Evolution and divergence of the role of plant ethylene receptor–related histidine kinases in abscisic acid signaling Marcos T. Miyabe; Taketo Sasaki; Tsukasa Toriyama; Rahul Sk; Daisuke Takezawa; Izumi Yotsui; Teruaki Taji; Yoichi Sakata
Biochemical and Biophysical Research Communications,
巻：747,
開始ページ：151295,
終了ページ：151295, 2025年02月,
［査読有り］Elsevier BV, 研究論文（学術雑誌）
DOI：https://doi.org/10.1016/j.bbrc.2025.151295DOI ID：10.1016/j.bbrc.2025.151295,
ISSN：0006-291X Targeted in vivo mutagenesis of a sensor histidine kinase playing an essential role in ABA signaling of the moss Physcomitrium patens Rahul Sk; Marcos Takeshi Miyabe; Daisuke Takezawa; Shunsuke Yajima; Izumi Yotsui; Teruaki Taji; Yoichi Sakata
Biochemical and Biophysical Research Communications,
巻：637,
開始ページ：93,
終了ページ：99, 2022年12月,
［査読有り］Elsevier BV, 研究論文（学術雑誌）
DOI：https://doi.org/10.1016/j.bbrc.2022.11.009DOI ID：10.1016/j.bbrc.2022.11.009,
ISSN：0006-291X Differential regulations of abscisic acid-induced desiccation tolerance and vegetative dormancy by group B3 Raf kinases in liverwortsAkida Jahan; Yuto Yamazaki; Mousona Islam; Totan Kumar Ghosh; Nami Yoshimura; Hirotaka Kato; Kimitsune Ishizaki; Akihisa Shinozawa; Yoichi Sakata; Daisuke Takezawa
Frontiers in Plant Science,
巻：13, 2022年07月,
［査読有り］Phytohormone abscisic acid (ABA) plays a key role in stomata closure, osmostress acclimation, and vegetative and embryonic dormancy. Group B3 Raf protein kinases (B3-Rafs) serve as positive regulators of ABA and osmostress signaling in the moss Physcomitrium patens and the angiosperm Arabidopsis thaliana. While P. patens has a single B3-Raf called ARK, specific members of B3-Rafs among six paralogs regulate ABA and osmostress signaling in A. thaliana, indicating functional diversification of B3-Rafs in angiosperms. However, we found that the liverwort Marchantia polymorpha, belonging to another class of bryophytes, has three paralogs of B3-Rafs, MpARK1, MpARK2, and MpARK3, with structural variations in the regulatory domains of the polypeptides. By reporter assays of the P. patens ark line and analysis of genome-editing lines of M. polymorpha, we found that these B3-Rafs are functionally redundant in ABA response, with respect to inhibition of growth, tolerance to desiccation and expression of stress-associated transcripts, the majority of which are under the control of the PYR/PYL/RCAR-like receptor MpPYL1. Interestingly, gemmae in gemma cups were germinating only in mutant lines associated with MpARK1, indicating that dormancy in the gametophyte is controlled by a specific B3-Raf paralog. These results indicated not only conservation of the role of B3-Rafs in ABA and osmostress response in liverworts but also functional diversification of B3-Rafs, which is likely to have occurred in the early stages of land plant evolution.
Frontiers Media SA, 研究論文（学術雑誌）
DOI：https://doi.org/10.3389/fpls.2022.952820DOI ID：10.3389/fpls.2022.952820,
eISSN：1664-462X Sensor histidine kinases mediate ABA and osmostress signaling in the moss Physcomitrium patens Tsukasa Toriyama; Akihisa Shinozawa; Yuki Yasumura; Masashi Saruhashi; Mayuka Hiraide; Shiori Ito; Hideyuki Matsuura; Keiko Kuwata; Mika Yoshida; Tadashi Baba; Izumi Yotsui; Teruaki Taji; Daisuke Takezawa; Yoichi Sakata
Current Biology,
巻：32,
号：1,
開始ページ：164,
終了ページ：175.e8, 2022年01月,
［査読有り］Elsevier BV, 研究論文（学術雑誌）
DOI：https://doi.org/10.1016/j.cub.2021.10.068DOI ID：10.1016/j.cub.2021.10.068,
ISSN：0960-9822 Activation of SnRK2 by Raf-like kinase ARK represents a primary mechanism of ABA and abiotic stress responsesMousona Islam; Takumi Inoue; Mayuka Hiraide; Nobiza Khatun; Akida Jahan; Keiko Kuwata; Sotaro Katagiri; Taishi Umezawa; Izumi Yotsui; Yoichi Sakata; Daisuke Takezawa
Plant Physiology,
巻：185,
号：2,
開始ページ：533,
終了ページ：546, 2021年12月,
［査読有り］Abstract
The Raf-like protein kinase abscisic acid (ABA) and abiotic stress-responsive Raf-like kinase (ARK) previously identified in the moss Physcomitrium (Physcomitrella) patens acts as an upstream regulator of subgroup III SNF1-related protein kinase2 (SnRK2), the key regulator of ABA and abiotic stress responses. However, the mechanisms underlying activation of ARK by ABA and abiotic stress for the regulation of SnRK2, including the role of ABA receptor-associated group A PP2C (PP2C-A), are not understood. We identified Ser1029 as the phosphorylation site in the activation loop of ARK, which provided a possible mechanism for regulation of its activity. Analysis of transgenic P. patens ark lines expressing ARK-GFP with Ser1029-to-Ala mutation indicated that this replacement causes reductions in ABA-induced gene expression, stress tolerance, and SnRK2 activity. Immunoblot analysis using an anti-phosphopeptide antibody indicated that ABA treatments rapidly stimulate Ser1029 phosphorylation in the wild type (WT). The phosphorylation profile of Ser1029 in ABA-hypersensitive ppabi1 lacking protein phosphatase 2C-A (PP2C-A) was similar to that in the WT, whereas little Ser1029 phosphorylation was observed in ABA-insensitive ark missense mutant lines. Furthermore, newly isolated ppabi1 ark lines showed ABA-insensitive phenotypes similar to those of ark lines. Therefore, ARK is a primary activator of SnRK2, preceding negative regulation by PP2C-A in bryophytes, which provides a prototype mechanism for ABA and abiotic stress responses in plants.
Oxford University Press (OUP), 研究論文（学術雑誌）
DOI：https://doi.org/10.1093/plphys/kiaa046DOI ID：10.1093/plphys/kiaa046,
ISSN：0032-0889,
eISSN：1532-2548 Arabidopsis group C Raf-like protein kinases negatively regulate abscisic acid signaling and are direct substrates of SnRK2Yoshiaki Kamiyama; Misaki Hirotani; Shinnosuke Ishikawa; Fuko Minegishi; Sotaro Katagiri; Conner J. Rogan; Fuminori Takahashi; Mika Nomoto; Kazuya Ishikawa; Yutaka Kodama; Yasuomi Tada; Daisuke Takezawa; Jeffrey C. Anderson; Scott C. Peck; Kazuo Shinozaki; Taishi Umezawa
Proceedings of the National Academy of Sciences,
巻：118,
号：30,
開始ページ：e2100073118,
終了ページ：e2100073118, 2021年07月,
［査読有り］The phytohormone abscisic acid (ABA) plays a major role in abiotic stress responses in plants, and subclass III SNF1-related protein kinase 2 (SnRK2) kinases mediate ABA signaling. In this study, we identified Raf36, a group C Raf-like protein kinase in
Arabidopsis, as a protein that interacts with multiple SnRK2s. A series of reverse genetic and biochemical analyses revealed that 1) Raf36 negatively regulates ABA responses during postgermination growth, 2) the N terminus of Raf36 is directly phosphorylated by SnRK2s, and 3) Raf36 degradation is enhanced in response to ABA. In addition, Raf22, another C-type Raf-like kinase, functions partially redundantly with Raf36 to regulate ABA responses. A comparative phosphoproteomic analysis of ABA-induced responses of wild-type and
raf22raf36-1 plants identified proteins that are phosphorylated downstream of Raf36 and Raf22 in planta. Together, these results support a model in which Raf36/Raf22 function mainly under optimal conditions to suppress ABA responses, whereas in response to ABA, the SnRK2 module promotes Raf36 degradation as a means of alleviating Raf36-dependent inhibition and allowing for heightened ABA signaling to occur.
