Search

植物構造が司る環境適応性を理解する上で，構成する個々の細胞の力学状態を知ることは本質的な課題である．原子間力顕微鏡法（AFM）は，細胞レベルの力学状態を評価するための有力な手段であり，近年注目されている．本稿では，AFMの基本原理と，その計測から細胞の弾性率を推定する方法について説明する．AFMにより植物細胞の力学状態を経時的に測定するためには，植物の成長を妨げずに，試料を基板に固定しなければならない．この問題解決のためにマイクロデバイスを固定基板として適用した我々の研究例も合わせて紹介する. Clarification of mechanical states of plants are essential for understanding structural inevitability and environmental adaptability of growing plants. Atomic force microscopy (AFM) has attracted attention as a promising measurement tool for evaluating mechanical states with resolution of single-cell level. This review describes principle of the AFM device and method to estimate elasticity of the cell from force curves obtained by the AFM measurement. When we apply the measurement to a growing plant, the sample has to be held on the measurement substrate without inhibiting the growth. We also introduce a solution for this problem, in which a glass micro device is applied to the substrate.

Meiosis is an essential process for the production of gametes, and it consists of one round of chromosome replication and two rounds of mitosis. During meiosis, the chromosome number must be accurately halved, or subsequent generations produced by fertilization of gametes with irregular numbers of chromosomes will become aneuploids, which are lethal in most cases. This accuracy is guaranteed by homologous chromosome synapsis and even segregation. Therefore, synapsis and the following segregation are prerequisites for the production of normal gametes and the next generation. The molecular mechanism of chromosome control during meiosis of plants remains unclear. Unknown components such as ncRNA may be involved. In order to elucidate components of this mechanism, a new method of intact chromosome preparation is necessary. We explored the Digest-and-Fix method, which consists of two steps. First, digestion of pollen mother cells with cell wall-digesting enzymes while keeping their plasma membrane intact, and second, simultaneous fixing and spreading of chromosomes with Farmer’s solution onto slide glasses., Departmental Bulletin Paper, 原著, 2018年度神奈川大学総合理学研究所共同研究助成論文

第35回宇宙環境利用シンポジウム(2021年1月19日-20日. オンライン開催), Space Utilization Research (January 19-20, 2021. Online Meeting), 著者人数: 13名, 資料番号: SA6000156011, レポート番号: G-03

第34回宇宙環境利用シンポジウム (2020年1月21日-22日. 宇宙航空研究開発機構宇宙科学研究所(JAXA)(ISAS)), 相模原市, 神奈川県, Space Utilization Research (January 21-22, 2020. Institute of Space and Astronautical Science, Japan Aerospace Exploration Agency(JAXA)(ISAS)), Sagamihara, Kanagawa Japan, 資料番号: SA6000145016, G-08

第32回宇宙環境利用シンポジウム (2018年1月15日-16日. 宇宙航空研究開発機構宇宙科学研究所(JAXA)(ISAS)), 相模原市, 神奈川県, Space Utilization Research (January 15-16, 2018. Institute of Space and Astronautical Science, Japan Aerospace Exploration Agency(JAXA)(ISAS)), Sagamihara, Kanagawa Japan, 著者人数: 16名, 資料番号: SA6000117006, レポート番号: G-01

In February 2008, 'Cell Wall Experiment' is scheduled to be launched and conducted using EMCS (European Modular Cultivation System) in ISS (International Space Station). The main aim of this experiment is to elucidate the molecular mechanism by which supporting tissues in land plants are constructed and maintained in response to gravitational conditions. In this paper, an outline of our space experiment and biological background are described, together with a perspective of how this project will contribute to both pure and applied life sciences., 資料番号: AA0063706109

