私たちは、優れた技術サービスとジョンラルフ、貴重なアドバイス、ディスカッション、およびポプラの木のサンプルのウィスコンシン大学のマシューロバートウェザーに感謝しています。この作品は、科学局、米国エネルギー省（DOE）五大湖バイオエネルギー研究センター（科学DE - FC02 - 07ER64494のDOE BER局）によって、化学科学、地球科学およびバイオサイエンス部門、基礎エネルギー科学局によって資金を供給された、米国エネルギー省（賞なし。DE - FG02 - 91ER20021）。

1。細胞壁の分離<ol><li> iWallを使用して2ミリリットルザルスタットスクリューキャップチューブ、研削及び調剤ロボット（30秒）で5.5 mm、ステンレス鋼ボールで空気または凍結乾燥植物の材料の約60 - 70mgを挽く。ボールミル（retschmill）を使用して代替の非ロボットの低スループットの手順は、第II部2に示す。 </li><li>徹底的に分注された地盤材料を70％エタノール水溶液を1.5 ml加え、ボルテックス。 </li><li>ペレットアルコール不溶性残渣への10分間10,000 rpmで遠心する。 </li><li>上清を吸引したり、デカントする。 </li><li>ペレットを再懸濁するために残渣にクロロホルム/メタノール（1:1 v / v）の溶液1.5mlを加え、よくチューブを振る。 </li><li> 10分と吸引または上清をデカントし、10,000 rpmで遠心。 </li><li>アセトン500μlのでペレットを再懸濁します。 </li><li>乾燥するまで35℃の空気の流れ° Cで溶媒を蒸発させる。 必要に応じて乾燥試料は、さらに処理するまで室温で保存することができます。 </li><li>サンプルから澱粉の除去を開始するには0.1 Mの酢酸ナトリウム緩衝液pH 5.0を1.5 mlでペレットを再懸濁します。 </li><li> 20分間ザルスタット管と熱にフタを。 80℃の加熱ブロックインチ</li><li>氷の上に、懸濁液を冷却する。 </li><li>ペレットには、次のエージェントを追加：0.01％アジ化ナトリウム（NaN3）の35μlの、35μlのアミラーゼ（50μg/ mlのH 2 O、 バチルス属から、シグマ）、17μlのプルラナーゼ（ 枯草acidopullulyticusから17.8単位;シグマ） 。チューブと徹底的にボルテックスにフタを。 </li><li>懸濁液を37℃で一晩インキュベート°回転シェーカーでC。管を配向させる水平側近は改良混合。 </li><li> 100℃の熱サスペンション° C消化を終了するために加熱ブロックに10分間。 </li><li>遠心（10,000 rpm、10分）とを含む上清を可溶化された澱粉を捨てる。 </li><li> 、1.5 mlの水を加えボルテックス、遠心分離、洗浄水からデカンテーションによって、残りのペレット3回洗浄する。 </li><li>アセトン500μlのでペレットを再懸濁します。 </li><li>乾燥するまで35℃の空気の流れ° Cで溶媒を蒸発させる。それはよりよい乾燥用スパチュラでチューブの材料を分割することも必要になる場合があります。 乾燥した材料は、単離された細胞壁（リグノセルロース）を提示。必要に応じて乾燥試料は、さらに処理するまで室温で保存することができます。 </li></ol> 2。リグニン含量このメソッドは、福島とハットフィールド3で報告された方法に基づいています。 <ol><li> 1量る - 調製した細胞壁の材料の1.5ミリグラム（1を参照）ブランクのための空のチューブを残し、2 mlのメスフラスコにする。 </li><li>チューブの底に細胞壁の材料を収集し、空気の流れの下で非常に優しいアセトンを蒸発させるためにアセトン250μlをチューブの壁を洗う。 </li><li>ゆっくりと水はねを防止するためにチューブの壁に沿ってたてのアセチルブロマイド溶液100μl（25％氷酢酸でV / Vアセチルブロマイド）を追加します。 </li><li> 50キャップのメスフラスコと熱° 2時間のためのC </li><li> 15分毎にボルテックスで追加の時間のための熱。 </li><li>室温に氷上で冷却してください。 </li><li> 2M水酸化ナトリウムと作りたての0.5 M塩酸ヒドロキシルアミン70μlの400μlを添加する。渦メスフラスコ。 </li><li>氷酢酸を持っている2.0 mlのマーク、キャップに正確にメスフラスコをいっぱいにして混在させることを数回転倒。 </li><li>ピペットUV特定の96ウェルプレートへの溶液200μlと280nmでELISAリーダーで読み取る。 </li><li> （;草= 17.75;ポプラ= 18.21 シロイヌナズナ = 15.69）、適切な係数を用いて臭化アセチル水溶性リグニン（％ABSL）の割合を決定し、次の式で： ％ABSL Calcの： <img src="/files/ftp_upload/1745/1745eq.jpg" alt="図1" /> UG / mgの細胞壁のユニットで10件の検索結果で％のABSLの乗算 それは、少なくとも3プレートは微粒子の吸光度値のわずかな変動を引き起こす可能性があるので、吸光度（ABS）を平均化する読み取り行うことができます。