Proceedings of the National Academy of Sciences, 研究論文（学術雑誌）
DOI：https://doi.org/10.1073/pnas.2100073118DOI ID：10.1073/pnas.2100073118,
ISSN：0027-8424,
eISSN：1091-6490 Arabidopsis Raf‐like kinases act as positive regulators of subclass III SnRK2 in osmostress signalingShohei Katsuta; Goro Masuda; Hyeokjin Bak; Akihisa Shinozawa; Yoshiaki Kamiyama; Taishi Umezawa; Daisuke Takezawa; Izumi Yotsui; Teruaki Taji; Yoichi Sakata
The Plant Journal,
巻：103,
号：2,
開始ページ：634,
終了ページ：644, 2020年07月,
［査読有り］Wiley, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1111/tpj.14756DOI ID：10.1111/tpj.14756,
ISSN：0960-7412,
eISSN：1365-313X Phosphoproteomic profiling reveals ABA-responsive phosphosignaling pathways in Physcomitrella patens. Amagai A; Honda Y; Ishikawa S; Hara Y; Kuwamura M; Shinozawa A; Sugiyama N; Ishihama Y; Takezawa D; Sakata Y; Shinozaki K; Umezawa T
The Plant journal : for cell and molecular biology,
巻：94,
号：4,
開始ページ：699,
終了ページ：708, 2018年05月,
［査読有り］DOI：https://doi.org/10.1111/tpj.13891DOI ID：10.1111/tpj.13891,
ISSN：0960-7412,
PubMed ID：29575231,
Web of Science ID：WOS:000432036300012 Abscisic acid-induced gene expression in the liverwort Marchantia polymorpha is mediated by evolutionarily conserved promoter elements Totan K. Ghosh; Midori Kaneko; Khaleda Akter; Shuhei Murai; Kenji Komatsu; Kimitsune Ishizaki; Katsuyuki T. Yamato; Takayuki Kohchi; Daisuke Takezawa
PHYSIOLOGIA PLANTARUM,
巻：156,
号：4,
開始ページ：407,
終了ページ：420, 2016年04月,
［査読有り］Abscisic acid (ABA) is a phytohormone widely distributed among members of the land plant lineage (Embryophyta), regulating dormancy, stomata closure and tolerance to environmental stresses. In angiosperms (Magnoliophyta), ABA-induced gene expression is mediated by promoter elements such as the G-box-like ACGT-core motifs recognized by bZIP transcription factors. In contrast, the mode of regulation by ABA of gene expression in liverworts (Marchantiophyta), representing one of the earliest diverging land plant groups, has not been elucidated. In this study, we used promoters of the liverwort Marchantia polymorpha dehydrin and the wheat Em genes fused to the -glucuronidase (GUS) reporter gene to investigate ABA-induced gene expression in liverworts. Transient assays of cultured cells of Marchantia indicated that ACGT-core motifs proximal to the transcription initiation site play a role in the ABA-induced gene expression. The RY sequence recognized by B3 transcriptional regulators was also shown to be responsible for the ABA-induced gene expression. In transgenic Marchantia plants, ABA treatment elicited an increase in GUS expression in young gemmalings, which was abolished by simultaneous disruption of the ACGT-core and RY elements. ABA-induced GUS expression was less obvious in mature thalli than in young gemmalings, associated with reductions in sensitivity to exogenous ABA during gametophyte growth. In contrast, lunularic acid, which had been suggested to function as an ABA-like substance, had no effect on GUS expression. The results demonstrate the presence of ABA-specific response mechanisms mediated by conserved cis-regulatory elements in liverworts, implying that the mechanisms had been acquired in the common ancestors of embryophytes.
WILEY-BLACKWELL, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1111/ppl.12385DOI ID：10.1111/ppl.12385,
ISSN：0031-9317,
eISSN：1399-3054,
PubMed ID：26456006,
Web of Science ID：WOS:000372974400004 Plant Raf-like kinase integrates abscisic acid and hyperosmotic stress signaling upstream of SNF1-related protein kinase2. Saruhashi M; Kumar Ghosh T; Arai K; Ishizaki Y; Hagiwara K; Komatsu K; Shiwa Y; Izumikawa K; Yoshikawa H; Umezawa T; Sakata Y; Takezawa D
Proceedings of the National Academy of Sciences of the United States of America,
巻：112,
号：46,
開始ページ：E6388, 2015年11月,
［査読有り］Proceedings of the National Academy of Sciences
DOI：https://doi.org/10.1073/pnas.1511238112DOI ID：10.1073/pnas.1511238112,
ISSN：0027-8424,
ORCID：31588702,
PubMed ID：26540727 Epoxycarotenoid‐mediated synthesis of abscisic acid in Physcomitrella patens implicating conserved mechanisms for acclimation to hyperosmosis in embryophytesDaisuke Takezawa; Naoki Watanabe; Totan Kumar Ghosh; Masashi Saruhashi; Atsushi Suzuki; Kanako Ishiyama; Shinnosuke Somemiya; Masatomo Kobayashi; Yoichi Sakata
New Phytologist,
巻：206,
号：1,
開始ページ：209,
終了ページ：219, 2014年12月,
［査読有り］Summary
Plants acclimate to environmental stress signals such as cold, drought and hypersalinity, and provoke internal protective mechanisms. Abscisic acid (ABA), a carotenoid‐derived phytohormone, which increases in response to the stress signals above, has been suggested to play a key role in the acclimation process in angiosperms, but the role of ABA in basal land plants such as mosses, including its biosynthetic pathways, has not been clarified.
Targeted gene disruption of PpABA1, encoding zeaxanthin epoxidase in the moss Physcomitrella patens was conducted to determine the role of endogenous ABA in acclimation processes in mosses.
The generated ppaba1 plants were found to accumulate only a small amount of endogenous ABA. The ppaba1 plants showed reduced osmotic acclimation capacity in correlation with reduced dehydration tolerance and accumulation of late embryogenesis abundant proteins. By contrast, cold‐induced freezing tolerance was less affected in ppaba1, indicating that endogenous ABA does not play a major role in the regulation of cold acclimation in the moss.
Our results suggest that the mechanisms for osmotic acclimation mediated by carotenoid‐derived synthesis of ABA are conserved in embryophytes and that acquisition of the mechanisms played a crucial role in terrestrial adaptation and colonization by land plant ancestors.
Wiley, 研究論文（学術雑誌）
DOI：https://doi.org/10.1111/nph.13231DOI ID：10.1111/nph.13231,
ISSN：0028-646X,
eISSN：1469-8137 Biochemical and structural characterization of an endoplasmic reticulum-localized late embryogenesis abundant (LEA) protein from the liverwort Marchantia polymorpha. Hatanaka R; Furuki T; Shimizu T; Takezawa D; Kikawada T; Sakurai M; Sugawara Y
Biochemical and biophysical research communications,
巻：454,
号：4,
開始ページ：588,
終了ページ：593, 2014年11月,
［査読有り］Hatanaka R, Furuki T, Shimizu T, Takezawa D, Kikawada T, Sakurai M, Sugawara Y, Biochemical and biophysical research communications, 2014, vol. 454, no. 4, pp. 588-593
DOI：https://doi.org/10.1016/j.bbrc.2014.10.130DOI ID：10.1016/j.bbrc.2014.10.130,
ISSN：0006-291X,
ORCID：14673271,
PubMed ID：25450698 Abscisic acid-induced rearrangement of intracellular structures associated with freezing and desiccation stress tolerance in the liverwort Marchantia polymorpha Khaleda Akter; Masahiro Kato; Yuki Sato; Yasuko Kaneko; Daisuke Takezawa
JOURNAL OF PLANT PHYSIOLOGY,
巻：171,
号：15,
開始ページ：1334,
終了ページ：1343, 2014年09月,
［査読有り］The plant growth regulator abscisic acid (ABA) is known to be involved in triggering responses to various environmental stresses such as freezing and desiccation in angiosperms, but little is known about its role in basal land plants, especially in liverworts, representing the earliest land plant lineage. We show here that survival rate after freezing and desiccation of Marchantia polymorpha gemmalings was increased by pretreatment with ABA in the presence of increasing concentrations of sucrose. ABA treatment increased accumulation of soluble sugars in gemmalings, and sugar accumulation was further increased by addition of sucrose to the culture medium. ABA treatment of gemmalings also induced accumulation of transcripts for proteins with similarity to late embryogenesis abundant (LEA) proteins, which accumulate in association with acquisition of desiccation tolerance in maturing seeds. Observation by light and electron microscopy indicated that the ABA treatment caused fragmentation of vacuoles with increased cytosolic volume, which was more prominent in the presence of a high concentration of external sucrose. ABA treatment also increased the density of chloroplast distribution and remarkably enlarged their volume. These results demonstrate that ABA induces drastic physiological changes in liverwort cells for stress tolerance, accompanied by accumulation of protectants against dehydration and rearrangement and morphological alterations of cellular organelles. (C) 2014 Elsevier GmbH. All rights reserved.
ELSEVIER GMBH, URBAN & FISCHER VERLAG, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1016/j.jplph.2014.05.004DOI ID：10.1016/j.jplph.2014.05.004,
ISSN：0176-1617,
eISSN：1618-1328,
Web of Science ID：WOS:000345631000003 Group A PP2Cs evolved in land plants as key regulators of intrinsic desiccation tolerance Kenji Komatsu; Norihiro Suzuki; Mayuri Kuwamura; Yuri Nishikawa; Mao Nakatani; Hitomi Ohtawa; Daisuke Takezawa; Motoaki Seki; Maho Tanaka; Teruaki Taji; Takahisa Hayashi; Yoichi Sakata
NATURE COMMUNICATIONS,
巻：4,
開始ページ：2219, 2013年07月,
［査読有り］Vegetative desiccation tolerance is common in bryophytes, although this character has been lost in most vascular plants. The moss Physcomitrella patens survives complete desiccation if treated with abscisic acid (ABA). Group A protein phosphatases type 2C (PP2C) are negative regulators of abscisic acid signalling. Here we show that the elimination of Group A PP2C is sufficient to ensure P. patens survival to full desiccation, without ABA treatment, although its growth is severely hindered. Microarray analysis shows that the Group A PP2C-regulated genes exclusively overlap with genes exhibiting a high level of ABA induction. Group A PP2C disruption weakly affects ABA-activated kinase activity, indicating Group A PP2C action downstream of these kinases in the moss. We propose that Group A PP2C emerged in land plants to repress desiccation tolerance mechanisms, possibly facilitating plants propagation on land, whereas ABA releases the intrinsic desiccation tolerance from Group A PP2C regulation.
