to the rapid acidification of the cytoplasm and the concomitant alkalization of the vacuoles, suggesting an important role of the enzyme in maintaining homeostasis of the cellular pH in a cold environment. The stability of the vacuolar H+-ATPase to cold both in vivo and in vitro is distinctly different between species sensitive and insensitive to cold. These findings provide further insight into the way in which the vacuolar H+-ATPase is involved in cold adaptation of plants. In addition, the temperature reduction and the concentration of the cytoplasm as a consequence of freeze-induced dehydration may also result in changes in the cellular pH. In fact, we demonstrate here that the cytoplasm is markedly acidified upon freezing; in particular, in cells of less hardy plants. Freeze-induced acidification is presumably due to changes in the physicochemical properties of the cytoplasm and the changes in the permeability of the vacuolar membrane both of which result from severe dehydration. The physiological significance of freeze-induced acidification of the cytoplasm is discussed.
  • M Nagao, K Arakawa, D Takezawa, S Yoshida, S Fujikawa
    JOURNAL OF PLANT RESEARCH 112 (1106) 163 - 174 0918-9440 1999/06 [Refereed][Not invited]
     
    The process of akinete formation in relation to the acquirement of freezing tolerance in a fresh water alga, Tribonema bombycinum (Xanthophyceae), was examined, T. bombycinum shifted from vegetative cells to akinetes with starving by a prolonged batch culture, by culture with a diluted medium, or by culture with a single nutrient-deficient medium. In addition, akinetes developed by desiccation, but cold treatment at 4 C did not facilitate akinete formation. During starving, the vegetative cells, which had a large central vacuole in the protoplasm and thin cell walls, finally changed to akinetes, which had many small vacuoles and oil droplets in the protoplasm and thick cell walls. During akinete formation by starving, the freezing tolerance (LT50) increased gradually from -3C in vegetative cells to far below -30C in akinetes. When vegetative cells were subjected to equilibrium freezing, their size shrank greatly and aparticulate domains accompanied by fracture-jump lesions developed in the plasma membranes. Akinetes subjected to equilibrium freezing showed little shrinkage, and freezing-induced ultrastructural changes did not occur in the plasma membranes. The morphological changes in the process of akinete formation and the responses to equilibrium freezing resembled those of cold-acclimated terrestrial plants.
  • N Ukaji, C Kuwabara, D Takezawa, K Arakawa, S Yoshida, S Fujikawa
    PLANT PHYSIOLOGY 120 (2) 481 - 489 0032-0889 1999/06 [Refereed][Not invited]
     
    Cortical parenchyma cells of mulberry (Morus bombycis Koidz.) trees acquire extremely high freezing tolerance in winter as a result of seasonal cold acclimation. The amount of total proteins in endoplasmic reticulum (ER)-enriched fractions isolated from these cells increased in parallel with the process of cold acclimation. Protein compositions in the ER-enriched fraction also changed seasonally, with a prominent accumulation of 20-kD (WAP20) and 27-kD (WAP27) proteins in winter. The N-terminal amino acid sequence of WAP20 exhibited homology to ER-localized small heat-shock proteins (smHSPs), whereas that of WAP27 did not exhibit homology to any known proteins. Like other smHSPs, WAP20 formed a complex of high molecular mass in native-polyacrylamide gel electrophoresis. Furthermore, not only WAP20 but also 21-kD proteins reacted with antibodies against WAP20. Fractionation of the crude microsomes by isopycnic sucrose-gradient centrifugation revealed that both WAP27 and WAP20 were distributed on a density corresponding to the fractions with higher activity of ER marker enzyme, suggesting localization of these proteins in the ER. When ER-enriched fractions were treated with trypsin in the absence of detergent, WAP20 and WAP27 were undigested, suggesting localization of these proteins inside the ER vesicle. The accumulation of a large quantity of smHSPs in the ER in winter as a result of seasonal cold acclimation indicates that these proteins may play a significant role in the acquisition of freezing tolerance in cortical parenchyma cells of mulberry trees.
  • C Kuwabara, K Arakawa, S Yoshida
    PLANT AND CELL PHYSIOLOGY 40 (2) 184 - 191 0032-0781 1999/02 [Refereed][Not invited]
     
