基本情報

所属

• 地球環境科学研究院　環境生物科学部門　環境分子生物学分野

職名

• 准教授

学位

• 博士（農学）（東京大学）

ホームページURL

http://noah.ees.hokudai.ac.jp/emb/miwalab/
http://noah.ees.hokudai.ac.jp/emb/miwalab/en/

ORCID ID

•  https://orcid.org/0000-0001-7328-3628

J-Global ID

• 200901022910810865

研究キーワード

• 変異株   ホウ素   シロイヌナズナ   トランスポーター   要求量   ペクチン   植物細胞壁   ホウ酸   遺伝子発現制御   ストレス耐性   輸送体   無機栄養   

研究分野

• 環境・農学 / 環境農学
• ライフサイエンス / 植物栄養学、土壌学
• ライフサイエンス / 植物分子、生理科学

担当教育組織

•  学士課程　理学部
•  修士課程　環境科学院
•  博士課程　環境科学院

職歴

• 2014年04月 - 現在 北海道大学 地球環境科学研究科(研究院) 准教授
• 2021年08月 - 2023年07月 文部科学省学術調査官
• 2009年09月 - 2014年03月 北海道大学 創成研究機構 特任助教
• 2009年04月 - 2009年09月 東京大学 生物生産工学研究センター 日本学術振興会特別研究員
• 2008年04月 - 2009年03月 東京大学 生物生産工学研究センター 特任研究員
• 2006年04月 - 2008年03月 東京大学 大学院農学生命科学研究科 日本学術振興会特別研究員

学歴

• 2002年04月 - 2007年03月   東京大学   大学院農学生命科学研究科   応用生命工学専攻
• 1998年04月 - 2002年03月   東京大学   農学部   生命化学専修

所属学協会

• 日本植物生理学会   日本土壌肥料学会   

研究活動情報

論文

• Mutations in type II Golgi-localized proton pyrophosphatase AVP2;1/VHP2;1 affect pectic polysaccharide rhamnogalacturonan-II and alter root growth under low boron condition in Arabidopsis thaliana.

  Amarachukwu Faith Onuh, Kyoko Miwa

  Frontiers in Plant Science 14 1255486 - 1255486 2023年 

  The essential plant nutrient boron is required for the crosslinking of the pectin polysaccharide, rhamnogalacturonan II (RG-II). The synthesis of the pectic polysaccharides takes place in the Golgi apparatus, acidified by proton pumps. AVP2;1/VHP2;1 is a type II proton pyrophosphatase localized in the Golgi apparatus, which possesses proton pumping activity coupled with pyrophosphate hydrolysis. Its activity and expression patterns have been previously revealed but its role in plants remains unknown. The aim of the present work therefore was to explore the physiological role of AVP2;1 in Arabidopsis thaliana. In the screening of mutants under low boron, a mutant carrying a missense mutation in AVP2;1 was isolated. This mutant showed increased primary root growth under low boron conditions but no significant difference under normal boron condition compared to wild type plants. T-DNA insertion caused similar growth, suggesting that reduced function of AVP2;1 was responsible. Root cell observation revealed an increase in meristematic zone length, cell number in meristem and length of matured cell in avp2;1 mutants compared to wild type under low boron. Calcium concentration was reduced in mutant root cell wall under low boron. RG-II specific sugars also tended to be decreased in mutant root cell wall under low and normal boron conditions. These results suggest that changes in cell wall component by mutations in AVP2;1 may possibly explain the increased root length of mutants under low boron. This supports the idea that AVP2;1 plays a role in pH homoeostasis in Golgi apparatus for pectin synthesis.
• Translational Landscape of a C4 plant, Sorghum bicolor, under Normal and Sulfur Deficient Conditions.

  Naoyuki Sotta, Yukako Chiba, Haruka Aoyama, Seidai Takamatsu, Takamasa Suzuki, Kyoko Miwa, Yui Yamashita, Satoshi Naito, Toru Fujiwara

  Plant & cell physiology 2022年02月14日 

  Recent accumulation of genomic and transcriptomic information has facilitated genetic studies. Increasing evidence has demonstrated that translation is an important regulatory step, and the transcriptome does not necessarily reflect the profile of functional protein production. Deep sequencing of ribosome-protected mRNA fragments (ribosome profiling, or Ribo-seq) has enabled genome-wide analysis of translation. Sorghum is a C4 cereal important not only as food, but also as forage and a bioenergy resource. Its resistance to harsh environments has made it an agriculturally important research subject. Yet genome-wide translational profiles in sorghum are still missing. In this study, we took advantage of Ribo-seq and identified actively translated reading frames throughout the genome. We detected translation of 4,843 main open reading frames (ORFs) annotated in the sorghum reference genome version 3.1 and revealed a number of unannotated translational events. A comparison of the transcriptome and translatome between sorghums grown under normal and sulfur-deficient conditions revealed that gene expression is modulated independently at transcript and translation levels. Our study revealed the translational landscape of sorghum's response to sulfur and provides datasets that could serve as a fundamental resource to extend genetic research on sorghum, including studies on translational regulation.
• Global analysis of boron‐induced ribosome stalling reveals its effects on translation termination and unique regulation by AUG‐stops in Arabidopsis shoots

  Naoyuki Sotta, Yukako Chiba, Kyoko Miwa, Seidai Takamatsu, Mayuki Tanaka, Yui Yamashita, Satoshi Naito, Toru Fujiwara

  The Plant Journal 106 5 1455 - 1467 2021年06月26日 [査読有り]
   

  We previously reported that ribosome stalling at AUG-stop sequences in the 5'-untranslated region plays a critical role in regulating the expression of Arabidopsis thaliana NIP5;1, which encodes a boron uptake transporter, in response to boron conditions in media. This ribosome stalling is triggered specifically by boric acid, but the mechanisms are unknown. Although upstream open reading frames (uORFs) are known in many cases to regulate translation through peptides encoded by the uORF, AUG-stop stalling does not involve any peptide synthesis. The unique feature of AUG-stops - that termination follows immediately after initiation - suggests a possible effect of boron on the translational process itself. However, the generality of AUG-stop-mediated translational regulation and the effect of boron on translation at the genome scale are not clear. Here, we conducted a ribosome profiling analysis to reveal the genome-wide regulation of translation in response to boron conditions in A. thaliana shoots. We identified hundreds of translationally regulated genes that function in various biological processes. Under high-boron conditions, transcripts with reduced translation efficiency were rich in uORFs, highlighting the importance of uORF-mediated translational regulation. We found 673 uORFs that had more frequent ribosome association. Moreover, transcripts that were translationally downregulated under high-boron conditions were rich in minimum uORFs (AUG-stops), suggesting that AUG-stops play a global role in the boron response. Metagene analysis revealed that boron increased the ribosome occupancy of stop codons, indicating that this element is involved in global translational termination processes.
• Golgi-localized membrane protein AtTMN1/EMP12 functions in the deposition of rhamnogalacturonan II and I for cell growth in Arabidopsis

  Akihiko Hiroguchi, Shingo Sakamoto, Nobutaka Mitsuda, Kyoko Miwa

  Journal of Experimental Botany 72 10 3611 - 3629 2021年05月04日 [査読有り]
   

  Appropriate pectin deposition in cell walls is important for cell growth in plants. Rhamnogalacturonan II (RG-II) is a portion of pectic polysaccharides; its borate crosslinking is essential for maintenance of pectic networks. However, the overall process of RG-II synthesis is not fully understood. To identify a novel factor for RG-II deposition or dimerization in cell walls, we screened Arabidopsis mutants with altered boron (B)-dependent growth. The mutants exhibited alleviated disorders of primary root and stem elongation, and fertility under low B, but reduced primary root lengths under sufficient B conditions. Altered primary root elongation was associated with cell elongation changes caused by loss of function in AtTMN1 (Transmembrane Nine 1)/EMP12, which encodes a Golgi-localized membrane protein of unknown function that is conserved among eukaryotes. Mutant leaf and root dry weights were lower than those of wild-type plants, regardless of B conditions. In cell walls, AtTMN1 mutations reduced concentrations of B, RG-II specific 2-keto-3-deoxy monosaccharides, and rhamnose largely derived from rhamnogalacturonan I (RG-I), suggesting reduced RG-II and RG-I. Together, our findings demonstrate that AtTMN1 is required for the deposition of RG-II and RG-I for cell growth and suggest that pectin modulates plant growth under low B conditions.
• Involvement of boron transporter BOR1 in growth under low boron and high nitrate conditions in Arabidopsis thaliana