NATURE PUBLISHING GROUP, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1038/ncomms3219DOI ID：10.1038/ncomms3219,
ISSN：2041-1723,
PubMed ID：23900426,
Web of Science ID：WOS:000323717600002 Cold acclimation in the moss Physcomitrella patens involves abscisic acid-dependent signaling. Bhyan SB; Minami A; Kaneko Y; Suzuki S; Arakawa K; Sakata Y; Takezawa D
Journal of plant physiology,
巻：169,
号：2,
開始ページ：137,
終了ページ：145, 2012年01月,
［査読有り］DOI：https://doi.org/10.1016/j.jplph.2011.08.004DOI ID：10.1016/j.jplph.2011.08.004,
ISSN：0176-1617,
PubMed ID：21958596 ABA in bryophytes: how a universal growth regulator in life became a plant hormone? Daisuke Takezawa; Kenji Komatsu; Yoichi Sakata
JOURNAL OF PLANT RESEARCH,
巻：124,
号：4,
開始ページ：437,
終了ページ：453, 2011年07月,
［査読有り］Abscisic acid (ABA) is not a plant-specific compound but one found in organisms across kingdoms from bacteria to animals, suggesting that it is a ubiquitous and versatile substance that can modulate physiological functions of various organisms. Recent studies have shown that plants developed an elegant system for ABA sensing and early signal transduction mechanisms to modulate responses to environmental stresses for survival in terrestrial conditions. ABA-induced increase in stress tolerance has been reported not only in vascular plants but also in non-vascular bryophytes. Since bryophytes are the key group of organisms in the context of plant evolution, clarification of their ABA-dependent processes is important for understanding evolutionary adaptation of land plants. Molecular approaches using Physcomitrella patens have revealed that ABA plays a role in dehydration stress tolerance in mosses, which comprise a major group of bryophytes. Furthermore, we recently reported that signaling machinery for ABA responses is also conserved in liverworts, representing the most basal members of extant land plant lineage. Conservation of the mechanism for ABA sensing and responses in angiosperms and basal land plants suggests that acquisition of this mechanism for stress tolerance in vegetative tissues was one of the critical evolutionary events for adaptation to the land. This review describes the role of ABA in basal land plants as well as non-land plant organisms and further elaborates on recent progress in molecular studies of model bryophytes by comparative and functional genomic approaches.
SPRINGER TOKYO, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1007/s10265-011-0410-5DOI ID：10.1007/s10265-011-0410-5,
ISSN：0918-9440,
Web of Science ID：WOS:000292448700002 Endoplasmic reticulum-localized small heat shock protein that accumulates in mulberry tree (Morus bombycis Koidz.) during seasonal cold acclimation is responsive to abscisic acid Norifumi Ukaji; Chikako Kuwabara; Yuri Kanno; Mitsunori Seo; Daisuke Takezawa; Keita Arakawa; Seizo Fujikawa
TREE PHYSIOLOGY,
巻：30,
号：4,
開始ページ：502,
終了ページ：513, 2010年04月,
［査読有り］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.
OXFORD UNIV PRESS, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1093/treephys/tpp125DOI ID：10.1093/treephys/tpp125,
ISSN：0829-318X,
PubMed ID：20100700,
Web of Science ID：WOS:000276305100007 Evolutionarily conserved regulatory mechanisms of abscisic acid signaling in land plants: characterization of ABSCISIC ACID INSENSITIVE1-like type 2C protein phosphatase in the liverwort Marchantia polymorpha. Tougane K; Komatsu K; Bhyan SB; Sakata Y; Ishizaki K; Yamato KT; Kohchi T; Takezawa D; Plant physiology, v
巻：152,
号：3,
開始ページ：1529,
終了ページ：1543, 2010年03月,
［査読有り］Tougane K, Komatsu K, Bhyan SB, Sakata Y, Ishizaki K, Yamato KT, Kohchi T, Takezawa D, Plant physiology, 2010, vol. 152, no. 3, pp. 1529-1543
DOI：https://doi.org/10.1104/pp.110.153387DOI ID：10.1104/pp.110.153387,
ORCID：10384760,
PubMed ID：20097789 アブシジン酸による耐凍性上昇に伴うヒメツリガネゴケの生理的変化の解析 南 杏鶴; 長尾 学; 荒川 圭太; 藤川 清三; 竹澤 大輔
低温生物工学会誌,
巻：49,
号：2,
開始ページ：179,
終了ページ：183, 2003年
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.
低温生物工学会, 日本語
DOI：https://doi.org/10.20585/cryobolcryotechnol.49.2_179DOI ID：10.20585/cryobolcryotechnol.49.2_179,
ISSN：1340-7902,
J-Global ID：200902284634086862,
CiNii Articles ID：110002914000,
CiNii Books ID：AN10448734 Roles of the plasma membrane and the cell wall in the responses of plant cells to freezing. Yamada T; Kuroda K; Jitsuyama Y; Takezawa D; Arakawa K; Fujikawa S
Planta,
巻：215,
開始ページ：770,
終了ページ：778, 2002年09月,
［査読有り］5
DOI：https://doi.org/10.1007/s00425-002-0814-5DOI ID：10.1007/s00425-002-0814-5,
ISSN：0032-0935,
PubMed ID：12244442 Abscisic acid- and cold-induced thaumatin-like protein in winter wheat has an antifungal activity against snow mould, Microdochium nivale C Kuwabara; D Takezawa; T Shimada; T Hamada; S Fujikawa; K Arakawa
PHYSIOLOGIA PLANTARUM,
巻：115,
号：1,
開始ページ：101,
終了ページ：110, 2002年05月,
［査読有り］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.
BLACKWELL MUNKSGAARD, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1034/j.1399-3054.2002.1150112.xDOI ID：10.1034/j.1399-3054.2002.1150112.x,
ISSN：0031-9317,
PubMed ID：12010473,
Web of Science ID：WOS:000175895400012 Cold acclimation-induced WAP27 localized in endoplasmic reticulum in cortical parenchyma cells of mulberry tree was homologous to group 3 late-embryogenesis abundant proteins N Ukaji; C Kuwabara; D Takezawa; K Arakawa; S Fujikawa
PLANT PHYSIOLOGY,
巻：126,
号：4,
開始ページ：1588,
終了ページ：1597, 2001年08月,
［査読有り］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.
AMER SOC PLANT PHYSIOLOGISTS, 英語, 研究論文（学術雑誌）
DOI：https://doi.org/10.1104/pp.126.4.1588DOI ID：10.1104/pp.126.4.1588,
ISSN：0032-0889,
CiNii Articles ID：80012556716,
PubMed ID：11500557,
Web of Science ID：WOS:000170413800026 A rapid induction by elicitors of the mRNA encoding CCD-1, a 14 kDa Ca2+-binding protein in wheat cultured cells
D Takezawa
PLANT MOLECULAR BIOLOGY, 巻：42, 号：6, 開始ページ：807, 終了ページ：817, 2000年04月, ［査読有り］
Intracellular Ca2+ has been implicated in the signal transduction processes during the development of the plant defense system against fungal pathogens. From wheat cultured cells that had been treated with the elicitor derived from Typhula ishikariensis, the ccd-1 gene encoding a 14 kDa Ca2+-binding protein with an acidic amphiphilic feature was isolated. The ccd-1-encoded protein (CCD-1) shares homology to the C-terminal half domain of centrin, a Ca2+-binding protein conserved in eukaryotes. Unlike typical eukaryotic centrins, CCD-1 contains only one Ca2+-binding loop, which corresponds to the one in the fourth EF-hand from the N-terminus of centrin. The recombinant CCD protein expressed in Escherichia coli bound to a phenyl-Sepharose column in the presence of Ca2+ and was eluted out by EGTA. It also showed a Ca2+-dependent electrophoretic mobility shift on the non-denaturing polyacrylamide gel. The ccd-1 mRNA expression was rapidly induced by treatment with fungal and chitosan oligosaccharide elicitors, implying that it might have a role in transducing Ca2+ signals provoked by the elicitors. The expression of the ccd-1 mRNA was induced by treatment with A23187, and the induction was suppressed by La3+ or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). This study suggests the involvement of intracellular Ca2+ in the elicitor-induced mRNA expression of a novel class of Ca2+-binding proteins conserved in higher plants.