植物は細胞壁の物理的な強度によって自らの体を支えている。我々は、シロイヌナズナ花茎の支持組織の細胞壁構築において重要な役割を担う遺伝子の解析を重点的に進めている。マイクロアレイ法を用いた2種類のスクリーニングによって、花茎支持組織の細胞壁構築に特に重要である考えられる数種の遺伝子を同定した。プロモーター::GUS(β-glucuronidase)形質転換体を用いた細胞レベルでの発現解析では、各遺伝子が独自の細胞特異的発現を有することを明らかにした。また同定された遺伝子について逆遺伝学的な機能解析を行なったところ、グリシンリッチタンパク質が原生木部の細胞壁の構造に関与していて、それ故、木部における物質輸送に必須なものであることを示唆する結果を得た。, Cell wall confers mechanical strength to the plant tissues, and thereby offer support to the aerial portion of the plant body. We have focused our research effort on a set of genes that play important roles in specific aspects of cell wall construction in supporting tissues of the inflorescence stem of Arabidopsis. Results from two microarray screenings identified several key candidate genes responsible for cell wall formation in supporting tissues of the inflorescence stem. We examined expression profiles of promoter::GUS (beta-glucuronidase) fusion constructs for each of the genes. The results indicated that each of the genes exhibited distinct expression patterns in terms of cell specificity. Reverse genetics analysis for function of the genes indicated that glycine-rich protein modifies mechanical strength of the cell wall in protoxylem in basal of the stem, thereby providing a conduit for the translocation of water and nutrients., 資料番号: AA0063338001, レポート番号: JAXA-SP-06-032

イネ科植物の細胞壁結合性フェルラ酸とジフェルラ酸の形成には、フェニルアラニンアンモニアリアーゼ(PAL: Phenylalanine Ammonia Lyase)とペルオキシダーゼ(POD: Peroxidase)が重要な役割を担っている。PALとPODには複数の遺伝子が存在し、ファミリーを形成していることから、今年度の研究では、解析対象となる遺伝子の絞り込みと、それら遺伝子の発現解析条件について検討した。また、細胞壁内に存在するシュウ酸酸化酵素(germin)がPOD活性に必須の過酸化水素の生成・供給を介して、ジフェルラ酸の形成に影響する可能性について、活性細胞壁標品を用いて検討した。, Phenylalanine Ammonia-Lyase (PAL) and cell wall Peroxidase (POD) have been shown to be involved in the formation of cell wall-bound ferulic and diferulic acids in graminaceous plants. In rice plants, PAL and POD are encoded by the multi-gene families. In the present study, we chose suitable genes for the analysis of expression level of PAL and POD genes in rice shoots and also determined their analytical conditions. In addition, the contribution of oxalate oxidase (germin) to the formation of diferulic acid in cell walls was examined using isolated active cell wall preparations, because the oxalate oxidase has been shown to produce hydrogen peroxide, a key substance for POD activity., 資料番号: AA0063339001, レポート番号: JAXA-SP-06-033

本研究の目的は、シロイヌナズナ突然変異系統を用いて、抗重力反応における微小管-原形質膜-細胞壁連絡の役割を検証し、抗重力のしくみを明らかにすることにある。本年度は、EMCS(European Modular Cultivation System)を利用した宇宙実験に備えて、播種方法、植物育成環境、植物観察方法、試料固定および回収条件などの軌道上操作の手順を検討し、確定した。また、多くのチューブリン並びにHMGR(3-Hydroxy-3-Methyl Glutaryl-CoA Reductase)変異体の中から、最も宇宙実験に適した系統としてtua6並びにhmg1を選定し、遠心過重力環境を用いた予備実験を実施した。過重力環境下では植物茎器官の伸長成長が抑制され肥大成長が促進されるが、これらの変異体は1g環境下で既に太く短い形状をしており、過重力環境でもそれ以上の伸長抑制や肥大促進が起こらなかった。すなわち、このような変異体は重力に抵抗する能力が低く、1gの重力にも耐えられないことがわかった。宇宙の微小重力環境では、これらの変異体の形質が回復することが期待される。, The present study aims to confirm the hypothesis that the structural or physiological continuum of microtubule-plasma membrane-cell wall is responsible for gravity resistance in plants, by the space experiment using Arabidopsis mutants. For this purpose, we have decided the procedure for space experiment in EMCS (European Modular Cultivation System), such as the optimal method of seed sowing, condition of plant cultivation, and methods of sample fixation and collection. We have also selected one tubulin mutant (tua 6) and one HMGR (3-Hydroxy-3-Methyl Glutaryl-CoA Reductase) mutant (hmg 1) in the Columbia background for space experiment. Hypergravity suppressed elongation growth, whereas it stimulated lateral expansion of stem organs. These mutants had thicker and shorter hypocotyls than wild-type even under 1 g conditions, and hypergravity did not affect their length or diameter, suggesting that the mutants are hypersensitive to the gravitational force. We expect that such defects of these mutants are rescued under microgravity conditions in space., 資料番号: AA0063338002, レポート番号: JAXA-SP-06-032