  Qing Wang, Wenna Zhang, Hua Xiao, Naoyuki Sotta, Marcel P. Beier, Junpei Takano, Kyoko Miwa, Lihong Gao, Toru Fujiwara

  Physiologia Plantarum 171 4 703 - 713 2021年04月
• Regulation, Diversity and Evolution of Boron Transporters in Plants

  Amarachukwu Faith Onuh, Kyoko Miwa

  Plant and Cell Physiology 62 4 590 - 599 2021年02月11日 [査読有り]
  
• Boron-dependent translational suppression of the borate exporter BOR1 contributes to the avoidance of boron toxicity

  Izumi Aibara, Tatsuya Hirai, Koji Kasai, Junpei Takano, Hitoshi Onouchi, Satoshi Naito, Toru Fujiwara, Kyoko Miwa

  Plant Physiology 177 2 759 - 774 2018年06月01日 [査読有り][通常論文]
   

  Boron (B) is an essential element for plants however, as high B concentrations are toxic, B transport must be tightly regulated. BOR1 is a borate exporter in Arabidopsis (Arabidopsis thaliana) that facilitates B translocation into shoots under B deficiency conditions. When the B supply is sufficient, BOR1 expression is down-regulated by selective degradation of BOR1 protein, while additional BOR1 regulatory mechanisms are proposed to exist. In this study, we identified a novel B-dependent BOR1 translational suppression mechanism. In vivo and in vitro reporter assays demonstrated that BOR1 translation was reduced in a B-dependent manner and that the 5′-untranslated region was both necessary and sufficient for this process. Mutational analysis revealed that multiple upstream open reading frames in the 5′-untranslated region were required for BOR1 translational suppression, and this process depended on the efficiency of translational reinitiation at the BOR1 open reading frame after translation of the upstream open reading frames. To understand the physiological significance of BOR1 regulation, we characterized transgenic plants defective in either one or both of the BOR1 regulation mechanisms. BOR1 translational suppression was induced at higher B concentrations than those triggering BOR1 degradation. Plants lacking both regulation mechanisms exhibited more severe shoot growth reduction under high-B conditions than did plants lacking BOR1 degradation alone, thus demonstrating the importance of BOR1 translational suppression. This study demonstrates that two mechanisms of posttranscriptional BOR1 regulation, each induced under different B concentrations, contribute to the avoidance of B toxicity in plants.
• Effect of Exogenous General Plant Growth Regulators on the Growth of the Duckweed Lemna minor.

  Utami D, Kawahata A, Sugawara M, Jog RN, Miwa K, Morikawa M

  Frontiers in chemistry 6 251  2018年 [査読有り][通常論文]
  
• The Minimum Open Reading Frame, AUG-Stop, Induces Boron-Dependent Ribosome Stalling and mRNA Degradation.

  Mayuki Tanaka, Naoyuki Sotta, Yusuke Yamazumi, Yui Yamashita, Kyoko Miwa, Katsunori Murota, Yukako Chiba, Masami Yokota Hirai, Tetsu Akiyama, Hitoshi Onouchi, Satoshi Naito, Toru Fujiwara

  The Plant cell 28 11 2830 - 2849 2016年11月 [査読有り][通常論文]
   

  Upstream open reading frames (uORFs) are often translated ahead of the main ORF of a gene and regulate gene expression, sometimes in a condition-dependent manner, but such a role for the minimum uORF (hereafter referred to as AUG-stop) in living organisms is currently unclear. Here, we show that AUG-stop plays an important role in the boron (B)-dependent regulation of NIP5;1, encoding a boric acid channel required for normal growth under low B conditions in Arabidopsis thaliana High B enhanced ribosome stalling at AUG-stop, which was accompanied by the suppression of translation and mRNA degradation. This mRNA degradation was promoted by an upstream conserved sequence present near the 5'-edge of the stalled ribosome. Once ribosomes translate a uORF, reinitiation of translation must take place in order for the downstream ORF to be translated. Our results suggest that reinitiation of translation at the downstream NIP5;1 ORF is enhanced under low B conditions. A genome-wide analysis identified two additional B-responsive genes, SKU5 and the transcription factor gene ABS/NGAL1, which were regulated by B-dependent ribosome stalling through AUG-stop. This regulation was reproduced in both plant and animal transient expression and cell-free translation systems. These findings suggest that B-dependent AUG-stop-mediated regulation is common in eukaryotes.
• Synthesis of borate cross-linked rhamnogalacturonan II

  Hiroya Funakawa, Kyoko Miwa

  FRONTIERS IN PLANT SCIENCE 6 223  2015年04月 [査読有り][通常論文]
   

  In the present review, we describe current knowledge about synthesis of borate crosslinked rhamnogalacturonan II (RG-II) and it physiological roles. RG-II is a portion of pectic polysaccharide with high complexity, present in primary cell wall. It is composed of homogalacturonan backbone and four distinct side chains (A-D). Borate forms ester bonds with the apiosyl residues of side chain A of two RG-II monomers to generate borate dimerized RG-II, contributing for the formation of networks of pectic polysaccharides. In plant cell walls, more than 90% of RG-II are dimerized by borate under boron (B) sufficient conditions. Borate crosslinking of RG-II in primary cell walls, to our knowledge, is the only experimentally proven molecular function of B, an essential trace-element. Although abundance of RG-II and B is quite small in cell wall polysaccharides, increasing evidence supports that RG-II and its borate crosslinking are critical for plant growth and development. Significant advancement was made recently on the location and the mechanisms of RG-II synthesis and borate cross-linking. Molecular genetic studies have successfully identified key enzymes for RG-II synthesis and regulators including B transporters required for efficient formation of RG-II crosslinking and consequent normal plant growth. The present article focuses recent advances on (i) RG-II polysaccharide synthesis, (ii) occurrence of borate crosslinking and (iii) B transport for borate supply to RG-II. Molecular mechanisms underlying formation of borate RG-II crosslinking and the physiological impacts are discussed.
• Strategies for Optimization of Mineral Nutrient Transport in Plants: Multilevel Regulation of Nutrient-Dependent Dynamics of Root Architecture and Transporter Activity

  Izumi Aibara, Kyoko Miwa

  PLANT AND CELL PHYSIOLOGY 55 12 2027 - 2036 2014年12月 [査読有り][通常論文]
   

  How do sessile plants cope with irregularities in soil nutrient availability? The uptake of essential minerals from the soil influences plant growth and development. However, most environments do not provide sufficient nutrients; rather nutrient distribution in the soil can be uneven and change temporally according to environmental factors. To maintain mineral nutrient homeostasis in their tissues, plants have evolved sophisticated systems for coping with spatial and temporal variability in soil nutrient concentrations. Among these are mechanisms for modulating root system architecture in response to nutrient availability. This review discusses recent advances in knowledge of the two important strategies for optimizing nutrient uptake and translocation in plants: root architecture modification and transporter expression control in response to nutrient availability. Recent studies have determined (i) nutrient-specific root patterns; (ii) their physiological consequences; and (iii) the molecular mechanisms underlying these modulation systems that operate to facilitate efficient nutrient acquisition. Another mechanism employed by plants in nutrient-heterogeneous soils involves modification of nutrient transport activities in a nutrient concentration-dependent manner. In recent years, considerable progress has been made in characterizing the diverse functions of transporters for specific nutrients; it is now clear that the expression and activities of nutrient transporters are finely regulated in multiple steps at both the transcriptional and post-transcriptional levels for adaptation to a wide range of nutrient conditions.
• Plant growth-promoting bacterium Acinetobacter calcoaceticus P23 increases the chlorophyll content of the monocot Lemna minor (duckweed) and the dicot Lactuca sativa (lettuce)