KLUWER ACADEMIC PUBL, 英語, 研究論文（学術雑誌）
ISSN：0167-4412, Web of Science ID：WOS:000087182000002
Elicitor- and A23187-induced expression of WCK-1, a gene encoding mitogen-activated protein kinase in wheat
D Takezawa
PLANT MOLECULAR BIOLOGY, 巻：40, 号：6, 開始ページ：921, 終了ページ：933, 1999年08月, ［査読有り］
Wheat cultured cells were used to study the role of Ca2+ in regulating protein kinases during the induction of defense-related genes by fungal elicitor treatments. Manipulation of intracellular Ca2+ concentrations by treatment with calcium ionophore A23187 in the presence of high extracellular Ca2+ resulted in the induction of mRNA expression of WCK-1, a gene encoding mitogen-activated protein (MAP) kinase. The induction of WCK-1 mRNA by A23187 did not occur when extracellular Ca2+ was chelated by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). The WCK-1 mRNA was also induced by Typhula ishikariensis-derived elicitors, suggesting a possible involvement of WCK-1 in the plant defense response against pathogens. BAPTA and a calcium channel blocker, La3+, inhibited the elicitor-induced expression of the WCK-1 mRNA. A recombinant fusion protein of WCK-1 (GST-WCK-1) autophosphorylated at the Tyr residue and exhibited an autophosphorylation-dependent protein kinase activity towards myelin basic protein. Alteration of Tyr-196 in the conserved 'TEY' motif in GST-WCK-1 to Phe by site-directed mutagenesis abolished the autophosphorylation. The GST-WCK-1 protein was activated by elicitor-treated wheat cell extracts but not by the control extract. These results suggest that fungal elicitors activate WCK-1, a specific MAP kinase in wheat. Furthermore, the results suggest a possible involvement of Ca2+ in enhancing the MAP kinase signaling cascade in plants by controlling the levels of the MAP kinase transcripts.
KLUWER ACADEMIC PUBL, 英語, 研究論文（学術雑誌）
ISSN：0167-4412, Web of Science ID：WOS:000082567400003
Akinete formation in Tribonema bombycinum derbes et solier (Xanthophyceae) in relation to freezing tolerance
M Nagao; K Arakawa; D Takezawa; S Yoshida; S Fujikawa
JOURNAL OF PLANT RESEARCH, 巻：112, 号：1106, 開始ページ：163, 終了ページ：174, 1999年06月, ［査読有り］
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.
BOTANICAL SOC JAPAN, 英語, 研究論文（学術雑誌）
ISSN：0918-9440, Web of Science ID：WOS:000087329700003
Alterations of intracellular pH in response to low temperature stresses
S Yoshida; K Hotsubo; Y Kawamura; M Murai; K Arakawa; D Takezawa
JOURNAL OF PLANT RESEARCH, 巻：112, 号：1106, 開始ページ：225, 終了ページ：236, 1999年06月, ［査読有り］
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.
BOTANICAL SOC JAPAN, 英語, 研究論文（学術雑誌）
ISSN：0918-9440, Web of Science ID：WOS:000087329700009
Accumulation of small heat-shock protein homologs in the endoplasmic reticulum of cortical parenchyma cells in mulberry in association with seasonal cold acclimation
N Ukaji; C Kuwabara; D Takezawa; K Arakawa; S Yoshida; S Fujikawa
PLANT PHYSIOLOGY, 巻：120, 号：2, 開始ページ：481, 終了ページ：489, 1999年06月, ［査読有り］
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.
AMER SOC PLANT PHYSIOLOGISTS, 英語, 研究論文（学術雑誌）
ISSN：0032-0889, Web of Science ID：WOS:000080936500015
Accumulation of 19-kDa plasma membrane polypeptide during induction of freezing tolerance in wheat suspension-cultured cells by abscisic acid
M Koike; D Takezawa; K Arakawa; S Yoshida
PLANT AND CELL PHYSIOLOGY, 巻：38, 号：6, 開始ページ：707, 終了ページ：716, 1997年06月, ［査読有り］
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.
JAPANESE SOC PLANT PHYSIOLOGISTS, 英語, 研究論文（学術雑誌）
ISSN：0032-0781, Web of Science ID：WOS:A1997XF27600009
Functional domains of plant chimeric calcium/calmodulin-dependent protein kinase: Regulation by autoinhibitory and visinin-like domains
S Ramachandiran; D Takezawa; W Wang; BW Poovaiah
JOURNAL OF BIOCHEMISTRY, 巻：121, 号：5, 開始ページ：984, 終了ページ：990, 1997年05月, ［査読有り］
A novel calcium-binding calcium/calmodulin-dependent protein kinase (CCaMK) with a catalytic domain, calmodulin-binding domain, and a neural visinin-like domain was cloned and characterized from plants [Patil et al., (1995) Proc. Natl. Acad. Sci. USA 92, 4797-4801; Takezawa et al. (1996) J. Biol. Chem. 271, 8126-8132]. The mechanisms of CCaMK activation by calcium and calcium/calmodulin were investigated using various deletion mutants. The use of deletion mutants of CCaMK lacking either one, two, or all three calcium-binding EF hands indicated that all three calcium-binding sites in the visinin-like domain were crucial for the full calcium/calmodulin-dependent kinase activity. As each calcium-binding EF hand was deleted, there was a gradual reduction in calcium/calmodulin-dependent kinase activity from 100 to 4%. Another mutant (amino acids 1-322) which lacks both the visinin-like domain containing three EF hands and the calmodulin-binding domain was constitutively active, indicating the presence of an autoinhibitory domain around the calmodulin-binding domain. By using various synthetic peptides and the constitutively active mutant, we have shown that CCaMK contains an autoinhibitory domain within the residues 322-340 which overlaps its calmodulin-binding domain. Kinetic studies with both ATP and the GS peptide substrate suggest that the autoinhibitory domain of CCaMK interacts only with the peptide substrate binding motif of the catalytic domain, but not with the ATP-binding motif.
JAPANESE BIOCHEMICAL SOC, 英語, 研究論文（学術雑誌）
ISSN：0021-924X, Web of Science ID：WOS:A1997WY02200028
Characterization of vacuolar H+-ATPases that are sensitive and tolerant to cold
K Hotsubo; Y Kawamura; D Takezawa; K Arakawa; S Yoshida
PLANT COLD HARDINESS, 開始ページ：237, 終了ページ：244, 1997年, ［査読有り］
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.
PLENUM PRESS DIV PLENUM PUBLISHING CORP, 英語, 研究論文（国際会議プロシーディングス）
Web of Science ID：WOS:000071892500021
Regulated expression of a calmodulin isoform alters growth and development in potato
BW Poovaiah; D Takezawa; G An; TJ Han
JOURNAL OF PLANT PHYSIOLOGY, 巻：149, 号：5, 開始ページ：553, 終了ページ：558, 1996年10月, ［査読有り］
A transgene approach was taken to study the consequences of altered expression of a calmodulin isoform on plant growth and development. Eight genomic clones of potato calmodulin (PCM1 to 8) have been isolated and characterized (Takezawa et al., 1995). Among the potato calmodulin isoforms studied, PCM1 differs from the other isoforms because of its unique amino acid substitutions. Transgenic potato plants were produced carrying sense construct of PCM1 fused to the CaMV 35S promoter. Transgenic plants showing a moderate increase in PCM 1 mRNA exhibited strong apical dominance, produced elongated tubers, and were taller than the controls. Interestingly, the plants expressing the highest level of PCM1 mRNA did not form underground tubers. Instead, these transgenic plants produced aerial tubers when allowed to grow for longer periods. The expression of different calmodulin isoforms (PCM 1, 5, 6, and 8) was studied in transgenic plants. Among the four potato calmodulin isoforms, only the expression of PCM 1 mRNA was altered in transgenic plants, while the expression of other isoforms was not significantly altered. Western analysis revealed increased PCM1 protein in transgenic plants, indicating that the expression of both mRNA and protein are altered in transgenic plants. These results suggest that increasing the expression of PCM 1 alters growth and development in potato plants.
ELSEVIER GMBH, URBAN & FISCHER VERLAG, 英語, 研究論文（学術雑誌）
ISSN：0176-1617, Web of Science ID：WOS:A1996VM27500012
Calcium-dependent protein kinase genes in corn roots
D Takezawa; S Patil; A Bhatia; BW Poovaiah
JOURNAL OF PLANT PHYSIOLOGY, 巻：149, 号：3-4, 開始ページ：329, 終了ページ：335, 1996年08月, ［査読有り］
Two cDNAs encoding Ca2+-dependent protein kinases (CDPKs), CRPK1 and CRPK2 (corn root protein kinase 1 and 2), were isolated from the root tip library of corn (Zea mays L., cv. Merit) and their nucleotide sequences were determined. Deduced amino acid sequences of both the clones have features characteristic of plant CDPKs, including all 11 conserved serine/threonine kinase subdomains, a junction domain and a calmodulin-like domain with four Ca2+-binding sites. Northern analysis revealed that CRPK1 mRNA is preferentially expressed in roots, especially in the root tip; whereas, the expression of CRPK2 mRNA was very low in all the tissues tested. In situ hybridization experiments revealed that CRPK1 mRNA is highly expressed in the root apex, as compared to other parts of the root; Partially purified CDPK from the root tip phosphorylates syntide-2, a common peptide substrate for plant CDPKs, and the phosphorylation was stimulated 7-fold by the addition of Ca2+. Our results show that two CDPK isoforms are expressed in corn roots and they may be involved in the Ca2+-dependent signal transduction process.