    In suspension-cultured cells of winter wheat (Triticum aestivum L. cv. Chihokukomugi), the accumulation of soluble secretory proteins in the culture medium was promoted by ABA treatment in comparison with non-treated cells. The total amount of secretory proteins in ABA-treated cells was 1.7-fold higher than that in nontreated cells. The analysis of two-dimensional electrophoresis revealed that at least twelve secretory proteins were induced by ABA, and these were named WAS (wheat ABA-induced secretory) proteins 1 to 12. N-terminal amino acid sequence analysts of WAS proteins revealed the sequences of WAS-2 and WAS-3. Homology searches showed that WAS-2 had 55% identity with the N-terminus of the wheat chemically induced gene (WCI-5 gene) product. WAS-8 was also shown to have 93% identify with the N-terminus of the barley protein R, a typical member of thaumatin-like proteins (TLPs). Immunoblot analysis also suggested that WAS3 was related to protein R. These results suggest that exogenous ABA induces some basic secretory proteins that are related to the plant defense system in wheat.
  • KUBOTA Masatoshi, ARAKAWA Keita, KURODA Katsushi, FUJIKAWA Seizo
    Cryobiology and Cryotechnology 低温生物工学会 45 (2) 124 - 127 1340-7902 1999 [Refereed][Not invited]
     
    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.
  • M Koike, D Takezawa, K Arakawa, S Yoshida
    PLANT AND CELL PHYSIOLOGY 38 (6) 707 - 716 0032-0781 1997/06 [Refereed][Not invited]
     
    Suspension-cultured cells derived from immature embryos of winter wheat (Triticum aestivum L. cv. Chihoku) were used in experiments designed to obtain clues to the mechanism of the ABA-induced development of freezing tolerance. Cultured cells treated with 50 mu M ABA for 5 d at 23 degrees C acquired the maximum level of freezing tolerance (LT50; -21.6 degrees C). The increased freezing tolerance of ABA-treated cells was closely associated with the remarkable accumulation of 19-kDa polypeptides in the plasma membrane. The 19-kDa polypeptide components were isolated by preparative gel electrophoresis and were further separated into one major (AWPM-19) and other minor polypeptide components by Tricine-SDS-PAGE, N-terminal amino acid sequence of AWPM-19 was determined, and a cDNA clone encoding AWPM-19 was isolated by PCR from the library prepared from the ABA-treated cultured cells. The cDNA clone (WPM-I) encoded a 18.9 kDa hydrophobic polypeptide with four putative membrane spanning domains and with a high pi value (10.2). Expression of WPM-I mRNA was dramatically induced by 50 mu M ABA within a few hours. These results suggest that the AWPM-19 might be closely associated with the ABA-induced increase in freezing tolerance in wheat cultured cells.
  • K Hotsubo, Y Kawamura, D Takezawa, K Arakawa, S Yoshida
    PLANT COLD HARDINESS 237 - 244 1997 [Refereed][Not invited]
     
    The susceptibility of the vacuolar H+-ATPase to cold in vivo differed markedly between leguminous species sensitive and tolerant to cold. In chilling-sensitive species such as mung bean (Vigna radiata L.), the vacuolar H+-ATPase exhibited a marked decline in activity during the early process of cold treatment. In chilling-tolerant species such as pea (Pisum sativm L.), by contrast, the H+-ATPase remained to be highly active for long periods of cold treatment. The obvious difference in the cold susceptibility of the vacuolar H+-ATPases in vivo between the two species was also reflected in the sensitivity to the MgATP-dependent cold inactivation in vitro, in particular, with respect to the sensitivity to chaotropic anions, such as Cl-, NO2-, and NO3-. The H+-ATPase from chilling-sensitive species is more susceptible to the anions, especially to the NO2-, than the enzyme from chilling-tolerant species. The vacuolar H+-ATPases were categorized into two types, namely, the cold-sensitive "mung bean-type" and the cold-stable "pea-type" enzymes, in terms of the susceptibility to the MgATP-dependent cold inactivation in the presence of NO2- ions. Immunoblot analyses using anti-mung bean H+-ATPase antibodies have revealed that the antigenicity of the 16 kD proteolipids is distinctly different between the two types of enzyme.
  • M ODAIRA, K ARAKAWA, S YOSHIDA, M MAESHIMA
    PLANT AND CELL PHYSIOLOGY 36 (6) 945 - 953 0032-0781 1995/09 [Refereed][Not invited]
     