Beta-1,3:1,4-Glucan is a major cell wall component and responsible for the regulation of elongation growth in rice. We examined the effects of water immersion as a microgravity simulator on beta-1,3:1,4-glucan synthase activity in rice seedlings. Water immersion significantly reduced both amount and molecular weight of beta-1,3:1,4-glucan in the cell walls, stimulating elongation growth of the coleoptiles. The beta-1,3:1,4-glucan synthase activity in the microsome fraction of coleoptiles underwater was decreased to less than 50 percent of that of the control. These results indicate that the growth stimulation underwater is mediated by decrease in activity of beta-1,3:1,4-glucan synthase as well as increase in activity of beta-1,3:1,4-glucan degrading enzymes in the cell walls., 資料番号: AA0063349106

植物は細胞壁の物理的な強度によって自らの体を支えている。我々は、マイクロアレイ解析によって、シロイヌナズナの花菜を支えている組織において細胞壁の構築に重要な役割を果たす遺伝子群を同定した。これらの中には、花茎木部の2次細胞壁の肥厚やリグニン化に関与すると報告されている遺伝子が含まれていた。花茎を横倒しにすることによって、これらの遺伝子の発現が顕著に抑制された。このことは、花茎の重さ自体が支持組織の細胞壁の強度を制御していることを示唆するものであった。同定された遺伝子について逆遺伝学的な機能解析を行なったところ、グリシンリッチタンパク質が原生木部の細胞壁の構造に関与していて、それ故、木部における物質輸送に必須なものであることを示唆する結果を得た。, Cell wall confers mechanical strength to the plant tissues, and thereby offer support to the aerial portion of the plant body. Using gene-specific oligonucleotide microarray system, we identified a set of genes that play important roles in specific aspects of cell wall construction in supporting tissues of the inflorescence stem of Arabidopsis. Some of the genes were reported to be involved in secondary wall deposition or lignification of xylem cells of the inflorescence stem. The expression of these genes sharply decreased when the inflorescence stem are placed horizontally. The results suggest that the weight carried by the upper part of the stem may be a signal for the induction of cell wall rigidification in supporting tissues of the stem. Reverse genetics analysis for function of the genes indicated that glycine-rich protein modifies mechanical strength of the cell wall in protoxylem in basal of the stem, thereby providing a conduit for the translocation of water and nutrients., 資料番号: AA0049799003, レポート番号: JAXA-SP-05-037

今年度の研究では、イネshootの成長過程でのフェニルアラニンアンモニアリアーゼ(PAL)と細胞壁結合型ペルオキシダーゼ(POD)の活性の変化について検討した。昨年度の研究で、イネshoot細胞壁結合性のフェルラ酸(FA)とジフェルラ酸(DFA)量は、播種後4日目から6日目にかけて大きく増加することを明らかにした(2004年度成果報告書、若林)。この4日目から6日目にかけてPAL活性は急速に増加し、6日目には4日目の5倍の活性を示した。PAL活性と細胞壁のFA量との間で非常に高い相関が見られた。一方、細胞壁POD活性も、PALと同様にこの期間に大きく増加した。POD活性は、細胞壁のDFA量と同じパターンの変化を示した。以上の結果から、PALと細胞壁PODの活性はイネshoot細胞壁のフェノール化合物量の調節に直接的に関与していると考えられる。, Changes in the activities of phenylalanine ammonia-lyase (PAL) and cell wall peroxidase (POD) were studied in dark-grown rice shoots. The amounts of cell wall-bound ferulic (FA) and diferulic acids (DFA) in rice shoots greatly increased from four to six days after planting (Annual Report; Wakabayashi 2004). The activity of PAL per shoot increased by 5-fold from day four to six. There was a close correlation between the FA level and PAL activity. Similarly, the activity of cell wall POD per shoot progressively increased during this period, and the activity changed in parallel with the increase in DFA content. These results suggest that both PAL and cell wall POD are involved in determining the level of wall-bound phenolic acids in rice shoots., 資料番号: AA0049799001, レポート番号: JAXA-SP-05-037