GUSTAV FISCHER VERLAG, 英語, 研究論文（学術雑誌）
ISSN：0176-1617, Web of Science ID：WOS:A1996VH80200016
A potato cDNA encoding a homologue of mammalian multidrug resistant P-glycoprotein
W Wang; D Takezawa; BW Poovaiah
PLANT MOLECULAR BIOLOGY, 巻：31, 号：3, 開始ページ：683, 終了ページ：687, 1996年06月, ［査読有り］
A homologue of the multidrug resistance (MDR) gene was obtained while screening a potato stolen tip cDNA expression library with S-35-labeled calmodulin. The mammalian MDR gene codes for a membrane-bound P-glycoprotein (170-180 kDa) which imparts multidrug resistance to cancerous cells. The potato cDNA (PMDR1) codes for a polypeptide of 1313 amino acid residues (ca. 144 kDa) and its structural features are very similar to the MDR P-glycoprotein. The N-terminal half of the PMDR1-encoded protein shares striking homology with its C-terminal half, and each half contains a conserved ATP-binding site and six putative transmembrane domains. Southern blot analysis indicated that potato has one or two MDR-like genes. PMDR1 mRNA is constitutively expressed in all organs studied with higher expression in the stem and stolen tip. The PMDR1 expression was highest during tuber initiation and decreased during tuber development.
KLUWER ACADEMIC PUBL, 英語, 研究論文（学術雑誌）
ISSN：0167-4412, Web of Science ID：WOS:A1996VE97300021
Dual regulation of a chimeric plant serine threonine kinase by calcium and calcium calmodulin
D Takezawa; S Ramachandiran; Paranjape, V; BW Poovaiah
JOURNAL OF BIOLOGICAL CHEMISTRY, 巻：271, 号：14, 開始ページ：8126, 終了ページ：8132, 1996年04月, ［査読有り］
A chimeric Ca2+/calmodulin dependent protein kinase (CCaMK) gene characterized by a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca2+-binding domain was recently cloned from plants (Patil, S., Takezawa, D., and Poovaiah, B. W. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4797-4801), The Escherichia coli-expressed CCaMK phosphorylates various protein and peptide substrates in a Ca2+/calmodulin-dependent manner, The calmodulin-binding region of CCaMK has similarity to the calmodulin-binding region of the alpha-subunit of multifunctional Ca2+/calmodulin-dependent protein kinase (CaMKII). CCaMK exhibits basal autophosphorylation at the threonine residue(s) (0.098 mol of P-32/mol) that is stimulated 3.4-fold by Ca2+ (0.339 mol of P-32/mol), while calmodulin inhibits Ca2+-stimulated autophosphorylation to the basal level, A deletion mutant lacking the visinin-like domain did not show Ca2+-stimulated autophosphorylation activity but retained Ca2+/calmodulin dependent protein kinase activity at a reduced level, Ca2+-dependent mobility shift assays using E. coli-expressed protein from residues 358-520 revealed that Ca2+ binds to the visinin-like domain, Studies with site-directed mutants of the visinin-like domain indicated that EF-hands II and III are crucial for Ca2+-induced conformational changes in the visinin-like do main, Autophosphorylation of CCaMK increases Ca2+/calmodulin-dependent protein kinase activity by about B-fold, whereas it did not affect its Ca2+-independent activity, This report provides evidence for the existence of a protein kinase in plants that is modulated by Ca2+ and Ca2+/calmodulin, The presence of a visinin-like Ca2+-binding domain in CCaMK adds an additional Ca2+-sensing mechanism not previously known to exist in the Ca2+/calmodulin-mediated signaling cascade in plants.
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 英語, 研究論文（学術雑誌）
ISSN：0021-9258, Web of Science ID：WOS:A1996UD60000040
CHIMERIC PLANT CALCIUM/CALMODULIN-DEPENDENT PROTEIN-KINASE GENE WITH A NEURAL VISININ-LIKE CALCIUM-BINDING DOMAIN
S PATIL; D TAKEZAWA; BW POOVAIAH
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 巻：92, 号：11, 開始ページ：4897, 終了ページ：4901, 1995年05月, ［査読有り］
Calcium, a universal second messenger, regulates diverse cellular processes in eukaryotes. Ca2+ and Ca2+/calmodulin-regulated protein phosphorylation play a pivotal role in amplifying and diversifying the action of Ca2+-mediated signals. A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) gene with a visinin-like Ca2+-binding domain was cloned and characterized from lily. The cDNA clone contains an open reading frame coding for a protein of 520 amino acids. The predicted structure of CCaMK contains a catalytic domain followed by two regulatory domains, a calmodulin-binding domain and a visinin-like Ca2+-binding domain. The amino-terminal region of CCaMK contains all 11 conserved subdomains characteristic of serine/threonine protein kinases, The calmodulin-binding region of CCaMK has high homology (79%) to alpha subunit of mammalian Ca2+/calmodulin-dependent protein kinase. The calmodulin-binding region is fused to a neural visinin-like domain that contains three Ca2+-binding EF-hand motifs and a biotin binding site. The Escherichia coli-expressed protein (approximate to 56 kDa) binds calmodulin in a Ca2+-dependent manner. Furthermore, Ca-45-binding assays revealed that CCaMK directly binds Ca2+. The CCaMK gene is preferentially expressed in developing anthers. Southern blot analysis revealed that CCaMK is encoded by a single gene. The structural features of the gene suggest that it has multiple regulatory controls and could play a unique role in Ca2+ signaling in plants.
NATL ACAD SCIENCES, 英語, 研究論文（学術雑誌）
ISSN：0027-8424, Web of Science ID：WOS:A1995RA15000037
The bHLH transcription factor MpHYPNOS regulates gemma dormancy in the liverwort Marchantia polymorpha Hirotaka Kato; Nami Yoshimura; Mikako Yoshikawa; Hideyuki Matsuura; Kosaku Takahashi; Daisuke Takezawa; Tomoyuki Furuya; Yuki Kondo; Hidehiro Fukaki; Tetsuro Mimura; Kimitsune Ishizaki
bioRxiv,
開始ページ：488978, 2022年04月25日
DOI：https://doi.org/10.1101/2022.04.25.488978DOI ID：10.1101/2022.04.25.488978 Long- and short-term freezing induce different types of injury in Arabidopsis thaliana leaf cells M. Nagao; K. Arakawa; D. Takezawa; S. Fujikawa
巻：227,
号：2,
開始ページ：477,
終了ページ：489, 2008年01月
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.
英語
DOI：https://doi.org/10.1007/s00425-007-0633-9DOI ID：10.1007/s00425-007-0633-9,
ISSN：0032-0935,
CiNii Articles ID：80017945202,
PubMed ID：17924137,
Web of Science ID：WOS:000251373400018 Gene expression associated with increased supercooling capability in xylem parenchyma cells of larch (Larix kaempferi) Naoki Takata; Jun Kasuga; Daisuke Takezawa; Keita Arakawa; Seizo Fujikawa
巻：58,
号：13,
開始ページ：3731,
終了ページ：3742, 2007年10月
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.
英語
DOI：https://doi.org/10.1093/jxb/erm223DOI ID：10.1093/jxb/erm223,
ISSN：0022-0957,
eISSN：1460-2431,
CiNii Articles ID：80019361940,
PubMed ID：18057043,
Web of Science ID：WOS:000251506300020 Gene expression associated with increased supercooling capability in xylem parenchyma cells of larch (Larix kaempferi) Naoki Takata; Jun Kasuga; Daisuke Takezawa; Keita Arakawa; Seizo Fujikawa
巻：58,
号：13,
開始ページ：3731,
終了ページ：3742, 2007年10月
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°C during summer to -60°C during winter as measured by freezing resistance. Artificial deacclimation of larch twigs collected in winter reduced the supercooling capability from -60°C to -30°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. © 2007 The Author(s).
英語
DOI：https://doi.org/10.1093/jxb/erm223DOI ID：10.1093/jxb/erm223,
ISSN：0022-0957,
CiNii Articles ID：80019361940,
PubMed ID：18057043,
SCOPUS ID：36849057130 Reduced sensitivity to freezing and dehydration in the abscisic acid-insensitive mutant in mosses
Daisuke Takezawa
巻：48, 開始ページ：S240, 終了ページ：S240, 2007年
英語, 研究発表ペーパー・要旨（国際会議）
ISSN：0032-0781, Web of Science ID：WOS:000245922701436
植物の脱水応答に関わるシグナル因子の探索
竹澤 大輔
総合研究機構研究プロジェクト研究成果報告書, 号：5(18年度), 開始ページ：653, 終了ページ：654, 2007年
植物の脱水応答に関わるシグナル因子の探索
竹澤 大輔
号：5(18年度), 開始ページ：653, 終了ページ：654, 2007年
Accumulation of theanderose in association with development of freezing tolerance in the moss Physcomitrella patens M Nagao; K Oku; A Minami; K Mizuno; M Sakurai; K Arakawa; S Fujikawa; D Takezawa
巻：67,
号：7,
開始ページ：702,
終了ページ：709, 2006年04月
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.
英語
DOI：https://doi.org/10.1016/j.phytochem.2006.01.031DOI ID：10.1016/j.phytochem.2006.01.031,
ISSN：0031-9422,
CiNii Articles ID：80019277810,
PubMed ID：16527318,
Web of Science ID：WOS:000236819800009 Alterered Freezing tolerance in teh Physcomitrella patens mutant with reduced sensitivity to abscisic acid.