    Nuclei from mung bean (Vigna radiata) hypocotyls contained two glycoproteins of 50 and 49 kDa, respectively, that reacted with concanavalin A. The glycoproteins were released from the nuclear envelope by treatment with 2 M KCI but not with nucleases. The glycoproteins, tentatively named gp50 and gp49, were isolated and characterized. Gel-permeation chromatography suggested that gp50 and gp49 seem to exist as a complex with other components. The glycoproteins could be detected only in the nuclear fraction by immunoblot analysis with specific antibodies, and they were not detected in endoplasmic reticulum, plasma membrane, vacuolar membrane or mitochondria. Agglutinin I from Ulex europeaus, peanut agglutinin, soybean agglutinin and wheat germ agglutinin all failed to bind to the glycoproteins. Treatment with glycopeptidase F removed all oligosaccharides from the glycoproteins and decreased their molecular masses by about one thousand daltons each. These results suggest that the glycoproteins contained N-linked, high-mannose-type oligosaccharides with six or seven hexose residues. gp50 and gp49 seemed to be isoforms of a single glycoprotein because the two proteins had some common properties. Nuclear fractions from azuki bean (Phaseolus angularis) and soybean (Glycine max) contained proteins that were immunologically similar to gp50 and gp49.
  • BL ZHOU, K ARAKAWA, S FUJIKAWA, S YOSHIDA
    PLANT AND CELL PHYSIOLOGY 35 (2) 175 - 182 0032-0781 1994/03 [Refereed][Not invited]
     
    The objective of this study was to identify plasma membrane proteins that are specifically induced by cold acclimation in wheat (Triticum aestivum L.). Two cultivars with a marked difference in the genetic ability to cold-acclimate, namely, spring wheat (cv. Chinese Spring) and winter wheat (cv. Norstar), were used as the experimental material. After four weeks of growth in a cold chamber, the freezing tolerance in the shoots of winter wheat increased to -18-degrees-C, whereas it increased only to - 8-degrees-C in the shoots of spring wheat. In the case of roots from both cultivars, freezing tolerance increased only slightly after the growth in the cold environment. Cold acclimation induced remarkable changes in the electrophoretic patterns of plasma membrane proteins which depended on both the cultivar and the tissue examined. Levels of polypeptides with molecular masses from 22 to 31 kDa decreased in both the root and shoot plasma membranes from both cultivars. Among these polypeptides, levels of those of 28 and 26 kDa decreased abruptly after one week of cold acclimation. By contrast, levels of polypeptides of 89, 83, 52, 23, 18 and 17 kDa increased specifically in the shoots of winter wheat. The increases in the levels of the 23-, 18- and 17-kDa polypeptides were proportional to the development of freezing tolerance. Freeze-fracture electron microscopy of plasma membranes from shoot cells revealed that the number of intramembrane particles on the fracture faces decreased markedly in winter wheat after cold acclimation, but to a lesser extent in spring wheat. These results suggest that the plasma membranes might undergo molecular reorganization during cold acclimation.
  • S. KISHITANI, K. WATANABE, S. YASUDA, K. ARAKAWA, T. TAKABE
    Plant, Cell and Environment 17 (1) 89 - 95 0140-7791 1994/01 [Refereed][Not invited]
  • ISHITANI, M, ARAKAWA, K, MIZUNO, K, KISHITANI, S, TAKABE, T
    PLANT AND CELL PHYSIOLOGY Japanese Society of Plant Physiologists 34 (3) 493 - 495 0032-0781 1993/04 [Refereed][Not invited]
     