注：0.539センチメートルは、光路長を表しますが、板によってはこれを決定する必要があります。 </li></ol> 3。リグニン組成このメソッドは、ロビンソンとマンスフィールド5で発表された最近の方法を採用したものになっています。 <ol><li> thioacidolysis用スクリューキャップ付きガラス管に（1を参照。）細胞壁の材料の約2 mgを転送する。 </li><li>慎重に2.5パーセント三フッ化ホウ素ジエチルエーテレート（BF 3）、10％エタンチオール（EtSH）溶液を調製。あなたは、窒素とジオキサンボトルで失われたボリュームを置換する窒素ガスを充填した気球を使用する必要があります。ジオキサンが非常に有害である、フードの中から試料や装置を取ることはありません。サンプルあたりの溶液の調製に必要なボリューム：ジオキサン175μL、20μLEtSH、5μlのBF 3。 </li><li> EtSH、BF 3、各サンプルのジオキサン溶液200μlを加え。 </li><li>すぐに窒素ガスとキャップ付きパージバイアルヘッドスペース。 </li><li> 100℃熱°だけ時間混合穏やかで4時間。 </li><li>氷上で5分間冷却することにより、エンド反応。 </li><li> 0.4M重炭酸ナトリウム150μlの、渦を追加</li><li>クリーンアップのために1mlの水と酢酸エチル0.5mlの、渦と相が分離することができます（上に酢酸エチル、底面の水）を追加します。 </li><li> 2ミリリットルザルスタットチューブに酢酸エチル層を150μlを移す。ない水が転送されていないことを確認します。 </li><li>空気とコンセントレータで溶剤を蒸発させる。 </li><li> （2回の合計のために繰り返す余分な水分を除去）200μlのアセトンを追加し、蒸発させる。 </li><li> TMS誘導体化のために酢酸エチル500μlの、ピリジン20μlを、そしてN、各チューブにO -ビス（トリメチルシリル）アセトアミドの100μlを加える。 </li><li> 25℃で2時間インキュベート℃に</li><li> GC / MSバイアル​​に反応液100μlを移し、アセトン100μlを加える。 </li><li>四重極質量分光器または炎イオン化検出器付きGCによるサンプルを分析する。 AgilentのHP - 5MSカラムは、（X 0.25ミリメートル× 0.25μmの膜厚30mm）をインストールされています。以下の温度勾配を30分間溶剤遅延および1.1 ml / minの流速で使用されています：130イニシャルホールド℃で3分間、3℃〜250℃/分のランプ℃、1分間保持;を許可130℃の初期温度に平衡化</li><li>ピークはそれぞれ、テトラコサン内部標準（オプション）を使用して相対保持時間によって、または299メートル/ zの、269のm / z、およびSの239メートル/ Z、G、およびHの単量体の特性マススペクトルのイオンによって識別されます（図を参照してください。 2）。リグニン成分の組成は、100％に、総ピーク面積を設定することによって定量化される</li></ol> 4。代表的な結果壁の解析の例を図2に示す。この場合にはポプラの幹（木）は、プロトコルのセクションで概説、様々な手順で分析した。 thioacidolysisとTMS -誘導体化後のリグニン成分の分離の例のクロマトグラムが表示されます。明らかに、syringyl -（S）の相対量、guaiacyl -（G）、およびp - hydroxyphenol -（H）の単位を決定することができます。臭化アセチル水溶性リグニンの含有量は自明であり、一つは壁乾燥重量の20〜50％の間の値を期待することができます。一つは、臭化アセチルを壁にリグニンの存在のすべてを可溶化しない、と可溶化の程度は材料によって異なることに注意する必要があります。しかし、この方法は、比較的実施しやすいと急速であり、リグノセルロース材料のリグニン含量の優れた近似を与える。 <img src="/files/ftp_upload/1745/1745fig1.jpg" alt="図1" /> 図1：。リグノセルロースの分析の概要細胞壁（リグノセルロース）は、原油乾燥植物材料から分離されています。壁材は、アリコートに重み付けをし、様々なアッセイのために分割されます。壁材は、臭化アセチルとUV -分光法により定量化可溶化したリグニンと扱われます。リグニンの組成の決定については、壁材はthioacidolysisに供される。可溶化されたフェノール類は、TMS誘導体化を受け、その後分離し、GC - MS分析により定量することができる。マトリックス多糖類組成と結晶セルロースのコンテンツのプロトコルは、パートII 2で説明されています。 <img src="/files/ftp_upload/1745/1745fig2.jpg" alt="図2" /> 図2：ポプラ（ ポプラtremoloides）からポプラ樹木木材チップの包括的なリグノセルロースの分析は、記載されているプロトコルに供した。 Ligin組成物; H のp -ヒドロキシフェニル、G guaiacyl、S syringylユニット。