  Wakako Suzuki, Masayuki Sugawara, Kyoko Miwa, Masaaki Morikawa

  JOURNAL OF BIOSCIENCE AND BIOENGINEERING 118 1 41 - 44 2014年07月 [査読有り][通常論文]
   

  Acinetobacter cakoaceticus P23 is a plant growth-promoting bacterium that was isolated from the surface of duckweed (Lemna aoubilmsa). The bacterium was observed to colonize on the plant surfaces and increase the chlorophyll content of not only the monocotyledon Lemna minor but also the dicotyledon Lactuca sativa in a hydroponic culture. This effect on the Lactuca sativa was significant in nutrient-poor ( x 1/100 dilution of H2 medium) and not nutrient-rich ( x 1 or x1/l0 dilutions of H2 medium) conditions. Strain P23 has the potential to play a part in the future development of fertilizers and energy-saving hydroponic agricultural technologies. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.
• Improved tolerance to boron deficiency by enhanced expression of the boron transporter BOR2

  Shigeki Takada, Kyoko Miwa, Hiroyuki Omori, Toru Fujiwara, Satoshi Naito, Junpei Takano

  SOIL SCIENCE AND PLANT NUTRITION 60 3 341 - 348 2014年06月 [査読有り][通常論文]
   

  Boron (B) cross-links the pectin polysaccharide rhamnogalacturonan II (RG-II) and thus is important for cell wall structure in plants. B deficiency is an agricultural problem that causes significant losses of crop productivity worldwide. To address this, B deficiency-tolerant plants have been generated using B transporters. With the goal of further improving plant tolerance to low-B conditions, we generated transgenic Arabidopsis thaliana (L.) Heynh. with enhanced expression of BOR2, a B transporter that promotes cross-linking of RG-II and root elongation under low B supply. We generated a DNA construct containing the cauliflower mosaic virus 35S RNA promoter, a native promoter of BOR2, a BOR2 gene and green fluorescent protein (GFP) (Pro35S-BOR2: BOR2-GFP), and obtained three independent transgenic lines with relatively high levels of BOR2-GFP expression. In the transgenic lines, BOR2-GFP was expressed mainly in the lateral root caps in the meristem zone and in the epidermal cells in the elongation zone, similar to its expression when driven only by its native promoter. In the Pro35S-BOR2: BOR2-GFP lines, BOR2-GFP was also expressed in various cells in the maturation zone of roots and epidermal cells of shoots, where the expression was hardly detectable in ProBOR2: BOR2-GFP lines. The transgenic lines were cultured under various B concentrations on solid media and it was found that root growth of three lines and shoot growth of two lines were enhanced compared to wild-type plants under low-B conditions. This finding established that enhanced expression of BOR2 leads to improved root growth under low-B conditions. We also examined growth of the transgenic lines under hydroponic conditions. One of the three lines showed better growth and fertility under a low-B condition, while the wild type did not set seeds under the same condition, suggesting the potential utility of BOR2 expression in agricultural applications.
• Arabidopsis thaliana BOR4 is upregulated under high boron conditions and confers tolerance to high boron

  Kyoko Miwa, Izumi Aibara, Toru Fujiwara

  SOIL SCIENCE AND PLANT NUTRITION 60 3 349 - 355 2014年06月 [査読有り][通常論文]
   

  Boron (B) is an essential micronutrient for plants, but a high concentration of B is toxic. Typical B toxicity symptoms include necrosis of marginal leaves and inhibition of root elongation. Reduction of crop productivity has been reported in high-B contaminated soils, present especially in semi-arid areas in the world. We have previously reported that overexpression of BOR4, an Arabidopsis thaliana (L.) Heynh. efflux-type B transporter, conferred high B tolerance in A. thaliana. In the present study, we characterized physiological roles and expression patterns of endogenous BOR4 in A. thaliana. Decreased shoot growths and increased B concentrations in shoots were found in A. thaliana T-DNA insertion mutants of BOR4 under toxic levels of B supply, compared to the wild type plants. beta-glucuronidase (GUS) staining in the transgenic ProBOR4-GUS plants was predominately detected in root meristems and endodermis of mature portion of the roots. Furthermore, mRNA levels of BOR4 in roots were increased by 2-fold upon 3-day treatment of the high B condition and analysis of the transgenic ProBOR4-GUS plants showed that this high B-dependent induction is controlled by the 5' flanking sequences of BOR4 ORF. We concluded that endogenous BOR4 is a high-B inducible gene that functions in high-B tolerance.
• Generation of boron-deficiency-tolerant tomato by overexpressing an Arabidopsis thaliana borte transporter AtBOR1

  Shimpei Uraguchi, Yuichi Kato, Hideki Hanaoka, Kyoko Miwa, Toru Fujiwara

  FRONTIERS IN PLANT SCIENCE 5 125  2014年04月 [査読有り][通常論文]
   

  Nutrient deficiency in soil poses a widespread agricultural problem. Boron (B) is an essential micronutrient in plants, and its deficiency causes defects in both vegetative and reproductive growth in various crops in the field. In Arabidopsis thaliana, increased expression of a major borate transporter gene AtBOR1 or boric acid channel gene AtNIP5;1 improves plant growth under B-deficient conditions. In this study, we examined whether high expression of a borate transporter gene increases B accumulation in shoots and improves the growth of tomato plant, a model of fruit-bearing crops, under B-deficient conditions. We established three independent transgenic tomato plants lines expressing AtBOR1 using Agrobacterium-mediated transformation of tomato (Solanum lycopersicum L. cv. Micro-Tom). Reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed that two lines (Line 1 and Line 2) more strongly expressed AtBOR1than Line 3. Wild-type plants and the transgenic plants were grown hydroponically under B-sufficient and B-deficient conditions. Wild-type and Line 3 (weakly expressing transgenic line) showed a defect in shoot growth under B-deficient conditions, especially in the development of new leaves. However, seedlings of Line 1 and Line 2, the transgenic lines showing strong AtBOR1 expression, did not show the B-deficiency phenotype in newly developing leaves. In agreement with this phenotype, shoot biomass under low-B conditions was higher in the strongly expressing AtBOR1line. B concentrations in leaves or fruits were also higher in Line 2 and Line 1. The present study demonstrates that strong expression of AtBOR1 improved growth in tomato under B-deficient conditions
• Difference in cesium accumulation among rice cultivars grown in the paddy field in Fukushima Prefecture in 2011 and 2012

  Yoshihiro Ohmori, Yayoi Inui, Masataka Kajikawa, Atsumi Nakata, Naoyuki Sotta, Koji Kasai, Shimpei Uraguchi, Nobuhiro Tanaka, Sho Nishida, Takahiro Hasegawa, Takuya Sakamoto, Yuko Kawara, Kayoko Aizawa, Haruka Fujita, Ke Li, Naoya Sawaki, Koshiro Oda, Ryuichiro Futagoishi, Takahiro Tsusaka, Satomi Takahashi, Junpei Takano, Shinji Wakuta, Akira Yoshinari, Masataka Uehara, Shigeki Takada, Hayato Nagano, Kyoko Miwa, Izumi Aibara, Takuya Ojima, Kaoru Ebana, Satoru Ishikawa, Kuni Sueyoshi, Hiroshi Hasegawa, Tetsuro Mimura, Mari Mimura, Natsuko I. Kobayashi, Jun Furukawa, Daisuke Kobayashi, Toshiyasu Okouchi, Keitaro Tanoi, Toru Fujiwara

  JOURNAL OF PLANT RESEARCH 127 1 57 - 66 2014年01月 [査読有り][通常論文]
   