    The accumulation of betaine and the distribution of betaine aldehyde dehydrogenase, which catalyzes the last step in the synthesis of betaine, were analyzed in leaves of control and salt-stressed cereal plants of the Gramineae. BADH protein was present in both betaine-accumulating and nonaccumulating leaves.
  • IMMUNOLOGICAL STUDIES OF BETAINE ALDEHYDE DEHYDROGENASE IN BARLEY
    ARAKAWA, K, MIZUNO, K, KISHITANI, S, TAKABE, T
    PLANT AND CELL PHYSIOLOGY 33 (7) 833 - 840 1992/10 [Refereed][Not invited]
  • ARAKAWA, K, KATAYAMA, M, TAKABE, T
    PLANT AND CELL PHYSIOLOGY 31 (6) 797 - 803 1990/09 [Refereed][Not invited]
  • ISHITANI Manabu, ARAKAWA Keita, TAKEBE Tetsuko
    Chemical Regulation of Plants 植物化学調節学会 25 149 - 162 0388-9130 1990 [Not refereed][Not invited]
  • Betaine accumulation and osmoregulation in higher plants and cyanobacteria
    Takabe T, Arakawa K
    Plant water relations and growth under stress 151 - 158 1989 [Not refereed][Not invited]
  • Tetsuko Takabe, Aran Incharoensakdi, Keita Arakawa, Sadaki Yokota
    Plant Physiology 88 (4) 1120 - 1124 0032-0889 1988/12/01 [Refereed][Not invited]
  • Keita Arakawa, Tetsuko Takabe, Tatsuo Sugiyama, Takashi Akazawa
    Journal of Biochemistry 101 (6) 1485 - 1488 0021-924X 1987 [Refereed][Not invited]
     
    Betaine-aldehyde dehydrogenase was purified from spinach leaves and characterized. The Molecular weight of the enzyme was estimated to be 120 kDa by a gel filtration chromatorgraphy. The enzyme was judged to consist of two identical pieces of the monomeric subunit with molecular weight of 60 kDa. A specific polyclonal antibody was raised against the enzyme subunit. © 1987 Oxford University Press.

MISC

  • 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
  • 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
  • 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
  • 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
  • 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
  • 小池 孝良, 菅井 徹人, 渡部 敏裕, 市川 一, 藤戸 永志, 佐々木 圭子, 曲 来葉, 渡辺 誠, 荒川 圭太, 山﨑 友紀, 佐藤 冬樹  北方森林保全技術  (37)  11  -17  2019/12  [Not refereed][Not invited]
  • 山岸 祐介, 宮﨑 淳子, 荒川 圭太, 河原﨑 政行  木材工業 = Wood industry  73-  (10)  406  -408  2018/10  [Not refereed][Not invited]
  • 荒川 圭太  グリーンテクノ情報  14-  (1)  13  -17  2018/06  [Not refereed][Not invited]
  • 堀内玲子, 実山豊, 荒川圭太, 鈴木卓  園芸学研究 別冊  17-  (2)  2018
  • 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
  • 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  2013/11  [Not refereed][Not invited]
  • 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
  • 鈴木伸吾, 遠藤圭太, 岡田香織, 荒川圭太, 高橋大輔, 上村松生  日本木材学会北海道支部講演集(Web)  (44)  WEB ONLY P-2  2012/11  [Not refereed][Not invited]
  • 坂本友陽, 岡田香織, 鈴木伸吾, 宇梶慎子, 荒川圭太  日本木材学会北海道支部講演集(Web)  (44)  WEB ONLY P-6  2012/11  [Not refereed][Not invited]
  • Chikako Kuwabara, Jun Kasuga, Donghui Wang, Yukiharu Fukushi, Keita Arakawa, Seizo Fujikawa  CRYOBIOLOGY  64-  (3)  308  -308  2012/06  [Not refereed][Not invited]
  • INADA Takaaki, KOYAMA Toshie, KUWABARA Chikako, ARAKAWA Keita, FUJIKAWA Seizo  The Proceedings of Mechanical Engineering Congress, Japan  2012-  (0)  _J024031  -1-_J024031-3  2012  [Not refereed][Not invited]
     
    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.
  • 森若元太, 宇梶槙子, 藤川清三, 荒川圭太  日本木材学会北海道支部講演集(Web)  (43)  WEB ONLY O-3  2011/11  [Not refereed][Not invited]
  • 板羽貴史, 砂留光利, 上出奈央, 佐野雄三, 藤川清三, 荒川圭太  日本木材学会北海道支部講演集(Web)  (42)  WEB ONLY O-2  2010/11  [Not refereed][Not invited]
  • 能美彩香, 森本和成, 藤川清三, 荒川圭太  日本木材学会北海道支部講演集(Web)  (42)  WEB ONLY O-1  2010/11  [Not refereed][Not invited]
  • 遠藤圭太, 荒川圭太, 藤川清三  日本木材学会北海道支部講演集(Web)  (42)  WEB ONLY O-3  2010/11  [Not refereed][Not invited]
  • 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  [Not refereed][Not invited]
  • 遠藤圭太, 荒川圭太, 藤川清三  日本木材学会北海道支部講演集  (41)  7  -10  2009/10/20  [Not refereed][Not invited]
  • Morimoto Kazunari, Mori Hitoshi, Ksuga Jun, Fujikawa Seizo, Arakawa Keita  Plant and Cell Physiology Supplement  2009-  (0)  512  -512  2009  [Not refereed][Not invited]
     