本研究の目的は、シロイヌナズナ突然変異系統を用いて、抗重力反応における微小管-原形質膜-細胞壁連絡の役割を検証し、抗重力のしくみを明らかにすることにある。本年度は、宇宙実験に適したColumbiaエコタイプの突然変異体を作出した。表層微小管の形成に関わる変異体に関しては、チュブリン遺伝子の1アミノ酸置換に起因し、表層微小管の配向が右巻きになって細胞が左巻きにねじれるlefty変異体15系統、および微小管が左巻きに配向し細胞が右巻きにねじれるspiral(spr)変異体25系統を、Columbiaエコタイプより新たに選抜した。一方、膜ステロールの合成に関わるHMGRのノックアウト変異体hmg(WSエコタイプ)に関しては、戻し交雑によりColumbiaバックグラウンドに置き換えた。また、細胞壁物性測定用の引っ張り試験機を更新するとともに、プログラムを改良して、宇宙実験に適した測定条件を設定した。, The present study aims to confirm the hypothesis that the structural or physiological continuum of microtubule-plasma membrane-cell wall is responsible for gravity resistance in plants, by the space experiment using Arabidopsis mutants. For this purpose, we isolated novel amino acid substitution mutants defective in formation of cortical microtubules (lefty & spr) in the Columbia background. The mutant defective in synthesis of membrane sterols (hmg) in the Columbia background was also produced by crossing the WS background mutant with wild type Columbia. In addition, we designed new software and interface for measuring the mechanical properties of the cell walls, suitable for space experiments., 資料番号: AA0049799002, レポート番号: JAXA-SP-05-037

In response to hypergravity treatment at 300 g for 12 h, the gene expression of CesA1, 6, 7 and 9 decreased in 3-day old seedlings, but did not change in the floral stem in 20 day-old plants. On the other hand, hypergravity treatment at 300 g for 1 h decreased the gene expression of XTH15, and increased that of XTH22. The effect of hypergravity on the gene expression of two XTHs was partially alleviated by 0.1 mM La(3+), suggesting the possible involvement of mechanosensor in hypergravity-induced changes in XTH gene expression. In addition, the morphology of the secondary cell wall was influenced by hypergravity. Xylem vessels showed intense staining with phloroglucinol-HC1 and autofluorescence under UV light at the basal region of floral stem 3 day after hypergravity treatment. The cross section area of the metaxylem element increased after hypergravity treatment, suggesting that lignification and the development of the secondary wall of xylem vessels are positively regulated under hypergravity., 資料番号: AA0046917040

Modifications of cell wall structure of wheat coleoptiles in response to continuous hypergravity (300 g) treatment were investigated. Growth of coleoptiles exposed to hypergravity for 2 to 4 days from germination stage was substantially reduced. The net amounts of cell wall polysaccharides of hypergravity-treated coleoptiles increased as much as those of 1 g control coleoptiles during the incubation period. As a result, the levels of cell wall polysaccharides per unit length of coleoptile, which mean the thickness of cell walls, largely increased under hypergravity conditions. In the hemicellulose fraction, a polysaccharide fraction extracted from cell walls with strong alkali, glucose (Glc), arabinose (Ara) and xylose (Xyl) were the major sugar components, and the ratios of Ara and Xyl to Glc were higher in hypergravity-treated coleoptiles than in control coleoptiles. Furthermore, the fractionation of hemicellulosic polymers into the neutral and acidic polymers by the anion-exchange column showed that the levels of acidic polymers (mainly composed of arabinoxylans) in cell walls of hypergravity-treated coleoptiles were higher than those of control coleoptiles. In addition to wall polysaccharides, the amounts of cell wall-bound phenolics, such as ferulic acid and diferulic acid, increased at the same extent in both hypergravity-treated and control coleoptiles. These results suggest that hypergravity stimuli from the germination stage do not restrict the net synthesis of cell wall constituents in wheat coleoptiles, although they bias the type of synthesized hemicellulosic polysaccharides. The stimulation of the synthesis of arabinoxylans may be involved in the structural changes, such as thickening of cell walls, under hypergravity conditions. These structural changes in cell walls may contribute to plant resistance to gravity stimuli., 資料番号: AA0046917039