  After the accident of the Fukushima 1 Nuclear Power Plant in March 2011, radioactive cesium was released and paddy fields in a wide area including Fukushima Prefecture were contaminated. To estimate the levels of radioactive Cs accumulation in rice produced in Fukushima, it is crucial to obtain the actual data of Cs accumulation levels in rice plants grown in the actual paddy field in Fukushima City. We herein conducted a two-year survey in 2011 and 2012 of radioactive and non-radioactive Cs accumulation in rice using a number of rice cultivars grown in the paddy field in Fukushima City. Our study demonstrated a substantial variation in Cs accumulation levels among the cultivars of rice.
• Roles of BOR2, a boron exporter, in cross linking of rhamnogalacturonan II and root elongation under boron limitation in arabidopsis

  Kyoko Miwa, Shinji Wakuta, Shigeki Takada, Koji Ide, Junpei Takano, Satoshi Naito, Hiroyuki Omori, Toshiro Matsunaga, Toru Fujiwara

  Plant Physiology 163 4 1699 - 1709 2013年12月 [査読有り][通常論文]
   

  Boron (B) is required for cross linking of the pectic polysaccharide rhamnogalacturonan II (RG-II) and is consequently essential for the maintenance of cell wall structure. Arabidopsis (Arabidopsis thaliana) BOR1 is an efflux B transporter for xylem loading of B. Here, we describe the roles of BOR2, the most similar paralog of BOR1. BOR2 encodes an efflux B transporter localized in plasma membrane and is strongly expressed in lateral root caps and epidermis of elongation zones of roots. Transfer DNA insertion of BOR2 reduced root elongation by 68%, whereas the mutation in BOR1 reduced it by 32% under low B availability (0.1 mM), but the reduction in shoot growth was not as obvious as that in the BOR1 mutant. A double mutant of BOR1 and BOR2 exhibited much more severe growth defects in both roots and shoots under B-limited conditions than the corresponding single mutants. All single and double mutants grew normally under B-sufficient conditions. These results suggest that both BOR1 and BOR2 are required under B limitation and that their roles are, at least in part, different. The total B concentrations in roots of BOR2 mutants were not significantly different from those in wild-type plants, but the proportion of cross-linked RG-II was reduced under low B availability. Such a reduction in RG-II cross linking was not evident in roots of the BOR1 mutant. Thus, we propose that under B-limited conditions, transport of boric acid/borate by BOR2 from symplast to apoplast is required for effective cross linking of RG-II in cell wall and root cell elongation. © 2013 American Society of Plant Biologists. All Rights Reserved.
• Expression of the Arabidopsis Borate Efflux Transporter Gene, AtBOR4, in Rice Affects the Xylem Loading of Boron and Tolerance to Excess Boron

  Masataka Kajikawa, Takahiro Fujibe, Shimpei Uraguchi, Kyoko Miwa, Toru Fujiwara

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 75 12 2421 - 2423 2011年12月 [査読有り][通常論文]
   

  Boron is an essential nutrient for plants, but it is toxic in excess. Transgenic rice plants expressing an Arabidopsis thaliana borate efflux transporter gene, AtBOR4, at a low level exhibited increased tolerance to excess boron. Those lines with high levels of expression exhibited reduced growth. These findings suggest a potential of the borate transporter BOR4 for the generation of high-boron tolerant rice.
• Role of overexpressed BOR4, a boron exporter, in tolerance to high level of boron in shoots

  Kyoko Miwa, Toru Fujiwara

  SOIL SCIENCE AND PLANT NUTRITION 57 4 558 - 565 2011年08月 [査読有り][通常論文]
   

  High boron (B) tolerance is an important trait for crop production in high-B soils. We previously reported that overexpression of BOR4, an Arabidopsis thaliana B exporter, conferred high B tolerance in A. thaliana. This improvement appeared to be mainly due to the decreased B concentrations in roots through BOR4-mediated exclusion of B. In the present study, we describe a novel role of overexpressed BOR4 in shoots for high B tolerance. We compared shoot growth of wild type A. thaliana and transgenic plants moderately overexpressing BOR4 in relation to B concentration in shoots. It was found that growths and chlorophyll accumulation of shoots containing similar levels of B are better in the transgenic plants than the wild type. This finding established that BOR4-overexpressing plants are more capable of expanding leaves and accumulating chlorophyll in the presence of high B in shoot tissues. BOR4-GFP was found to be localized to plasma membrane in leaf epidermis cells. We suggest that overexpressed BOR4 alters B distribution in leaves by exporting B from the cytoplasm to the apoplast, leading to enhancement of high B tolerance in shoots.
• High Boron-induced Ubiquitination Regulates Vacuolar Sorting of the BOR1 Borate Transporter in Arabidopsis thaliana

  Koji Kasai, Junpei Takano, Kyoko Miwa, Atsushi Toyoda, Toru Fujiwara

  JOURNAL OF BIOLOGICAL CHEMISTRY 286 8 6175 - 6183 2011年02月 [査読有り][通常論文]
   

  Boron homeostasis is important for plants, as boron is essential but is toxic in excess. Under high boron conditions, the Arabidopsis thaliana borate transporter BOR1 is trafficked from the plasma membrane (PM) to the vacuole via the endocytic pathway for degradation to avoid excess boron transport. Here, we show that boron-induced ubiquitination is required for vacuolar sorting of BOR1. We found that a substitution of lysine 590 with alanine (K590A) in BOR1 blocked degradation. BOR1 was mono-or diubiquitinated within several minutes after applying a high concentration of boron, whereas the K590A mutant was not. The K590A mutation abolished vacuolar transport of BOR1 but did not apparently affect polar localization to the inner PM domains. Furthermore, brefeldin A and wortmannin treatment suggested that Lys-590 is required for BOR1 translocation from an early endosomal compartment to multivesicular bodies. Our results show that boron-induced ubiquitination of BOR1 is not required for endocytosis from the PM but is crucial for the sorting of internalized BOR1 to multivesicular bodies for subsequent degradation in vacuoles.
• Boron transport in plants: co-ordinated regulation of transporters

  Kyoko Miwa, Toru Fujiwara

  ANNALS OF BOTANY 105 7 1103 - 1108 2010年06月 [査読有り][通常論文]
   

  The essentiality of boron (B) for plant growth was established > 85 years ago. In the last decade, it has been revealed that one of the physiological roles of B is cross-linking the pectic polysaccharide rhamnogalacturonan II in primary cell walls. Borate cross-linking of pectic networks serves both for physical strength of cell walls and for cell adhesion. On the other hand, high concentrations of B are toxic to plant growth. To avoid deficiency and toxicity problems, it is important for plants to maintain their tissue B concentrations within an optimum range by regulating transport processes. Boron transport was long believed to be a passive, unregulated process, but the identification of B transporters has suggested that plants sense and respond to the B conditions and regulate transporters to maintain B homeostasis. Transporters responsible for efficient B uptake by roots, xylem loading and B distribution among leaves have been described. These transporters are required under B limitation for efficient acquisition and utilization of B. Transporters important for tolerating high B levels in the environment have also been identified, and these transporters export B from roots back to the soil. Two types of transporters are involved in these processes: NIPs (nodulin-26-like intrinsic proteins), boric acid channels, and BORs, B exporters. It is demonstrated that the expression of genes encoding these transporters is finely regulated in response to B availability in the environment to ensure tissue B homeostasis. Furthermore, plants tolerant to stress produced by low B or high B in the environment can be generated through altered expression of these transporters. The identification of the first B transporter led to the discovery that B transport was a process mediated not only by passive diffusion but also by transporters whose activity was regulated in response to B conditions. Now it is evident that plants sense internal and external B conditions and regulate B transport by modulating the expression and/or accumulation of these transporters. Results obtained in model plants are applicable to other plant species, and such knowledge may be useful in designing plants or crops tolerant to soils containing low or high B.
• Polar localization and degradation of Arabidopsis boron transporters through distinct trafficking pathways

  Junpei Takano, Mayuki Tanaka, Atsushi Toyoda, Kyoko Miwa, Koji Kasai, Kentaro Fuji, Hitoshi Onouchi, Satoshi Naito, Toru Fujiwara