    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.
  • 遠藤圭太, 荒川圭太, 藤川清三  日本木材学会北海道支部講演集  (40)  11  -14  2008/10/22  [Not refereed][Not invited]
  • Morimoto Kazunari, Arakawa Keita, Fujikawa Seizo  Plant and Cell Physiology Supplement  2008-  (0)  874  -874  2008  [Not refereed][Not invited]
     
    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: <-50oC) in winter. After the artificial deacclimation of larch twigs of winter, DSC ability was promptly decreased to -34oC 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.
  • 森本和成, 藤川清三, 荒川圭太  日本木材学会北海道支部講演集  (39)  11  -14  2007/10/22  [Not refereed][Not invited]
  • 葭葉恵, 春日純, 荒川圭太, 藤川清三  日本木材学会北海道支部講演集  (39)  19  -22  2007/10/22  [Not refereed][Not invited]
  • 春日純, 葭葉恵, 橋床泰之, 荒川圭太, 藤川清三  日本木材学会北海道支部講演集  (39)  15  -18  2007/10/22  [Not refereed][Not invited]
  • 春日純, 荒川圭太, 藤川清三  日本顕微鏡学会北海道支部学術講演会プログラム・予稿集  2006-  15  2007/02  [Not refereed][Not invited]
  • 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
  • Inada Hidetoshi, Fujikawa Seizo, Arakawa Keita  Plant and Cell Physiology Supplement  2007-  (0)  479  -479  2007  [Not refereed][Not invited]
     
    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.
  • 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
  • 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  [Not refereed][Not invited]
  • 水野薫, 春日純, 荒川圭太, 藤川清三  日本木材学会北海道支部講演集  (37)  13  -16  2005/10/21  [Not refereed][Not invited]
  • Kuwabara Chikako, Kondo Hidemasa, Noro Natsuko, Takezawa Daisuke, Arakawa Keita, Tsuda Sakae  Plant and Cell Physiology Supplement  2005-  (0)  848  -848  2005  [Not refereed][Not invited]
     
    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.
  • Minami Anzu, Nagao Manabu, Arakawa Keita, Fujikawa Seizo, Takezawa Daisuke  Plant and Cell Physiology Supplement  2005-  (0)  222  -222  2005  [Not refereed][Not invited]
     
    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.
  • Nagao Manabu, Ukaji Norifumi, Takezawa Daisuke, Arakawa Keita, Fujikawa Seizo  Plant and Cell Physiology Supplement  2005-  (0)  221  -221  2005  [Not refereed][Not invited]
     
    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, -20oC. The cryoprotective activity of bovine serum albumin was nearly equal to that of rWAP27 during prolonged freezing at -20oC, but decreased with freezing time at -2 and -4oC. The cryoprotective activity of ovalbumin was also decreased with freezing time at -2 to -20oC. The rWAP27 has a high cryoprotective activity to freeze-labile proteins against prolonged freezing.
  • Inada Hidetoshi, Ito Toshiaki, Nagao Manabu, Fujikawa Seizo, Arakawa keita  Plant and Cell Physiology Supplement  2005-  (0)  381  -381  2005  [Not refereed][Not invited]
     
    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.
  • 古俣寛隆, 伊藤利章, 佐野雄三, 荒川圭太, 藤川清三  日本木材学会北海道支部講演集  (36)  27  -30  2004/10/28  [Not refereed][Not invited]
  • 山根健一, 伊藤利章, 佐野雄三, 荒川圭太, 藤川清三  日本木材学会北海道支部講演集  (36)  23  -26  2004/10/28  [Not refereed][Not invited]
  • 高島寛, 荒川圭太, 藤川清三  日本木材学会北海道支部講演集  (36)  31  -34  2004/10/28  [Not refereed][Not invited]
  • 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  [Not refereed][Not invited]
  • Inada Hidetoshi, Arakawa Keita  Plant and Cell Physiology Supplement  2004-  (0)  359  -359  2004  [Not refereed][Not invited]
     