The changes in growth and cell wall properties of roots of rice (Oryza sativa L. cv. Koshihikari) grown for 68.5, 91.5, and 136 h during the Space Shuttle STS-95 mission were analyzed, for clarifying the effects of microgravity on root growth. In space, elongation growth of rice coleoptiles was stimulated and the cell wall extensibility increased. Elongation growth of rice roots was also stimulated in space and the effect was greater than in coleoptiles. However, there were no differences in the cell wall extensibility of rice roots between in space and on the ground. Then analyzed were the mechanical properties of the cell wall of rice roots more precisely with the creep-extension method by adopting the Kelvin-Voigt-Burger's six element model. Some of elasticity moduli and viscosity coefficients were higher in roots grown in space than on the ground, suggesting that the cell wall of space-grown roots has a lower capacity to expand than the controls. The amounts of the cell wall polysaccharides per unit length were smaller in rice coleoptiles and roots grown in space. The effect was greater in roots. On the other hand, in rice coleoptiles grown in space, the ratio of high molecular mass region of hemicellulose decreased as compared with the controls, whereas the ratio increased in space-grown roots. The prominent thinning of the cell wall could overwhelm the disadvantageous changes in the cell wall mechanical properties, leading to the stimulation of elongation growth in rice roots in space. Thus, although elongation growth of both rice coleoptiles and roots was stimulated in space, the mechanism of stimulation was different between two organs., 資料番号: AA0045438025

Schizophyllan secured from fungus, Shizophyllum commune, is a water-soluble (1,3)-β-D-glucan with side chains consisting (1,6)-β-D-glucosyl residues attached to O-6 of the backbone residues, at has a triple helical conformation and shows antitumor activity foward Sarcoma 180. Schizophyllan is effective for promoting protoplast division of Saccharomyces cerevisiae. The frequency of regeneration of yeast protoplast with schizophyllan is 1,000 times times the control. The effect of stability mechanism was examined and found to improve plant protoplast stability. Various water-soluble-polysaccharides were examined stability to adhere to cellulose and effects on protoplast stability.

Growth of terrestrial plant is influenced by the magnitude of gravity. Hypergravity stimulus has been shown to suppress plant shoot growth by increasing the molecular mass of cell wall xyloglucans (Soga et al. 1999). And, it was found that mechanoreceptors are involved in perception of gravity stimulus in hypergravity-induced increase in the molecular mass of xyloglucans (Soga et al. 2004). These results suggest that hypergravity perceived by mechanoreceptors modifies the metabolism of cell wall xyloglucans by affecting the expression of xyloglucan endotransglucosylase/hydrolase genes. In the present study, the examinations, whether hypergravity affects the expression of XTH (Xyloglucan Translocase/Hydrolase) genes in Arabidopsis floral stem, and whether XTH genes are under the control of the mechanoreceptor, are conducted. It is demonstrated that hypergravity influenced the expression of some of XTH genes in Arabidopsis floral stems. The effect of hypergravity stimulus on the expression of these XTH genes was nullified by lanthanum chloride, which is a blocker of mechanoreceptors. These results suggest that the expression of these XTH genes in Arabidopsis is under the control of the mechanoreceptor. Further, spatial distribution patterns of these XTH mRNA in floral stem were analyzed by in situ hybridization., 資料番号: AA0048095066

In order to identify genes involved in the cell wall dynamics responsible for plant morphogenesis as regulated by gravity conditions, an oligo DNA microarray has been prepared that consists of 762 Arabidopsis cell-wall related genes. By use of this microarray, groups of genes committed to construction of supporting tissues in Arabidopsis inflorescence stems growing under 1 g gravity conditions have been identified., 資料番号: AA0046917041