Minami, A; Togawa, S; Nagao, M; Takezawa D
Cryobioloby and Cryotechnology, 巻：52, 号：2, 開始ページ：135, 終了ページ：139, 2006年
Many types of plant cells accumulate low molecular-weight soluble sugars when they acquire freezing tolerance in the process of cold acclimation. Protonema cells of the moss Physcomitrella patens develop freezing tolerance upon treatment with the phytohormone abscisic acid (ABA). We previously reported that the ABA-treated protonema cells accumulate low molecular-weight soluble sugars such as sucrose and theanderose. To clarify mechanisms underlying physiological processes leading to development of freezing tolerance, we isolated F patens mutant lines, AR1 to AR7, with reduced sensitivity to ABA. Among these lines, AR7 showed growth similar to wild type but did not develop freezing tolerance by ABA treatment. Analysis of low molecular-weight soluble sugars indicated that the AR7 cells accumulated sucrose upon ABA treatment but very little of theanderose.
低温生物工学会, 英語
DOI ID：10.20585/cryobolcryotechnol.52.2_135, ISSN：1340-7902, CiNii Articles ID：110006783031, CiNii Books ID：AN10448734
植物の脱水ストレス耐性機構の解明
竹澤 大輔
総合研究機構研究プロジェクト研究成果報告書, 号：4(17年度), 2006年
Alterered Freezing tolerance in teh Physcomitrella patens mutant with reduced sensitivity to abscisic acid. Minami, A; Togawa, S; Nagao, M; Takezawa D
巻：52,
開始ページ：135,
終了ページ：139, 2006年
DOI：https://doi.org/10.20585/cryobolcryotechnol.52.2_135DOI ID：10.20585/cryobolcryotechnol.52.2_135 Physiological and morphological alteration associated with freezing tolerance in the moss Physcomitrella patens.
Minami, A; Nagao, M; Arakawa, K; Fujikawa, S; Takezawa, D
開始ページ：138, 終了ページ：152, 2006年
植物の脱水ストレス耐性機構の解明
竹澤 大輔
号：4(17年度), 2006年
Rapid degradation of starch in chloroplasts and concomitant accumulation of soluble sugars associated with ABA-induced freezing tolerance in the moss Physcomitrefla patens M Nagao; A Minami; K Arakawa; S Fujikawa; D Takezawa
巻：162,
号：2,
開始ページ：169,
終了ページ：180, 2005年02月
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.
英語
DOI：https://doi.org/10.1016/j.jplph.2004.06.012DOI ID：10.1016/j.jplph.2004.06.012,
ISSN：0176-1617,
Web of Science ID：WOS:000227721100005 冬小麦PR-5タンパク質(WAS-3)の構造解析 桑原 慎子; 近藤 英昌; 野呂 奈津子; 竹澤 大輔; 荒川 圭太; 津田 栄
日本植物生理学会年会およびシンポジウム 講演要旨集,
巻：2005,
号：0,
開始ページ：848,
終了ページ：848, 2005年
WAS-3は、病原菌関連タンパク質PR-5に属し、冬小麦の低温馴化によってアポプラストに蓄積する。私たちは、WAS-3の機能を明らかにするために、小麦培養細胞を用いた組み換えタンパク質発現系を作成し、組み換えWAS-3タンパク質（rWAS-3）が
in vitroにおいて雪腐れ病菌やフザリウム菌の菌糸の成長を阻害する活性を持つことを明らかにしてきた。また、rWAS-3は雪腐れ病菌やフザリウム菌の菌糸の細胞壁画分に対し親和性を持ち、菌糸の成分であるβ-1,3-グルカンに結合することを示唆してきた。しかし、抗菌活性の詳細なメカニズムは、まだ明らかになっていない。そこで、本研究では、抗菌活性のメカニズムに関する知見を得るために、rWAS-3の結晶を作成し、結晶構造の解析を行った。ハンギングドロップ蒸気拡散法によって結晶化条件のスクリーニングを行った後、至適化した結果、0.8×0.5×0.01 mmの薄い板状結晶が得られた。構造解析は分子置換法を用いて行い、1.8オングストローム分解能の立体構造を決定した。その結果、rWAS-3は3個のドメインを構成しており、ドメインAとドメインBの間に「くぼみ」があることが明らかとなった。分子表面の静電ポテンシャルに基づきモデルを作成すると、ドメインAとドメインBの間のくぼみは、負に荷電した領域であることが示されたことから、この領域にβ-1,3-グルカンが結合する可能性が考えられた。
日本植物生理学会
DOI：https://doi.org/10.14841/jspp.2005.0.848.0DOI ID：10.14841/jspp.2005.0.848.0,
CiNii Articles ID：130006988465 ヒメツリガネゴケのアブシジン酸低感受性変異株における浸透圧及び凍結耐性の低下 南 杏鶴; 長尾 学; 荒川 圭太; 藤川 清三; 竹澤 大輔
日本植物生理学会年会およびシンポジウム 講演要旨集,
巻：2005,
号：0,
開始ページ：222,
終了ページ：222, 2005年
アブシジン酸（ABA）によるシグナル伝達は、植物の低温や浸透圧ストレスに対する応答や耐性の獲得に関与している。ヒメツリガネゴケ（
Physcomitrella patens）原糸体細胞ではABA処理によってストレス応答性遺伝子の発現誘導や可溶性糖の蓄積などがおこり、凍結耐性（耐凍性）が向上する。また、高張のマンニトール処理では内生ABA量が増加し、生理学的変化を伴った耐凍性の上昇がみられる。
ABAによる様々な生理的変化のなかで耐凍性を促進させる要因を探るため、ヒメツリガネゴケABA低感受性変異株AR1～7を紫外線照射処理によって単離した。すべてのAR変異株は、野生株が生育阻害をおこすABA濃度下でも生育することができ、ABA処理後の耐凍性は野生株よりも低い値を示した。特にABAによる耐凍性の上昇程度が低いAR4株では、ABA誘導性遺伝子の発現量やLEA様タンパク質、可溶性糖の蓄積において著しい減少がみられた。この変異株では、マンニトールによる浸透圧ストレスに対して強い感受性を示した。以上の結果は、ABA依存的な浸透圧シグナル伝達経路によって、凍結や高浸透圧溶液による脱水ストレス耐性が調節されていることを示唆している。
日本植物生理学会
DOI：https://doi.org/10.14841/jspp.2005.0.222.0DOI ID：10.14841/jspp.2005.0.222.0,
CiNii Articles ID：130006988346 クワ皮層柔細胞に冬季特異的に蓄積する蛋白質WAP27の長期凍結下での凍害保護活性 長尾 学; 宇梶 徳史; 竹澤 大輔; 荒川 圭太; 藤川 清三
日本植物生理学会年会およびシンポジウム 講演要旨集,
巻：2005,
号：0,
開始ページ：221,
終了ページ：221, 2005年
ヤマグワ（
Morus bombycis）の皮層柔細胞の耐凍性は夏季に。比べて冬季に大幅に上昇することが知られている。これまでにヤマグワの皮層柔細胞内のERにGroup 3 LEA蛋白質である分子量27-kDaのWAP27蛋白質 が冬季特異的に蓄積することがわかっている。
植物は野外では一定範囲の凍結温度下に長く置かれる。特に氷点に近い凍結温度下では低温や脱水のみでなく、未凍結溶液中の塩類濃縮に長期間さらされる。そこでWAP27の耐凍性における機能を知るために一定の凍結温度で長期間保持した凍結感受性蛋白質、乳酸脱水素酵素（LDH）に対する組み替えWAP27（rWAP27）凍害保護活性を調べた。rWAP27を添加し-2、-4、-10、-20℃で長期間保持したLDHはすべての凍結温度で80％以上の高い活性を維持した。それに対し牛血清アルブミン（BSA）は、-20℃ではrWAP27と同等の保護活性を維持したが、-2、-4℃では凍結時間に依存して活性が低下する傾向が見られた。卵白アルブミン（Ovalbumin）ではすべての凍結温度で活性が経時的に低下した。今回の結果からrWAP27は長期凍結下で凍結感受性蛋白質に対し、他の凍結保護物質より高い保護活性を持つことが明らかになった。
日本植物生理学会
DOI：https://doi.org/10.14841/jspp.2005.0.221.0DOI ID：10.14841/jspp.2005.0.221.0,
CiNii Articles ID：130006988344 Cold acclimation in bryophytes: low-temperature-induced freezing tolerance in Physcomitrella patens is associated with increases in expression levels of stress-related genes but not with increase in level of endogenous abscisic acid A Minami; M Nagao; K Ikegami; T Koshiba; K Arakawa; S Fujikawa; D Takezawa
巻：220,
号：3,
開始ページ：414,
終了ページ：423, 2005年01月
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.
英語
DOI：https://doi.org/10.1007/s00425-004-1361-zDOI ID：10.1007/s00425-004-1361-z,
ISSN：0032-0935,
CiNii Articles ID：80017121813,
PubMed ID：15349781,
Web of Science ID：WOS:000226810700006 Accumulation of pathogenesis-related (PR) 10/Bet v 1 protein homologues in mulberry (Morus bombycis Koidz.) tree during winter N Ukaji; C Kuwabara; D Takezawa; K Arakawa; S Fujikawa
巻：27,
号：9,
開始ページ：1112,
終了ページ：1121, 2004年09月
Seasonal evaluation of total soluble protein fractions extracted from cortical parenchyma cells of mulberry (Morus bombycis Koidz.) tree identified a predominant 18 kDa protein that was directly correlated to periods of cold acclimation. The 18 kDa protein, designated as WAP18 (winter accumulating 18 kDa proteins) increased from September to December and then gradually decreased until June. The maximum levels of WAP18 were detected in mid-winter, which corresponds to the maximum freeze tolerance in cortical parenchyma cells of mulberry tree. Two-dimensional gel electrophoresis confirmed that WAP18 consists of at least three proteins that range between an isoelectric point of 5.0 and 6.0. All three proteins reacted with anti-WAP18 antibodies, thereby suggesting that they represent individual isoforms. Furthermore, N-terminal amino acid sequence analysis demonstrated that all three proteins contain high sequence similarity to each other and high homology to pathogenesis-related (PR) -10/Bet v 1 protein families. The purified WAP18 exhibited in vitro cryoprotective activity for the freeze labile <smallcapitals>l</smallcapitals>-lactate dehydrogenase (LDH) enzyme. These results suggest that WAP18 may function in the freezing tolerance mechanism of cortical parenchyma cells of mulberry tree during winter.