<ol>
	<li>Carroll, A., Somerville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulosic+Biofuels.">Cellulosic Biofuels.</a> Annual Review of Plant Biology. 60, 165-165 (2009).</li><li>Foster, C. E., Martin, T., Pauly, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+compositional+analysis+of+Plant+Cell+Walls+(Lignocellulosic+biomass),+Part+II:+Carbohydrates.">Comprehensive compositional analysis of Plant Cell Walls (Lignocellulosic biomass), Part II: Carbohydrates.</a> J Vis Exp. , (2010).</li><li>Fukushima, R. S., Hatfield, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+isolation+of+lignin+for+utilization+as+a+standard+to+determine+lignin+concentration+using+the+acetyl+bromide+spectrophotometric+method.">Extraction and isolation of lignin for utilization as a standard to determine lignin concentration using the acetyl bromide spectrophotometric method.</a> J. Agric. Food Chem. 49 (7), 3133-3133 (2001).</li><li>Pauly, M., Keegstra, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-wall+carbohydrates+and+their+modification+as+a+resource+for+biofuels.">Cell-wall carbohydrates and their modification as a resource for biofuels.</a> Plant J. 54 (4), 559-559 (2008).</li><li>Robinson, A. R., Mansfield, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+analysis+of+poplar+lignin+monomer+composition+by+a+streamlined+thioacidolysis+procedure+and+near-infrared+reflectance-based+prediction+modeling.">Rapid analysis of poplar lignin monomer composition by a streamlined thioacidolysis procedure and near-infrared reflectance-based prediction modeling.</a> Plant J. 58 (4), 706-706 (2009).</li><li>Somerville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+systems+approach+to+understanding+plant-cell+walls.">Toward a systems approach to understanding plant-cell walls.</a> Science. 306 (5705), 2206-2206 (2004).</li><li>Teeri, T. T., Brumer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery,+characterization+and+applications+of+enzymes+from+the+wood-forming+tissues+of+poplar:+Glycosyl+transferases+and+xyloglucan+endo-transglycosylases.">Discovery, characterization and applications of enzymes from the wood-forming tissues of poplar: Glycosyl transferases and xyloglucan endo-transglycosylases.</a> Biocatalysis and Biotransformation. 21, 173-173 (2003).</li></ol>

記載された方法は、リグニン含量とリグノセルロース植物バイオマスの組成の迅速な定量的評価を有効にしてください。 iWallのロボットを使用して約350試料を粉砕し、日ごとに分配することができます。お一人様、様々な分析手法のスループットが異なります。プロトコルを使用すると、30サンプルを一日あたりのリグニン組成物のためリグニン含量のために処理し、15することができる、ここで説明する。データに最適な原料作物の定量的な性質のために、様々なまたは遺伝子型は、バイオ燃料生産のためのその適合性の観点で評価することができる。