第26回宇宙利用シンポジウム(2010年1月25日-26日, 宇宙航空研究開発機構宇宙科学研究本部相模原キャンパス), The Twenty-sixth Space Utilization Symposium (January 25-26, 2010: ISAS/JAXA Sagamihara, Japan), Resistance to the gravitational force is a principal graviresponse in plants,comparable to gravitropism. However, only limited information has been obtained for this graviresponse. To clarify the nature and mechanisms of gravity resistance, we have organized a research team, consisting 16 members. In the current year, we have continued discussion and exchange of information on strategy for understanding gravity resistance. We have also carried out ground-based experiments to clarify the role of cortical microtubules in gravity resistance using Arabidopsis mutants, and the effects of hypergravity on the positioning of the nucleus, osmoregulation, and stress reactions. The knowledge obtained by these activities was utilized in part to apply for the Announce of Opportunity for space experiments in Kibo during the 2nd phase., 共催: 日本学術会議, 著者人数: 16人, joint hosting: The Science Council of Japan, 資料番号: AA0064730064

本研究開発報告は、第4回国際公募採択テーマ「重力によるイネ芽生え細胞壁のフェルラ酸形成の制御機構(Ferulate)」および第5回国際公募採択テーマ「微小重力下における根の水分屈性とオーキシン制御遺伝子の発現(Hydro Tropi)」に関する2006年度の研究開発成果をまとめたものである。, 資料番号: AA0063339000, レポート番号: JAXA-SP-06-033

本研究開発報告は、第5回国際公募採択テーマ「微小重力環境下におけるシロイヌナズナの支持組織形成に関わる遺伝子群の逆遺伝学的解析(Cell Wall)」および「植物の抗重力反応における微小管-原形質膜-細胞壁連絡の役割(Resist Wall)」に関する2006年度の研究開発成果をまとめたものである。, 資料番号: AA0063338000, レポート番号: JAXA-SP-06-032

本研究開発報告は、第4回国際公募採択テーマ「重力によるイネ芽生え細胞壁のフェルラ酸形成の制御機構」、第5回国際公募採択テーマ「植物の抗重力反応における微小管-原形質膜細胞壁連絡の役割」、「微小重力環境下におけるシロイヌナズナの支持組織形成に関わる遺伝子群の逆遺伝学的解析」、「微小重力下における根の水分屈性とオーキシン制御遺伝子の発現」および宇宙科学研究本部ISS科学プロジェクト室においてすすめている「プロテオミクス解析から探る植物の重力応答機構」に関する平成17年度の研究開発成果をまとめたものである。とくに、第5回国際公募採択テーマは、JEM打ち上げ以前の2007年後半にESAの開発した宇宙実験装置(European Modular Cultivation System: EMCS)を利用したフライト実現を目指して、現在準備を進めている。各々のテーマは、それぞれ、(1)微小管や細胞膜ステロール合成に関わる突然変異体を用いた植物の重力抵抗メカニズムの解明、(2)植物の姿勢支持制御に関与する細胞壁関連遺伝子群の網羅的発現解析、(3)植物の水分屈性反応におけるオーキシン制御遺伝子の発現解析および分子メカニズムの解明を最終目標としている。また、第4回国際公募採択テーマ、JEMを利用して2010年の宇宙実験実現を目指しており、重力による高等植物細胞壁の構築およびその強度制御の仕組みを解明することを目的としている。さらに、ISS科学プロジェクト室で進めているプロテオミクス研究は、植物の重力応答をタンパク質レベルから網羅的に解明しようとする試みである。, 資料番号: AA0049799000, レポート番号: JAXA-SP-05-037

There are some cases of symbiotic relation such as allelopathy, endphyte and symbiosis of Fabaseae plant. The growth and distribution of endophyte, Neothyphodium, in its host plant, tall fescue, Festuca arundinacea, were investigated during the seed germination stage under pseudo-microgravity. The hypheae of endophyte in the seed part were gradually unclear during the seed germination. Hyphae might be autolysis during seed germination. The wall component of endphyte was different during the earth control and pseudo-microgravity. A change in the carbohydrate metabolism of the fungus and the plant due to the pseudo-microgravity was suggested. We found the different functions of the endogenous enzyme grown under normal gravity and pseudo-microgravity. The mechanisms related to gravity during early germination stage of endophyte infected plant might be very complicated., 資料番号: AA0063349111

研究科: 千葉大学大学院薬学研究院, 修了年:2009年, 学位:千大院医薬博乙第薬24号

研究科: 千葉大学大学院薬学研究院, 修了年:2009年, 学位:千大院医薬博乙第薬24号