英語
DOI：https://doi.org/10.1111/j.1365-3040.2004.01216.xDOI ID：10.1111/j.1365-3040.2004.01216.x,
ISSN：0140-7791,
eISSN：1365-3040,
Web of Science ID：WOS:000223661900004 Calmodulin-binding proteins in bryophytes: identification of abscisic acid-, cold-, and osmotic stress-induced genes encoding novel membrane-bound transporter-like proteins D Takezawa; A Minami
巻：317,
号：2,
開始ページ：428,
終了ページ：436, 2004年04月
Plant responses to environmental stresses are mediated in part by signaling processes involving cytosolic Ca2+ and a Ca2+- binding protein, calmodulin. Screening with radiolabeled calmodulin of a cDNA library of the moss Physcomitrella patens resulted in identification of genes encoding novel membrane transporter-like proteins, MCamb1 and MCamb2. These proteins each had a central hydrophobic domain with two putative membrane spans and N- and C-terminal hydrophilic domains, and showed sequence similarity to mammalian inward rectifier potassium channels. Calmodulin binds to MCamb1 and MCamb2 via interaction with basic amphiphilic amino acids in the C-terminal domain. Levels of MCamb1 and MCamb2 transcripts increased dramatically following treatment with low temperature, hyperosmotic solutes, and the stress hormone abscisic acid, all of which were previously shown to increase cellular tolerance to freezing stress. These results suggest that calmodulin participates in cellular signaling events leading to enhancement of stress resistance through regulation of novel transporter-like proteins. (C) 2004 Elsevier Inc. All rights reserved.
英語
DOI：https://doi.org/10.1016/j.bbrc.2004.03.052DOI ID：10.1016/j.bbrc.2004.03.052,
ISSN：0006-291X,
CiNii Articles ID：80016610378,
PubMed ID：15063776,
Web of Science ID：WOS:000220922200020 コケ植物の耐凍性獲得過程で発現する遺伝子の同定 南 杏鶴; 長尾 学; 荒川 圭太; 藤川 清三; 竹澤 大輔
日本植物生理学会年会およびシンポジウム 講演要旨集,
巻：2004,
号：0,
開始ページ：803,
終了ページ：803, 2004年
コケ植物は熱帯から極地まで幅広く生育し、その中には極端な乾燥や凍結に対して耐性を持つ種が少なからず存在する。蘚類ヒメツリガネゴケ（
Physcomitrella patens）の原糸体は、通常の培養条件では耐凍性を持たないが、アブシジン酸（ABA）処理によって高い耐凍性を獲得し、-80℃での凍結保存も可能になる。このことから、ABAが細胞の凍結脱水ストレス耐性に重要な役割を持つと考えられた。私たちは、ABA処理したヒメツリガネゴケ原糸体細胞において葉緑体や液胞の小型化、細胞内浸透濃度の増加および熱安定性タンパク質や可溶性糖の蓄積がおこり、凍結傷害の主要因である細胞膜の損傷が緩和されることを明らかにした。ABAで誘導される耐凍性上昇はシクロヘキシミド処理で著しく抑制されることから、この過程には核コードタンパク質の合成が必須であることが示された。
ヒメツリガネゴケの耐凍性上昇に関わる遺伝子を同定するため、フィルターアレイを用いたディファレンシャルスクリーニングによりABA処理で増加する60以上の遺伝子を単離した。その結果、これらの中にはLEAや水チャネルなどのように高等植物のストレス応答性遺伝子と相同性のあるものが多数含まれる一方、高等植物には保存されていない葉緑体タンパク質LI818や、UVI-1など原核生物遺伝子のホモログが新たに同定された。
日本植物生理学会
DOI：https://doi.org/10.14841/jspp.2004.0.803.0DOI ID：10.14841/jspp.2004.0.803.0,
CiNii Articles ID：130006987517 Characterization of a novel plant PP2C-like protein Ser/Thr phosphatase as a calmodulin-binding protein Daisuke Takezawa
巻：278,
号：39,
開始ページ：38076,
終了ページ：38083, 2003年09月26日
Protein phosphatases regulated by calmodulin (CaM) mediate the action of intracellular Ca2+ and modulate functions of various target proteins by dephosphorylation. In plants, however, the role of Ca2+ in the regulation of protein dephosphorylation is not well understood due to a lack of information on characteristics of CaM-regulated protein phosphatases. Screening of a cDNA library of the moss Physcomitrella patens by using 35S-labeled calmodulin as a ligand resulted in identification of a gene, PCaMPP, that encodes a protein serine/threonine phosphatase with 373 amino acids. PCaMPP had a catalytic domain with sequence similarity to type 2C protein phosphatases (PP2Cs) with six conserved metal-associating amino acid residues and also had an extra C-terminal domain. Recombinant GST fusion proteins of PCaMPP exhibited Mn2+-dependent phosphatase activity, and the activity was inhibited by pyrophosphate and 1 mM Ca2+ but not by okadaic acid, orthovanadate, or β-glycerophosphate. Furthermore, the PCaMPP activity was increased 1.7-fold by addition of CaM at nanomolar concentrations. CaM binding assays using deletion proteins and a synthetic peptide revealed that the CaM-binding region resides within the basic amphiphilic amino acid region 324-346 in the C-terminal domain. The CaM-binding region had sequence similarity to amino acids in one of three α-helices in the C-terminal domain of human PP2Cα, suggesting a novel role of the C-terminal domains for the phosphatase activity. These results provide the first evidence showing possible regulation of PP2C-related phosphatases by Ca2+/CaM in plants. Genes similar to PCaMPP were found in genomes of various higher plant species, suggesting that PCaMPP-type protein phosphatases are conserved in land plants.
英語
DOI：https://doi.org/10.1074/jbc.M301369200DOI ID：10.1074/jbc.M301369200,
ISSN：0021-9258,
CiNii Articles ID：80016236736,
PubMed ID：12860996,
SCOPUS ID：0141620376 Characterization of a novel plant PP2C-like protein Ser/Thr phosphatase as a calmodulin-binding protein D Takezawa
巻：278,
号：39,
開始ページ：38076,
終了ページ：38083, 2003年09月
Protein phosphatases regulated by calmodulin (CaM) mediate the action of intracellular Ca2+ and modulate functions of various target proteins by dephosphorylation. In plants, however, the role of Ca2+ in the regulation of protein dephosphorylation is not well understood due to a lack of information on characteristics of CaM-regulated protein phosphatases. Screening of a cDNA library of the moss Physcomitrella patens by using S-35-labeled calmodulin as a ligand resulted in identification of a gene, PCaMPP, that encodes a protein serine/ threonine phosphatase with 373 amino acids. PCaMPP had a catalytic domain with sequence similarity to type 2C protein phosphatases (PP2Cs) with six conserved metal-associating amino acid residues and also had an extra C-terminal domain. Recombinant GST fusion proteins of PCaMPP exhibited Mn2+-dependent phosphatase activity, and the activity was inhibited by pyrophosphate and 1 mM Ca2+ but not by okadaic acid, orthovanadate, or beta-glycerophosphate. Furthermore, the PCaMPP activity was increased 1.7-fold by addition of CaM at nanomolar concentrations. CaM binding assays using deletion proteins and a synthetic peptide revealed that the CaM-binding region resides within the basic amphiphilic amino acid region 324 - 346 in the C-terminal domain. The CaM-binding region had sequence similarity to amino acids in one of three alpha-helices in the C-terminal domain of human PP2Calpha, suggesting a novel role of the C-terminal domains for the phosphatase activity. These results provide the first evidence showing possible regulation of PP2C-related phosphatases by Ca2+/CaM in plants. Genes similar to PCaMPP were found in genomes of various higher plant species, suggesting that PCaMPP-type protein phosphatases are conserved in land plants.
英語
DOI：https://doi.org/10.1074/jbc.M301369200DOI ID：10.1074/jbc.M301369200,
ISSN：0021-9258,
CiNii Articles ID：80016236736,
PubMed ID：12860996,
Web of Science ID：WOS:000185437200129 Abscisic acid-induced freezing tolerance in the moss Physcomitrella patens is accompanied by increased expression of stress-related genes A Minami; M Nagao; K Arakawa; S Fujikawa; D Takezawa
巻：160,
号：5,
開始ページ：475,
終了ページ：483, 2003年05月
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.