  Proceedings of the National Academy of Sciences of the United States of America 107 11 5220 - 5225 2010年03月16日 [査読有り][通常論文]
   

  Boron (B) is essential for plant growth but is toxic when present in excess. In the roots of Arabidopsis thaliana under B limitation, a boric acid channel, NIP5 1, and a boric acid/borate exporter, BOR1, are required for efficient B uptake and subsequent translocation into the xylem, respectively. However, under high-B conditions, BOR1 activity is repressed through endocytic degradation, presumably to avoid B toxicity. In this study, we investigated the localization of GFP-tagged NIP5 1 and BOR1 expressed under the control of their native promoters. Under B limitation, GFP-NIP5 1 and BOR1-GFP localized preferentially in outer (distal) and inner (proximal) plasma membrane domains, respectively, of various root cells. The polar localization of the boric acid channel and boric acid/borate exporter indicates the radial transport route of B toward the stele. Furthermore, mutational analysis revealed a requirement of tyrosine residues, in a probable cytoplasmic loop region of BOR1, for polar localization in various cells of the meristem and elongation zone. The same tyrosine residues were also required for vacuolar targeting upon high B supply. The present study of BOR1 and NIP5 1 demonstrates the importance of selective endocytic trafficking in polar localization and degradation of plant nutrient transporters for radial transport and homeostasis of plant mineral nutrients.
• Molecular mechanism and regulation of boric acid transport in plants

  Mayuki Tanaka, Kyoko Miwa, Toru Fujiwara

  Seikagaku 82 5 367 - 377 2010年 [査読有り][通常論文]
  
• Molecular Mechanisms of Boron Transport in Plants: Involvement of Arabidopsis NIP5;1 and NIP6;1

  Kyoko Miwa, Mayuki Tanaka, Takehiro Kamiya, Toru Fujiwara

  MIPS AND THEIR ROLE IN THE EXCHANGE OF METALLOIDS 679 83 - 96 2010年 [査読有り][通常論文]
   

  Understanding of the molecular mechanisms of boron (B) transport has been greatly advanced in the last decade. BOR1, the first B transporter in living systems, was identified by forward genetics using Arabidopsis mutants. Genes similar to BOR1 have been reported to share different physiological roles in plants. NIPS;1, a member of aquaporins in Arabidopsis, was then identified as a boric acid channel gene responsible for the B uptake into roots. NIP6;1, the most similar gene to NIP5;1, encodes a B channel essential for B distribution to young leaves. In the present chapter, recent advancement of the understanding of molecular mechanisms of B transport and roles of NIP genes are discussed.
• The intracellular transport of transporters: membrane trafficking of mineral transporters

  Kentaro Fuji, Kyoko Miwa, Toru Fujiwara

  CURRENT OPINION IN PLANT BIOLOGY 12 6 699 - 704 2009年12月 [査読有り][通常論文]
   

  For mineral nutrients to be used by plants, they must be taken up from soil solutions into root cells and then transported to shoots. Mineral nutrient transporters play a central role in this process, and their expression and accumulation are known to be strictly regulated in response to change in nutrient conditions. Roots are cylindrically shaped organs with various types of cells. For the nutrients to move from soil solution toward the xylem they have to be transported across various types of cells. Nutrient condition-dependent accumulation and polar distributions of transporters in plant cells are established by membrane trafficking systems. The present article provides an overview of current findings regarding the membrane trafficking of mineral nutrient transporters and a discussion of future perspectives in this field of research.
• Homeostasis of the structurally important micronutrients, B and Si

  Kyoko Miwa, Takehiro Kamiya, Toru Fujiwara

  CURRENT OPINION IN PLANT BIOLOGY 12 3 307 - 311 2009年06月 [査読有り][通常論文]
   

  This review focuses on recent advances in understanding the transport mechanisms of two elements, B and Si in plants. Both are present as noncharged molecules in soil solution as boric acid and silicic acid. Both function in apoplast: pectic polysaccharides crosslinked with borate and polymers of hydrated silica are important for the physical strength of plant cells. In recent years, molecular genetics revealed analogous transport systems of B and Si. Combinations of NIP channels and exporters localized to distal and proximal sides, allow efficient trans-cellular transport of the nutrients. Polar localization, observed in these transport molecules, is likely to be a key to regulate directional transport of nutrients.
• 植物の無機栄養(第4部門 植物栄養)

  三輪 京子, 藤原 徹

  日本土壌肥料学雑誌 79 6 568 - 572 一般社団法人 日本土壌肥料学会 2009年
• Highly Boron Deficiency-Tolerant Plants Generated by Enhanced Expression of NIP5;1, a Boric Acid Channel

  Yuichi Kato, Kyoko Miwa, Junpei Takano, Motoko Wada, Toru Fujiwara

  PLANT AND CELL PHYSIOLOGY 50 1 58 - 66 2009年01月 [査読有り][通常論文]
   

  Boron (B) is an essential element for plants, and B deficiency is a worldwide agricultural problem. In B-deficient areas, B is often supplied as fertilizer, but excess B can be toxic to both plants and animals. Generation of B deficiency-tolerant plants could reduce B fertilizer use. Improved fertility under B-limiting conditions in Arabidopsis thaliana by overexpression of BOR1, a B transporter, has been reported, but the root growth was not improved by the BOR1 overexpression. In this study, we report that enhanced expression of NIP5;1, a boric acid channel for efficient B uptake, resulted in improved root elongation under B-limiting conditions in A. thaliana. An NIP5;1 activation tag line, which has a T-DNA insertion with enhancer sequences near the NIP5;1 gene, showed improved root elongation under B limitation. We generated a construct which mimics the tag line: the cauliflower mosaic virus 35S RNA promoter was inserted at 1,357bp upstream of the NIP5;1 transcription initiation site. Introduction of this construct into the nip5;1-1 mutant and the BOR1 overexpresser resulted in enhanced expression of NIP5;1 and improved root elongation under low B supply. Furthermore, one of the transgenic lines exhibited improved fertility and short-term B uptake. Our results demonstrate successful improvement of B deficiency tolerance and the potential of enhancing expression of a mineral nutrient channel gene to improve growth under nutrient-limiting conditions.
• Boron transport mechanisms: collaboration of channels and transporters

  Junpei Takano, Kyoko Miwa, Toru Fujiwara

  TRENDS IN PLANT SCIENCE 13 8 451 - 457 2008年08月 [査読有り][通常論文]
   

  Boron (B) is an essential element for plants, but is also toxic when present in excess. B deficiency and toxicity are both major agricultural problems worldwide, and elucidating the molecular mechanisms of B transport should allow us to develop technology to alleviate B deficiency and toxicity problems. Recent milestones include the identification of a boric acid channel, NIP5;1, and a boric acid/borate exporter, BOR1, from Arabidopsis thaliana. Both proteins were shown to be required for plant growth under B limitation. In addition, BOR1 homologs are required for B homeostasis in mammalian cells and B-toxicity tolerance in yeast and plants. Here, we discuss how transgenic approaches show promise for generating crops that are tolerant of B deficiency and toxicity.
• [Molecular mechanisms of boron transport in plants and its modification for plant growth improvement].