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Hydroxylamine Hydrochloride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>255580</td>
<td>&nbsp;</td>
<tr>
<td>Acetyl Bromide</td>
<td>&nbsp;</td>
<td>Aldrich</td>
<td>135968</td>
<td>&nbsp;</td>
<tr>
<td>Ethanethiol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>E3708</td>
<td>&nbsp;</td>
<tr>
<td>Borontrifluoride diethyl etherate</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>15719</td>
<td>&nbsp;</td>
<tr>
<td>N,O,-Bis(trimethylsilyl) acetimide</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>15241</td>
<td>&nbsp;</td>
<tr>
<td>Dioxane</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>296309</td>
<td>&nbsp;</td>
<tr>
<td>Spectromax Plus 384</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>Plus384</td>
<td>&nbsp;</td>
<tr>
<td>GC-MS</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>6890 GC/5975B MSD</td>
<td>(lignin composition)</td>
</tr>
<tr>
<td>5.5mm Stainless Steel Balls</td>
<td>&nbsp;</td>
<td>Salem Ball Company</td>
<td>(N/A)</td>
<td>&nbsp;</td>
<tr>
<td>96 well plate heat spreader</td>
<td>&nbsp;</td>
<td>Biocision</td>
<td>Coolsink 96F</td>
<td>&nbsp;</td>
<tr>
<td>Heating block</td>
<td>&nbsp;</td>
<td>Techne</td>
<td>Dri-block DB-3D</td>
<td>&nbsp;</td>
<tr>
<td>Sample concentrator</td>
<td>&nbsp;</td>
<td>Techne</td>
<td>FSC400D</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

comprehensive compositional analysis of plant cell walls (lignocellulosic biomass) part i: lignin

産業と社会のための再生可能エネルギー、カーボンニュートラル、そして持続可能な原材料の必要性は、21世紀の最重要課題の一つとなっている。これは輸送用液体燃料の生産のための工業用原料としての植物製品の使用に関心を再燃させたている<sup&gt; 1</sup&gt;このようなbiocomposite材料として他の製品<sup&gt; 7</sup&gt;。植物バイオマスは地球上で最大の未開発の埋蔵量の一つである<sup&gt; 4</sup&gt;。それは主にセルロース、様々なヘミセルロース（マトリックス多糖類、およびポリフェノールリグニンを含むエネルギーリッチなポリマーで構成されている細胞壁で構成されています<sup&gt; 6</sup&gt;そのため、時にはリグノセルロースと呼ばれる。しかし、植物の細胞壁は、壁は細胞やプラント全体への引張強度を提供するように病原体を撃退、そして水は植物全体に輸送できるように、劣化への反抗的になるように進化して、最大の木の場合には上記より100メートルにグランドレベル。壁の様々な機能が原因で、単一工場内の異なる植物種や細胞タイプの城壁内に巨大な構造の多様性がある<sup&gt; 4</sup&gt;。したがって、どのような作物の種の種類により、作物の品種、又は植物組織がバイオリファイナリーのために使用され、液体バイオ燃料の化学的/酵素的プロセスと様々な糖のその後の発酵による解重合のための処理ステップを調整し最適化する必要があります。この事実を支える植物のバイオマス原料の徹底した特性評価のための必要性。ここでは、リグノセルロースの組成の決定を可能にし、ハイスループット分析への培地に適している包括的な分析方法を説明します。この最初の部分では、ポリフェノールリグニン（図1）の分析に焦点を当てる。このメソッドはdestarched細胞壁の材料を準備してから始まります。結果として得られるリグノセルロースは、acetylbromideの可溶化により、そのリグニン含量を決定するために分割されます<sup&gt; 3</supそのsyringyl、guaiacyl -およびp -ヒドロキシフェニル単位の面で&gt;、及びそのリグニン組成<sup&gt; 5</sup&gt;。セルロースの含有量とマトリックス多糖組成物を含むリグノセルロース系バイオマス中の炭水化物を分析するためのプロトコルは、パートIIで説明されています<sup&gt; 2</sup&gt;。

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Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part I: Lignin

植物バイオマスは、バイオ燃料の生産に使用できる主要なカーボンニュートラルな再生可能な資源です。植物バイオマスは、主に細胞壁、リグノセルロースと呼ばれる構造的に複雑な複合材料で構成されています。ここでは、コンテンツとポリフェノールリグニンの組成の包括的な分析のためのプロトコルについて説明します。

植物細胞壁の包括的な組成分析（リグノセルロース系バイオマス）パートI：リグニン

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Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part I: Lignin

Unit 1

Plant Cell Structure and Organization

SCIENTISTS IN ACTION

32.0K ビュー.  Michigan State University (MSU). 植物バイオマスは、バイオ燃料の生産に使用できる主要なカーボンニュートラルな再生可能な資源です。植物バイオマスは、主に細胞壁、リグノセルロースと呼ばれる構造的に複雑な複合材料で構成されています。ここでは、コンテンツとポリフェノールリグニンの組成の包括的な分析のためのプロトコルについて説明します。

ビデオ: Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part I: Lignin

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

人間の膵島単離：パートI：膵組織の消化とコレクション

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

生物学

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 1 of 3.