    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.
  • Minami Anzu, Nagao Manabu, Arakawa Keita, Fujikawa Seizo, Takezawa Daisuke  Plant and Cell Physiology Supplement  2004-  (0)  803  -803  2004  [Not refereed][Not invited]
     
    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.
  • 高田直樹, 藤川清三, 荒川圭太, 南杏鶴, 竹沢大輔  日本木材学会北海道支部講演集  (35)  1  -4  2003/10/15  [Not refereed][Not invited]
  • Fujibe Takahiro, Saji Hikaru, Arakawa Keita, Takeuchi Yuichi, Yamamoto Kotaro T.  Plant and Cell Physiology Supplement  2003-  (0)  347  -347  2003  [Not refereed][Not invited]
     
    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.
  • Ukaji Norifumi, Takezawa Daisuke, Arakawa Keita, Fujikawa Seizo  Plant and Cell Physiology Supplement  2003-  (0)  308  -308  2003  [Not refereed][Not invited]
     
    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 4oC 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.
  • Arakawa Keita, Kasuga Jun, Fujikawa Seizo  Plant and Cell Physiology Supplement  2003-  (0)  307  -307  2003  [Not refereed][Not invited]
     
    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.
  • Minami Anzu, Nagao Manabu, Arakawa Keita, Fujikawa Seizo, Takezawa Daisuke  Plant and Cell Physiology Supplement  2003-  (0)  459  -459  2003  [Not refereed][Not invited]
     
    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.
  • N Ukaji, D Takezawa, K Arakawa, S Fujikawa  PLANT AND CELL PHYSIOLOGY  43-  S116  -S116  2002  [Not refereed][Not invited]
  • M Nagao, A Minami, K Arakawa, S Fujikawa, D Takezawa  PLANT AND CELL PHYSIOLOGY  43-  S168  -S168  2002  [Not refereed][Not invited]
  • NAGAO Manabu, MINAMI Anzu, TAKEZAWA Daisuke, ARAKAWA Keita, FUJIKAWA Seizo  Plant and cell physiology  42-  s121  2001
  • KUWABARA Chikako, TAKEZAWA Daisuke, Fujikawa Seizo, ARAKAWA Keita  Plant and cell physiology  42-  s142  2001
  • Ukaji Norifumi, Takezawa Daisuke, Arakawa Keita, Fujikawa Seizo  Plant and cell physiology  42-  s143  2001
  • ARAKAWA Keita, TAKEZAWA Daisuke, KUWABARA Chikako, KURODA Katsushi, YOSHIDA Shizuo, FUJIKAWA Seizo  Plant and cell physiology  42-  s12  2001
  • KUWABARA Chikako, ARAKAWA Keita, TAKEZAWA Daisuke, FUJIKAWA Seizo  Plant and cell physiology  41-  s50  2000
  • UKAJI Norifumi, TAKEZAWA Daisuke, ARAKAWA Keita, FUJIKAWA Seizo  Plant and cell physiology  41-  s48  2000
  • TAKEZAWA Daisuke, ARAKAWA Keita, YOSHIDA Shizuo, FUJIKAWA Seizo  Plant and cell physiology  41-  s10  2000
  • KUWABARA Chikako, ARAKAWA Keita, TAKEZAWA Daisuke, FUJIKAWA Seizo  Plant and cell physiology  40-  s75  -s75  1999/03
  • UKAJI Norifumi, TAKEZAWA Daisuke, ARAKAWA Keita, FUJIKAWA Seizo  Plant and cell physiology  40-  s104  -s104  1999/03
  • ARAKAWA Keita, HANAZAKI Mitsuru, YOSHIDA Shizuo  Plant and cell physiology  40-  s104  -s104  1999/03
  • KUWABARA Chikako, TAKEZAWA Daisuke, ARAKAWA Keita, YOSHIDA Shizuo  Plant and cell physiology  39-  S92  -S92  1998/05
  • UKAJI Norifumi, TAKEZAWA Daisuke, ARAKAWA Keita, FUJIKAWA Seizo, YOSHIDA Shizuo  Plant and cell physiology  39-  S139  -S139  1998/05
  • KOIKE Michiya, TAKEZAWA Daisuke, ARAKAWA Keita, YOSHIDA Shizuo  Plant and cell physiology  38-  s97  1997/03
  • ARAKAWA Keita, YOSHIDA Shizuo  Plant and cell physiology  38-  s97  1997/03
  • KUWABARA Chikako, ARAKAWA Keita, YOSHIDA Shizuo  Plant and cell physiology  38-  s42  1997/03
  • 荒川 圭太, 吉田 静夫  日本植物学会大会研究発表記録 = Proceedings of the annual meeting of the Botanical Society of Japan  60-  114  -114  1996/10  [Not refereed][Not invited]
  • 村井 麻理, 荒川 圭太, 吉田 静夫  日本植物学会大会研究発表記録 = Proceedings of the annual meeting of the Botanical Society of Japan  60-  261  -261  1996/10  [Not refereed][Not invited]
  • MURAI Mari, ARAKAWA Keita, YOSHIDA Shizuo  Plant and cell physiology  36-  S141  1995/03
  • KAWAMURA Yukio, ARAKAWA Keita, YOSHIDA Shizuo  Plant and cell physiology  36-  S141  1995/03
  • KOIKE Michiya, ARAKAWA Keita, YOSHIDA Shizuo  Plant and cell physiology  36-  S127  1995/03
  • 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  [Not refereed][Not invited]
  • BETAINE ALDEHYDE DEHYDROGENASE PROTEIN IS PRESENT IN LEAVES OF BOTH BETAINE ACCUMULATORS AND NONACCUMULATORS IN VARIOUS CEREAL PLANTS
    ARAKAWA, K, MIZUNO, K, TAKABE, T  PHOTOSYNTHESIS RESEARCH  34-  (1)  218  -218  1992/10  [Not refereed][Not invited]
  • IMMUNOLOGICAL STUDIES OF BETAINE ALDEHYDE DEHYDROGENASE IN BARLEY
    ARAKAWA, K, MIZUNO, K, TAKABE, T  PHOTOSYNTHESIS RESEARCH  34-  (1)  217  -217  1992/10  [Not refereed][Not invited]