  Miwa K, Takano J, Fujiwara T

  Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 53 9 1173 - 1179 共立出版 2008年07月 [査読有り][通常論文]
  
• Plants tolerant of high boron levels

  Kyoko Miwa, Junpei Takano, Hiroyuki Omori, Motoaki Seki, Kazuo Shinozaki, Toru Fujiwara

  SCIENCE 318 5855 1417 - 1417 2007年11月 [査読有り][通常論文]
  
• Cell-type specificity of the expression of os BOR1, a rice efflux boron transporter gene, is regulated in response to boron availability for efficient boron uptake and xylem loading

  Yuko Nakagawa, Hideki Hanaoka, Masaharu Kobayashi, Kazumaru Miyoshi, Kyoko Miwa, Toru Fujiwara

  PLANT CELL 19 8 2624 - 2635 2007年08月 [査読有り][通常論文]
   

  We describe a boron ( B) transporter, Os BOR1, in rice ( Oryza sativa). Os BOR1 is a plasma membrane-localized efflux transporter of B and is required for normal growth of rice plants under conditions of limited B supply (referred to as -B). Disruption of Os BOR1 reduced B uptake and xylem loading of B. The accumulation of Os BOR1 transcripts was higher in roots than that in shoots and was not affected by B deprivation; however, Os BOR1 was detected in the roots of wild-type plants under -B conditions, but not under normal conditions, suggesting regulation of protein accumulation in response to B nutrition. Interestingly, tissue specificity of Os BOR1 expression is affected by B treatment. Transgenic rice plants containing an Os BOR1 promoter-beta-glucuronidase (GUS) fusion construct grown with a normal B supply showed the strongest GUS activity in the steles, whereas after 3 d of -B treatment, GUS activity was elevated in the exodermis. After 6 d of -B treatment, GUS activity was again strong in the stele. Our results demonstrate that Os BOR1 is required both for efficient B uptake and for xylem loading of B. Possible roles of the temporal changes in tissue-specific patterns of Os BOR1 expression in response to B condition are discussed.
• Isolation of Arabidopsis thaliana cDNAs that confer yeast boric acid tolerance

  Akira Nozawa, Kyoko Miwa, Masaharu Kobayashi, Toru Fujiwara

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 70 7 1724 - 1730 2006年07月 [査読有り][通常論文]
   

  An Arabidopsis thaliana cDNA library was introduced into a Saccharomyces cerevisiae mutant that lacks ScBOR1 (YNL275W), a boron (B) efflux transporter. Five cDNAs were identified that confer tolerance to high boric acid. The nucleotide sequence analysis identified the clones as a polyadenylate-binding protein, AtPAB2; a ribosomal small subunit protein, AtRPS20B; an RNA-binding protein, AtRBP47c'; and two Myb transcription factors, AtMYB13 and AtMYB68. The expression of these five genes also conferred boric acid tolerance on wild-type yeast. Two yeast genes, ScRPS20 and ScHRB1, that are similar to the isolated clones, were necessary for this boric acid tolerance. The possible roles of these A. thaliana and S. cerevisiae genes in boric acid tolerance are discussed.
• Improvement of seed yields under boron-limiting conditions through overexpression of BOR1, a boron transporter for xylem loading, in Arabidopsis thaliana

  K Miwa, J Takano, T Fujiwara

  PLANT JOURNAL 46 6 1084 - 1091 2006年06月 [査読有り][通常論文]
   

  Soil fertilization is a common practice in modern agriculture, undertaken to prevent nutrient deficiency in crops. However, fertilization is costly and causes environmental pollution. The cultivation of plants that tolerate low nutrient supplies may circumvent this problem. Here, we report the generation of Arabidopsis thaliana plants that tolerate boron (B)-deficient conditions due to the overexpression of BOR1, an efflux B transporter that is required for efficient xylem loading of B. In several independently generated transgenic plants expressing BOR1 or BOR1-GFP under the control of the cauliflower mosaic virus 35S RNA promoter, root-to-shoot translocation of B was enhanced and shoot growth was greater under B-limiting conditions compared with wild-type plants. In addition, the transgenic plants showed increased translocation of B, especially to the shoot apex, and set seed normally under B-limiting conditions, under which wild-type plants failed to set seed. This study therefore reports plants that show improved seed yields compared with wild-type under nutrient-deficient conditions as a result of increased production of an essential mineral nutrient transporter.
• Endocytosis and degradation of BOR1, a boron transporter of Arabidopsis thaliana, regulated by boron availability

  J Takano, K Miwa, LX Yuan, N von Wiren, T Fujiwara

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 102 34 12276 - 12281 2005年08月 [査読有り][通常論文]
   

  Boron (B) is essential for plants but toxic when present in excess. Arabidopsis thaliana BOR1 is a B exporter for xylem loading and is essential for efficient B translocation from roots to shoots under B limitation. B translocation to shoots was enhanced under B limitation in WT but not in bor1-1 mutant plants. The enhanced translocation was suppressed upon resupply of high levels of B within several hours. Unlike a number of transporters for essential mineral nutrients, BOR1 mRNA accumulation was not strongly affected by B conditions. However, accumulation of a constitutively expressed BOR1-GFP fusion protein was elevated under conditions of limited B supply. Upon resupply of high levels of B, BOR1-GFP was degraded within several hours. These findings demonstrate that posttranscriptional mechanisms play a major role in regulation of BOR1 accumulation. Confocal laser scanning microscopy of root tip cells showed that BOR1-GFP is localized to the plasma membrane under B limitation. Shortly after B application, the protein was observed in dot-like structures in the cytoplasm before degradation. Colocalization studies of the fusion protein with an endocytic tracer FM4-64 and an endosomal Rab-GTPase Ara7 fused to monomeric red fluorescent protein suggested that BOR1 is transferred from the plasma membrane via the endosomes to the vacuole for degradation. These results establish that endocytosis and degradation of BOR1 are regulated by B availability, to avoid accumulation of toxic levels of B in shoots under high-B supply, while protecting the shoot from B deficiency under B limitation.
• Cloning of cDNAs encoding isopropylmalate dehydrogenase from Arabidopsis thaliana and accumulation patterns of their transcripts

  A Nozawa, J Takano, K Miwa, Y Nakagawa, T Fujiwara

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 69 4 806 - 810 2005年04月 [査読有り][通常論文]
   

  Isopropylmalate dehydrogenase (IPMDH) is an enzyme in the leucine biosynthetic pathway. We isolated three IPMDH ORF sequences from Arabidopsis thaliana, and genes corresponding to these ORF sequences were designated AtIMD1, AtIMD2, and AtIMD3. Deduced amino acid sequences of the three genes contain a putative transit-peptide for plastidic localization. AtIMD1, AtIMD2, and AtIMD3 were able to complement a leu2 mutant of yeast, suggesting that these genes encode functional IPMDH. RT-PCR analysis revealed different tissue specificity of transcript accumulation for the three genes.
• Arabidopsis boron transporter for xylem loading

  J Takano, K Noguchi, M Yasumori, M Kobayashi, Z Gajdos, K Miwa, H Hayashi, T Yoneyama, T Fujiwara

  NATURE 420 6913 337 - 340 2002年11月 [査読有り][通常論文]
   

  Boron deficiency hampers the productivity of 132 crops in more than 80 countries(1). Boron is essential in higher plants primarily for maintaining the integrity of cell walls(2-4) and is also beneficial and might be essential in animals(5) and in yeast(6). Understanding the molecular mechanism(s) of boron transport is crucial for alleviating boron deficiency. Here we describe the molecular identification of boron transporters in biological systems. The Arabidopsis thaliana mutant bor1-1 is sensitive to boron deficiency(7,8). Uptake studies indicated that xylem loading is the key step for boron accumulation in shoots with a low external boron supply and that the bor1-1 mutant is defective in this process. Positional cloning identified BOR1 as a membrane protein with homology to bicarbonate transporters in animals. Moreover, a fusion protein of BOR1 and green fluorescent protein (GFP) localized to the plasma membrane in transformed cells. The promoter of BOR1 drove GFP expression in root pericycle cells. When expressed in yeast, BOR1 decreased boron concentrations in cells. We show here that BOR1 is an efflux-type boron transporter for xylem loading and is essential for protecting shoots from boron deficiency.