英語
DOI：https://doi.org/10.1078/0176-1617-00888DOI ID：10.1078/0176-1617-00888,
ISSN：0176-1617,
Web of Science ID：WOS:000183064100006 Characterization of Physcomitrella PP2C-like protein phosphatase that binds calmodulin
D Takezawa
巻：44, 開始ページ：S40, 終了ページ：S40, 2003年
英語, 研究発表ペーパー・要旨（国際会議）
ISSN：0032-0781, Web of Science ID：WOS:000181914300157
PR-10/Bet v 1ファミリーは細胞質と核に局在する 宇梶 徳史; 竹澤 大輔; 荒川 圭太; 藤川 清三
日本植物生理学会年会およびシンポジウム 講演要旨集,
巻：2003,
号：0,
開始ページ：308,
終了ページ：308, 2003年
冬季に-80℃以下の高い凍結耐性を獲得するクワ(
Morus bombycis Koidz.)皮層柔細胞では、秋から冬にかけての季節的低温馴化に伴い、可溶性画分に18kDの分子サイズを有するタンパク質（WAP18）の蓄積が見出される。精製したWAP18は、in vitroに於いて凍結・融解による乳酸脱水素酵素活性低下を防止したことから、WAP18はクワ皮層柔細胞が獲得する冬季の凍結耐性獲得に、何らかの形で貢献するものと推測される。cDNAクローニングにより、WAP18はPR-10/Bet v 1ファミリーと高い相同性を有することが示された。ノーザン解析を行ったところ、WAP18遺伝子は、4℃の低温処理のみならず、wounding、エテフォン、およびサリチル酸による誘導が見られた。
一方、PR-10/Bet v 1ファミリーはその推定されるアミノ酸配列から、細胞質に局在すると推測されているが、正確な細胞内局在は示されていない。そこで、WAP18のクワ皮層柔細胞における細胞内局在を、免疫電子顕微鏡法を用いて観察したところ、細胞質と核に金コロイド標識が見いだされた。このことから、PR-10/Bet v 1ファミリーは細胞質と核に局在することが示唆された。
日本植物生理学会
DOI：https://doi.org/10.14841/jspp.2003.0.308.0DOI ID：10.14841/jspp.2003.0.308.0,
CiNii Articles ID：130006989896 ヒメツリガネゴケの耐凍性に関わるABA・低温誘導性プロテインキナーゼの機能解析 南 杏鶴; 長尾 学; 荒川 圭太; 藤川 清三; 竹澤 大輔
日本植物生理学会年会およびシンポジウム 講演要旨集,
巻：2003,
号：0,
開始ページ：459,
終了ページ：459, 2003年
植物が低温馴化により耐凍性を獲得する過程では、タンパク質のリン酸化や脱リン酸化が関わっていると考えられている。我々は、蘚類ヒメツリガネゴケ（
Physcomitrella patens）原糸体が、ABAや低温、高浸透圧処理によって凍結耐性を上昇することを以前に示した。ABA・低温処理によってmRNAの顕著な発現誘導が起こる
PARK(
P. patens ABA-responsive protein kinase)遺伝子は、セリン・スレオニンプロテインキナーゼをコードしていた。PARKはシロイロナズナのS-ドメインレセプター様プロテインキナーゼとキナーゼドメインにおいて56.4％の相同性があるが、細胞外ドメインを持たず、N末端は膜貫通ドメインと思われる疎水領域から始まっていた。GST融合タンパク質を用いたPARKのリン酸化活性の解析では、ミエリン塩基性タンパク質やヒストンIIISの基質をリン酸化し、PARK自身も自己リン酸化することが分かった。また、GFP融合遺伝子による解析では、PARKの細胞膜周辺への局在が示された。
PARK遺伝子破壊株の耐凍性を調べた結果、ABAや低温処理した破壊株では、野生株より低い凍結耐性を示した。これらの結果から、PARKが細胞膜結合型プロテインキナーゼとして、ヒメツリガネゴケのABAや低温による凍結耐性上昇のシグナル伝達の制御に関与している可能性が示唆された。
日本植物生理学会
DOI：https://doi.org/10.14841/jspp.2003.0.459.0DOI ID：10.14841/jspp.2003.0.459.0,
CiNii Articles ID：130006987934 Molecular chaperone activity of ER-localized small heat shock proteins that accumulated in cortical parenchyma cells of mulberry tree during winter in vitro
N Ukaji; D Takezawa; K Arakawa; S Fujikawa
巻：43, 開始ページ：S116, 終了ページ：S116, 2002年
英語, 研究発表ペーパー・要旨（国際会議）
ISSN：0032-0781, Web of Science ID：WOS:000174726400420
Analyses of calmodulin-dependent protein kinase in moss Physcomitrella patens
D Takezawa
巻：43, 開始ページ：S53, 終了ページ：S53, 2002年
英語, 研究発表ペーパー・要旨（国際会議）
ISSN：0032-0781, Web of Science ID：WOS:000174726400188
ABA-induced freezing tolerance in moss Physcomitrella patens and physiological changes
M Nagao; A Minami; K Arakawa; S Fujikawa; D Takezawa
巻：43, 開始ページ：S168, 終了ページ：S168, 2002年
英語, 研究発表ペーパー・要旨（国際会議）
ISSN：0032-0781, Web of Science ID：WOS:000174726400601
ABA-INDUCED FREEZING TOLERANCE IN Physcomitrella patens AND GENE EXPRESSION :
NAGAO Manabu; MINAMI Anzu; TAKEZAWA Daisuke; ARAKAWA Keita; FUJIKAWA Seizo
Plant and cell physiology, 巻：42, 開始ページ：s121, 2001年
Japanese Society of Plant Physiologists, 英語
ISSN：0032-0781, CiNii Articles ID：110003715616, CiNii Books ID：AA0077511X
Accumulation of 18kD Intracellular Pathogenesis-Related Proteins in Cortical Parenchyma Cells of Mulberry Tree during Seasonal Cold Acclimation :
Ukaji Norifumi; Takezawa Daisuke; Arakawa Keita; Fujikawa Seizo
Plant and cell physiology, 巻：42, 開始ページ：s143, 2001年
Japanese Society of Plant Physiologists, 英語
ISSN：0032-0781, CiNii Articles ID：110003715703, CiNii Books ID：AA0077511X
The Function of the ABA-induced Thaumatin-like Protein in Winter Wheat Cells :
KUWABARA Chikako; ARAKAWA Keita; TAKEZAWA Daisuke; FUJIKAWA Seizo
Plant and cell physiology, 巻：41, 開始ページ：s50, 2000年
Japanese Society of Plant Physiologists, 英語
ISSN：0032-0781, CiNii Articles ID：110003722122, CiNii Books ID：AA0077511X
Gene expression of WAP20 and WAP27 :
UKAJI Norifumi; TAKEZAWA Daisuke; ARAKAWA Keita; FUJIKAWA Seizo
Plant and cell physiology, 巻：41, 開始ページ：s48, 2000年
Japanese Society of Plant Physiologists, 英語
ISSN：0032-0781, CiNii Articles ID：110003722116, CiNii Books ID：AA0077511X
DIVERSITY OF PROTEINS EXPRESSED IN OVERWINTERING PLANTS UNDER THE LOW TEMPERATURE ENVIRONMENT :
TAKEZAWA Daisuke; ARAKAWA Keita; YOSHIDA Shizuo; FUJIKAWA Seizo
Plant and cell physiology, 巻：41, 開始ページ：s10, 2000年
Japanese Society of Plant Physiologists, 英語
ISSN：0032-0781, CiNii Articles ID：110003721982, CiNii Books ID：AA0077511X
WAS-3, the ABA-induced Thaumatin-like Protein, in Suspension-cultured Cells of Winter Wheat
KUWABARA Chikako; ARAKAWA Keita; TAKEZAWA Daisuke; FUJIKAWA Seizo
Plant and cell physiology, 巻：40, 開始ページ：s75, 終了ページ：s75, 1999年03月
英語
ISSN：0032-0781, CiNii Articles ID：10003758018, CiNii Books ID：AA0077511X
Molecular Cloning of ABA-induced Secretory Protein (WAS-2) in Winter Wheat Cultured Cells
KUWABARA Chikako; TAKEZAWA Daisuke; ARAKAWA Keita; YOSHIDA Shizuo
Plant and cell physiology, 巻：39, 開始ページ：S92, 終了ページ：S92, 1998年05月
英語
ISSN：0032-0781, CiNii Articles ID：10003751843, CiNii Books ID：AA0077511X
ABA-INDUCED ACCUMULATION OF A 19-kDa PLASMA MEMBRANE POLYPEPTIDE IN SUSPENSION CULTURED-CELLS OF WINTER WHEAT AND ITS RELATIONSHIP TO THE INDUCTION OF FREEZING TOLERANCE
KOIKE Michiya; TAKEZAWA Daisuke; ARAKAWA Keita; YOSHIDA Shizuo
Plant and cell physiology, 巻：38, 開始ページ：s97, 1997年03月
英語
ISSN：0032-0781, CiNii Articles ID：10004345773, CiNii Books ID：AA0077511X
Dual regulation of a chimeric plant serine/threonine kinase by calcium/calmodulin
D Takezawa; S Ramachandiran; Paranjape, V; BW Poovaiah
巻：111, 号：2, 開始ページ：155, 終了ページ：155, 1996年06月
英語, 研究発表ペーパー・要旨（国際会議）
ISSN：0032-0889, Web of Science ID：WOS:A1996UR53400199