Books etc

  • 小池, 孝良, 北尾, 光俊, 市栄, 智明, 渡辺, 誠(農学) (Contributor11.5 低温応答)
    共立出版 2020/11 (ISBN: 9784320058125) xvi, 235p, 図版 [8] p
  • 河村, 公隆 (Contributor樹木細胞の凍結挙動)
    朝倉書店 2016/07 (ISBN: 425416128X) 432
  • 日本木材学会 (Contributor第３章３ 環境ストレスと形成層活動)
    文永堂出版 2011/04 (ISBN: 9784830041204) xii, 313p

Association Memberships

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

Research Projects

  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2021/04 -2024/03 
    Author : 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：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2015/04 -2018/03 
    Author : 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：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
    Date (from‐to) : 2015/04 -2017/03 
    Author : 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：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2011 -2013 
    Author : 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：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2008 -2010 
    Author : 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：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2008 -2010 
    Author : 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：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
    Date (from‐to) : 2007 -2010 
    Author : 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：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2005 -2007 
    Author : 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：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2004 -2005 
    Author : 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：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2002 -2004 
    Author : 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.
  • 樹木木部柔細胞における深過冷却機構の解析
    Date (from‐to) : 2003
  • Study on the mechanism of deep supercooling in xylem cells of woody plants
    Date (from‐to) : 2003
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2000 -2002 
    Author : 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.
  • 越冬性植物の酸性雪ストレス耐性機構
    Date (from‐to) : 2002
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 1999 -2001 
    Author : 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：Grants-in-Aid for Scientific Research Grant-in-Aid for Encouragement of Young Scientists (A)
    Date (from‐to) : 1995 -1995 
    Author : 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：Grants-in-Aid for Scientific Research Grant-in-Aid for General Scientific Research (B)
    Date (from‐to) : 1993 -1994 
    Author : 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.
  • 木本ならびに草本植物の寒冷適応機構の研究
    Date (from‐to) : 1992
  • Study on plant cold hardiness
    Date (from‐to) : 1992

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  • 特許6655414:排気浄化システム    2020/02/05
    布施卓哉, 藤川清三, 荒川圭太, 宇梶慎子
  • 特許6319932:不凍性液体  
    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
  • Supercooling promoting agents
    WO2008/007684

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