その他活動・業績

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  折山 徹郎, 坂上 景子, 三輪 京子, 森川 正章 日本生物工学会大会講演要旨集 66 222 -222 2014年
• 3P-083 ウキクサに付着する能力の高い細菌の探索とその特性(環境浄化,修復,保全技術,一般講演)

  山川 祐介, 菅原 雅之, 尾島 拓也, 三輪 京子, 森川 正章 日本生物工学会大会講演要旨集 66 215 -215 2014年
• 3P-140 The Effects of General Plant Growth-Promoting Compounds on the Growth of duckweed Lemna minor :

  Desi Utami, Masayuki Sugawara, Kyoko Miwa, Masaaki Morikawa 日本生物工学会大会講演要旨集 66 229 -229 2014年
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  加藤雄大, 漆畑亘, 三輪京子, 森川正章 日本生物工学会大会講演要旨集 66th 2014年
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  森川正章, 菅原雅之, 鈴木和歌子, 三輪京子 化学と生物 52 (12) 799 -804 2014年 [査読無し][通常論文]
  
• 1P-163 有用物質生産を行うウキクサ根圏細菌の探索および諸特性解析(一般講演(バイオマス,資源,エネルギー工学))

  森 庄平, 倉科 光紀, 菅原 雅之, 三輪 京子, 森川 正章 日本生物工学会大会講演要旨集 65 58 -58 2013年
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  鈴木 和歌子, 菅原 雅之, 三輪 京子, 森川 正章 日本生物工学会大会講演要旨集 65 244 -244 2013年
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  加藤雄大, 漆畑亘, 三輪京子, 森川正章, 森川正章 環境バイオテクノロジー学会大会プログラム講演要旨集 2013 2013年
• P10-15 ホウ酸トランスポーターのアクチベーションによるホウ素欠乏耐性シロイヌナズナの作出(10.植物の微量栄養素,2012年度鳥取大会)

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  Li Ke, Kamiya Takehiro, Miwa Kyoko, Tanaka Mayuki, Fujiwara Toru 日本土壌肥料学会講演要旨集 (57) 75 -75 2011年08月08日
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  平井達也, 浦口晋平, 藤原徹, 三輪京子 日本土壌肥料学会講演要旨集 57 2011年
• P10-7 ホウ酸トランスポーターBOR1の蓄積に異常を示すシロイヌナズナ変異体の解析(ポスター紹介,10.植物の微量栄養素,2010年度北海道大会)

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• 10-8 ホウ素過剰に応答したユビキチン化によるホウ酸トランスポーターBOR1の分解制御(10.植物の微量栄養素,2010年度北海道大会)

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• 10-13 花粉管で発現するホウ素トランスポーターBOR6およびBOR7の花粉管伸長における働き(10.植物の微量栄養素,2009年度京都大会)

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• 10-15 ホウ素条件に対する応答に異常を示すシロイヌナズナの新規変異株(10.植物の微量栄養素,2009年度京都大会)

  樋口 みなみ, 三輪 京子, 乾 弥生, 藤原 徹 日本土壌肥料学会講演要旨集 (55) 84 -84 2009年09月15日
• 10-12 シロイヌナズナのホウ酸トランスポーターBOR1のホウ素依存的分解に関与するリジン残基の同定(10.植物の微量栄養素,2009年度京都大会)

  笠井 光治, 高野 順平, 豊田 敦至, 三輪 京子, 藤原 徹 日本土壌肥料学会講演要旨集 (55) 83 -83 2009年09月15日
• 植物養分吸収系の分子改良による植物バイオマス増産 (特集 植物バイオマスデザイン--統合バイオリファイナリーの実現に向けて)

  三輪 京子, 藤原 徹 バイオインダストリー 25 (4) 32 -41 2008年04月
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  加藤諭一, 三輪京子, 浦口晋平, 藤原徹, 藤原徹 日本土壌肥料学会講演要旨集 54 2008年
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  加藤諭一, 三輪京子, 浦口晋平, 藤原徹, 藤原徹 日本植物細胞分子生物学会大会・シンポジウム講演要旨集 26th 2008年
• P9-26 シロイヌナズナchl2変異株の解析(9.植物の無機栄養,2007年度東京大会)

  鈴木 小牧, 井出 曜子, 戸松 創, 三輪 京子, 藤原 徹 日本土壌肥料学会講演要旨集 (53) 85 -85 2007年08月22日
• P9-11 BOR1タンパク質の蓄積に異常を示すシロイヌナズナ変異株の検索(9.植物の無機栄養,2007年度東京大会)

  樋口 みなみ, 三輪 京子, 藤原 徹 日本土壌肥料学会講演要旨集 (53) 80 -80 2007年08月22日
• ホウ酸輸送の分子メカニズム

  高野順平, 三輪京子, 藤原徹 化学と生物 44 (12) 823 -830 2006年12月 [査読有り][招待有り]
  
• P9-7 トランスポーターを用いた植物のホウ素欠乏耐性植物の作出と解析(9. 植物の無機栄養, 2006年度秋田大会講演要旨)

  三輪 京子, 高野 順平, 藤原 徹 日本土壌肥料学会講演要旨集 (52) 76 -76 2006年09月05日
• P9-9 シロイヌナズナホウ素トランスポーターBOR1の局在とホウ素に依存したエンドサイトーシス(9. 植物の無機栄養, 2006年度秋田大会講演要旨)

  高野 順平, 三輪 京子, 藤原 徹 日本土壌肥料学会講演要旨集 (52) 76 -76 2006年09月05日
• シロイヌナズナホウ素トランスポータ-BOR1の制御機構(9. 植物の無機栄養, 2004年度大会講演要旨集)

  高野 順平, 三輪 京子, 小林 正治, 林 浩昭, 米山 忠克, von Wiren Nicolaus, 藤原 徹 日本土壌肥料学会講演要旨集 (50) 91 -91 2004年09月14日
• 9-72 ホウ素トランスポーターBOR1とYNL275wの輸送特性の解析(9.植物の無機栄養)

  小林 正治, 高野 順平, 三輪 京子, 林 浩昭, 米山 忠克, 藤原 徹 日本土壌肥料学会講演要旨集 (49) 88 -88 2003年08月20日

共同研究・競争的資金等の研究課題

• 受精遅延が促進する開花時期多様化と種分化メカニズムの解明

  日本学術振興会：科学研究費助成事業 基盤研究(A)

  研究期間 : 2021年04月 -2025年03月 

  代表者 : 佐竹 暁子, 金岡 雅浩, 三輪 京子
• 植物の栄養感知機構の解明と栄養応答統御

  日本学術振興会：科学研究費助成事業 基盤研究(S)

  研究期間 : 2019年06月 -2024年03月 

  代表者 : 藤原 徹, 三輪 京子, 高野 順平
• 植物細胞壁ペクチン生合成糖転移酵素の同定とペクチンの機能解明

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2019年04月 -2024年03月 

  代表者 : 石水 毅, 三輪 京子
• 周囲環境応答としての植物成長特性の力学的最適化の柔軟性

  日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)

  研究期間 : 2018年06月 -2023年03月 

  代表者 : 藤原 徹, 三輪 京子

   

  植物は栄養条件に応じて成長のパターンを変化させる。植物の根は栄養の獲得と併せて地上部を支え水分を吸収する役割があり、このような栄養に応じた成長パターン変化は根の持つ様々な機能の発現の中で起こるものと考えられ。本研究は植物の栄養や水分環境に対する応答について分子遺伝学的解析に加えて植物組織や細胞の力学的な側面からの理解を深め、これらの解析を統合することを通じて、植物が持つ環境に応じた応答能力の力学的な構造を理解し、新たなモジュール構造等を提案することを目的として進めている。 本年度の研究実績を列挙する。植物の栄養条件に応じた応答研究としては、昨年度までの研究で見出した栄養条件に応じた根の成長パターン変化についての通常の株と異なるパターンを示す変異候補株について、その表現型を確認し、交配などを進め原因となる遺伝子を推定することができた。栄養に対する屈性現象と水分屈性や重力屈性との関連を調べるために、根に栄養濃度の勾配と水ポテンシャル勾配の両方を与えて挙動を調べたところ、水分屈性を示す条件では栄養に対する応答が弱くなる傾向が見出された。また側根のタイプによる応答の違いも見られた。根の力学的な測定系については、樹脂製の細い柱の間に根を伸長させて柱の変位をもとに根のヤング率を測定することができるようになり、栄養条件や変異による影響を調査した。力学的刺激に対する植物の応答を見るために、ポプラを毎日５分間振動させ成長に及ぼす影響を見たところ、刺激を与えた場合の方が太い根が増える傾向がみられた。
• 生物におけるホウ素の新機能の検証－ビタミンB6代謝に対するホウ酸の役割－