ワクシニアウイルス感染とウイルスの遺伝子発現の時間的解析：その1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells.  Members of ...

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 2

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3.

ワクシニアウイルス感染とウイルスの遺伝子発現の時間的解析：その2

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 3

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 3 describes the process of fluorescently labeling the amplified RNA from both host and viral samples by amino allyl coupling of dyes. Part 3 of 3.

ワクシニアウイルス感染とウイルスの遺伝子発現の時間的解析：その3

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

植物プロトプラストの蛍光活性化細胞選別

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part II: Carbohydrates

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation2 and other products such as biocomposite materials6. Plant biomass remains one of the greatest untapped reserves on the planet4. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses, and the polyphenol lignin5 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls contribute extensively to the strength and structural integrity of the entire plant. Despite its necessary rigidity, the cell wall is a highly dynamic entity that is metabolically active and plays crucial roles in numerous cell activities such as plant growth and differentiation5. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant4. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerisation by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its monosaccharide composition of the hemicelluloses and other matrix polysaccharides1, and its content of crystalline cellulose7. The protocol for analyzing the lignin components in lignocellulosic biomass is discussed in Part I3.

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part II: Carbohydrates

Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of wall derived carbohydrates.

植物細胞壁の包括的な組成分析（リグノセルロース系バイオマス）パートII：炭水化物

Label-free in situ Imaging of Lignification in Plant Cell Walls

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find cost-effective and sustainable solutions has become a topical and critical task. Lignocellulosic biomass is poised to become the primary source of biomass for the conversion to liquid biofuels1-6. However, the recalcitrance of these plant cell wall materials to cost-effective and efficient degradation presents a major impediment for their use in the production of biofuels and chemicals4. In particular, lignin, a complex and irregular poly-phenylpropanoid heteropolymer, becomes problematic to the postharvest deconstruction of lignocellulosic biomass. For example in biomass conversion for biofuels, it inhibits saccharification in processes aimed at producing simple sugars for fermentation7. The effective use of plant biomass for industrial purposes is in fact largely dependent on the extent to which the plant cell wall is lignified. The removal of lignin is a costly and limiting factor8 and lignin has therefore become a key plant breeding and genetic engineering target in order to improve cell wall conversion.
Analytical tools that permit the accurate rapid characterization of lignification of plant cell walls become increasingly important for evaluating a large number of breeding populations. Extractive procedures for the isolation of native components such as lignin are inevitably destructive, bringing about significant chemical and structural modifications9-11. Analytical chemical in situ methods are thus invaluable tools for the compositional and structural characterization of lignocellulosic materials. Raman microscopy is a technique that relies on inelastic or Raman scattering of monochromatic light, like that from a laser, where the shift in energy of the laser photons is related to molecular vibrations and presents an intrinsic label-free molecular "fingerprint" of the sample. Raman microscopy can afford non-destructive and comparatively inexpensive measurements with minimal sample preparation, giving insights into chemical composition and molecular structure in a close to native state. Chemical imaging by confocal Raman microscopy has been previously used for the visualization of the spatial distribution of cellulose and lignin in wood cell walls12-14. Based on these earlier results, we have recently adopted this method to compare lignification in wild type and lignin-deficient transgenic Populus trichocarpa (black cottonwood) stem wood15. Analyzing the lignin Raman bands16,17 in the spectral region between 1,600 and 1,700 cm-1, lignin signal intensity and localization were mapped in situ. Our approach visualized differences in lignin content, localization, and chemical composition. Most recently, we demonstrated Raman imaging of cell wall polymers in Arabidopsis thaliana with lateral resolution that is sub-&mu;m18. Here, this method is presented affording visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species without staining or labeling of the tissues.

Label-free in situ Imaging of Lignification in Plant Cell Walls

A method based on confocal Raman microscopy is presented that affords label-free visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species.

ラベルフリーその場で</em植物細胞壁の木化の>イメージング

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

マイクロアレイを用いた植物細胞壁ポリマーの糖鎖プロファイリング

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

比較プロテオーム解析のための代謝標識および膜分画<em&gt;シロイヌナズナ</em&gt;サスペンション細胞培養

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

ホールマウント胚珠内でのクロマチン修飾や核構造の定量的、単一細胞分析のための効率的な方法<em&gt;シロイヌナズナ</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...