  日本学術振興会：科学研究費助成事業 挑戦的研究(萌芽)

  研究期間 : 2018年06月 -2021年03月 

  代表者 : 三輪 京子

   

  必須元素とは、生物のライフサイクルを完結させるために欠くことのできない元素を指す。ホウ素は植物の微量必須元素であり、植物細胞壁のペクチン質多糖を架橋する。近年、動物など植物細胞壁ペクチンをもたない生物においてもホウ素の有用性が報告されてきている。ホウ素の未知の生理機能があることが示唆されるが、植物細胞壁以外のホウ素の生理機能は明らかにされていない。 先行研究において、高濃度のホウ酸が植物（シロイヌナズナ）においてビタミンB6欠乏障害を緩和する現象を見出した。本研究では、本現象の解明を通じて、生物に普遍的なホウ素の役割の解明を目指した。 本年度はホウ酸の効果の普遍性を明らかにするため、酵母（Saccharomyces cerevisiae）における成長に対するホウ酸の効果の検証を進めた。高濃度ホウ酸がビタミンB6欠乏障害を緩和する現象が酵母で観察されるかを明らかにするため、野生型酵母とビタミンB6合成酵素欠損酵母（snz1株）を対象として、異なるホウ酸濃度条件における成長比較を主に進めた。 液体培地での成長試験では、ビタミンB6を含まない最少培地（SD培地）ではsnz1においてホウ酸添加区（2 mM）では無添加区と比較して、濁度の立ち上がりの時間が短くなる傾向が観察された。しかし、複数回の試験を実施したものの、必ずしも再現されず、現象の確定には至らなかった。ビタミンB6を含まない最少完全培地（SC培地）を用いたところ、野生型とsnz1ともに濁度の最終到達点がホウ酸添加区（2 mM）で無添加区と比較して上昇する傾向が観察された。この結果は、酵母の成長、定常期での分裂停止にホウ酸が影響する可能性を示唆するものである。
• 植物体内のホウ素要求量を低下させる分子基盤

  文部科学省：科学研究費補助金（若手研究(A)）

  研究期間 : 2016年04月 -2020年03月 

  代表者 : 三輪 京子
• ペクチンのホウ酸架橋による細胞伸長の制御

  文部科学省：科学研究費補助金(新学術領域研究(研究領域提案型))

  研究期間 : 2015年 -2016年 

  代表者 : 三輪 京子
• 細胞伸長を制御するホウ酸によるペクチン架橋の形成

  文部科学省：科学研究費補助金(新学術領域研究(研究領域提案型))

  研究期間 : 2013年 -2014年 

  代表者 : 三輪 京子
• 植物のホウ素要求量を低下させる分子基盤

  文部科学省：科学研究費補助金(挑戦的萌芽研究)

  研究期間 : 2012年 -2013年 

  代表者 : 三輪 京子

   

  今後の食糧・植物バイオマスの持続的な増産には、肥沃度の低い土壌環境で少ない肥料投入で高生産する植物の開発が必要である。これまで、低ミネラル環境に耐性を付与する遺伝子は複数同定されているが、植物個体の生育量に対する必須元素の要求量を低下させた例はない。本研究では、植物の微量必須元素のホウ素を対象に、植物個体のホウ素要求量を低下させる新規遺伝子の同定と解析を目指した。そこで、対照株がホウ素欠乏のために生育抑制を示す低ホウ素栄養条件において、生育抑制が緩和された変異株を新たに単離し、原因遺伝子の同定とその詳細な解析を行うことを研究の目的とした。平成24年にはシロイヌナズナホウ素ホウ素輸送体機能欠損株bor1-1に塩基置換を誘発するEMS処理を行い、1万9000のM1種子からM2種子を獲得した。変異株探索のため、ロックウールを用いて低ホウ素栄養条件で1万8000のM2種子を水耕栽培した。この一次スクリーニングの結果、対照株と比較してホウ素欠乏による葉の展開抑制の緩和を示した株が約100株得られた。二次スクリーニングとしてM3種子が取得できた72株を対象に、ロックウールを用いた水耕栽培と固形培地でのホウ素栄養に依存した生育を観察した。その結果、7株が対照株と比較して低ホウ素栄養条件での葉の展開抑制の緩和を示し、ホウ素通常条件での生育に違いは認められなかった。また、原因遺伝子がどの染色体に座上しているかを調べる遺伝子マッピングを進めるため、Landsberg erecta とbor1-1の交配を進めた。単離されたホウ素欠乏症状が緩和された新規変異株の変異の原因遺伝子はホウ素の利用・感知に関わるものと考察され、ホウ素要求量を低下させる遺伝子の同定につながると期待された。
• 植物細胞壁ペクチン質多糖のホウ酸による架橋に必要な分子の同定

  文部科学省：科学研究費補助金(若手研究(A))

  研究期間 : 2010年 -2012年 

  代表者 : 三輪 京子

   

  ホウ素は植物の必須元素であり、その欠乏は農業生産に重大な影響を与える。ホウ素の役割は植物細胞壁のペクチン質多糖ラムノガラクツロナンンII（RG-II）間を架橋することであるが、その架橋反応の分子機構は明らかにされていない。そこで、本研究では「RG-IIのホウ酸による架橋に必要な分子の同定」を目指した。RG-IIのホウ酸による架橋に関わる分子を単離するため、分子遺伝学手法による高ホウ素要求性変異株の単離を進めた。低濃度でのホウ酸存在下においてRG-IIとの架橋反応を促進する分子の存在を仮定すると、この分子の機能が低下した場合、通常の細胞内のホウ酸濃度ではRG-IIの架橋がうまく起きないと予想される。一方、過剰のホウ酸を加えた条件では反応基質が過剰となり、RG-IIの架橋が部分的には回復すると考えられる。つまり、機能欠損株は通常のホウ酸濃度条件では、野生型株と比較してホウ素欠乏症状を示し、ホウ素を過剰に添加した条件ではその生育が回復するという、ホウ素依存的な生育を示すことが期待される。そこで、塩基置換を誘発する変異誘起剤で処理を行ったシロイヌナズナCol-0の種子を準備し、ホウ酸通常条件の培地で生育させ、地上部ロゼット葉の展開または根の伸長を指標に生育抑制を示す株を選抜した。高濃度のホウ酸を含む培地へ移植後、生育が回復した株を「高ホウ素要求性変異株」として選抜した。その結果、ロゼット葉の展開を指標として1万4000株のM2から46株、根の伸長を指標として1万8000株のM2から15株の候補株を得られた。遺伝子マッピングの結果、既報の遺伝子の変異に加え、報告のない染色体1番と2番に原因遺伝子の存在が明らかになり、新規変異株の獲得が確認された。加えて、昨年度までに作出した低ホウ酸馴化シロイヌナズナ培養細胞が低いRG-II架橋率を示すことを見出し、網羅的な遺伝子発現解析を進めた。
• 植物の環境中の無機元素濃度に対する応答機構の解明と不良環境耐性植物の作出

  研究期間 : 2009年 

  発芽した場所から移動することができない植物は、土壌中から生育に必要な無機元素を選択的に吸収し、毒性を持つ元素を排出する機構を進化の過程で発達させてきた。無機元素の輸送という視点から生物の環境応答機構の解明を目標としている。分子レベルでは、モデル植物シロイヌナズナを用いて応答の第一段階を担う「無機元素のセンサー」の同定を目指す。また、異なる土壌環境で生育してきた個体集団を対象として無機元素の環境応答を担う遺伝子の多様性を検証し、遺伝子レベルの知見を生態学・進化学へ結びつけた統合的な理解を試みる。加えて、上記の研究をもとに、無機元素輸送に重要な遺伝子の発現改変を通じて、不良な環境（無機栄養欠乏や過剰）に耐性な植物の作出に取り組む。

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