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。

<table><tbody><tr><td>Hydroxylamine Hydrochloride</td><td>Sigma-Aldrich</td><td>255580</td><td></td></tr><tr><td>Acetyl Bromide</td><td>Aldrich</td><td>135968</td><td></td></tr><tr><td>Ethanethiol</td><td>Sigma-Aldrich</td><td>E3708</td><td></td></tr><tr><td>Borontrifluoride diethyl etherate</td><td>Fluka</td><td>15719</td><td></td></tr><tr><td>N,O,-Bis(trimethylsilyl) acetimide</td><td>Fluka</td><td>15241</td><td></td></tr><tr><td>Dioxane</td><td>Sigma-Aldrich</td><td>296309</td><td></td></tr><tr><td>Spectromax Plus 384</td><td>Molecular Devices</td><td>Plus384</td><td></td></tr><tr><td>GC-MS</td><td>Agilent Technologies</td><td>6890 GC/5975B MSD</td><td>(lignin composition)</td></tr><tr><td>5.5mm Stainless Steel Balls</td><td>Salem Ball Company</td><td>(N/A)</td><td></td></tr><tr><td>96 well plate heat spreader</td><td>Biocision</td><td>Coolsink 96F</td><td></td></tr><tr><td>Heating block</td><td>Techne</td><td>Dri-block DB-3D</td><td></td></tr><tr><td>Sample concentrator</td><td>Techne</td><td>FSC400D</td><td></td></tr></tbody></table>

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;。

Watch this Scientific Journal Video about Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part I: Lignin at JoVE.com

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

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 ...

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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

32.1K 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

Combining Raman Imaging and Multivariate Analysis to Visualize Lignin, Cellulose, and Hemicellulose in the Plant Cell Wall

The application of Raman imaging to plant biomass is increasing because it can offer spatial and compositional information on aqueous solutions. The analysis does not usually require extensive sample preparation; structural and chemical information can be obtained without labeling. However, each Raman image contains thousands of spectra; this raises difficulties when extracting hidden information, especially for components with similar chemical structures. This work introduces a multivariate analysis to address this issue. The protocol establishes a general method to visualize the main components, including lignin, cellulose, and hemicellulose within the plant cell wall. In this protocol, procedures for sample preparation, spectral acquisition, and data processing are described. It is highly dependent upon operator skill at sample preparation and data analysis. By using this approach, a Raman investigation can be performed by a non-specialist user to acquire high-quality data and meaningful results for plant cell wall analysis.

This protocol aims to present a general method to visualize lignin, cellulose, and hemicellulose in plant cell walls using Raman imaging and multivariate analysis.

ラマンイメージングと多変量解析を組み合わせて、植物細胞壁のリグニン、セルロース、およびヘミセルロースを視覚化する

The application of Raman imaging to plant biomass is increasing because it can offer spatial and compositional information on aqueous solutions. The ...

生化学

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is needed to open up the structure of the plant cell wall, increasing the accessibility of the cell wall carbohydrates. Lignin, a polyphenolic material present in many cell wall types, is known to be a significant hindrance to enzyme access. Reduction in lignin content to a level that does not interfere with the structural integrity and defense system of the plant might be a valuable step to reduce the costs of bioethanol production. In this study, we have genetically down-regulated one of the lignin biosynthesis-related genes, cinnamoyl-CoA reductase (ZmCCR1) via a double stranded RNA interference technique. The ZmCCR1_RNAi construct was integrated into the maize genome using the particle bombardment method. Transgenic maize plants grew normally as compared to the wild-type control plants without interfering with biomass growth or defense mechanisms, with the exception of displaying of brown-coloration in transgenic plants leaf mid-ribs, husks, and stems. The microscopic analyses, in conjunction with the histological assay, revealed that the leaf sclerenchyma fibers were thinned but the structure and size of other major vascular system components was not altered. The lignin content in the transgenic maize was reduced by 7-8.7%, the crystalline cellulose content was increased in response to lignin reduction, and hemicelluloses remained unchanged. The analyses may indicate that carbon flow might have been shifted from lignin biosynthesis to cellulose biosynthesis. This article delineates the procedures used to down-regulate the lignin content in maize via RNAi technology, and the cell wall compositional analyses used to verify the effect of the modifications on the cell wall structure.

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed.

のリグニンダウンレギュレーション<em&gt;トウモロコシ</em&gt; dsRNAiとクラソンリグニン分析によって

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is ...

生物工学

Visualizing Lignification Dynamics in Plants with Click Chemistry: Dual Labeling is BLISS!

Lignin is one of the most prevalent biopolymers on the planet and a major component of lignocellulosic biomass. This phenolic polymer plays a vital structural and protective role in the development and life of higher plants. Although the intricate mechanisms regulating lignification processes in vivo strongly impact the industrial valorization of many plant-derived products, the scientific community still has a long way to go to decipher them. In a simple three-step workflow, the dual labeling protocol presented herein enables bioimaging studies of actively lignifying zones of plant tissues. The first step consists in the metabolic incorporation of two independent chemical reporters, surrogates of the two native monolignols that give rise to lignin H- and G-units. After incorporation into growing lignin polymers, each reporter is then specifically labeled with its own fluorescent probe via a sequential combination of bioorthogonal SPAAC/CuAAC click reactions. Combined with lignin autofluorescence, this approach leads to the generation of three-color localization maps of lignin within plant cell walls by confocal fluorescence microscopy and provides precise spatial information on the presence or absence of active lignification machinery at the scale of plant tissues, cells and different cell wall layers.

BLISS, a dual labeling protocol for studying lignification dynamics, was developed. Using synthetic monolignol reporters and a sequential combination of SPAAC and CuAAC bioorthogonal click reactions, this methodology paves the way to in-depth analysis of the factors that regulate the biogenesis of lignins in planta.

化学のをクリックして、植物の木質化のダイナミクスを可視化する: 二重分類は至福である!

Lignin is one of the most prevalent biopolymers on the planet and a major component of lignocellulosic biomass. This phenolic polymer plays a vital ...

化学

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid (TGA)

Lignin is a natural polymer that is the second most abundant polymer on Earth after cellulose. Lignin is mainly deposited in plant secondary cell walls and is an aromatic heteropolymer primarily composed of three monolignols with significant industrial importance. Lignin plays an important role in plant growth and development, protects&#160;from biotic and abiotic stresses, and in the quality of animal fodder, the wood, and industrial lignin products. Accurate estimation of lignin content is essential for both fundamental understanding of the lignin biosynthesis and industrial applications of biomass. The thioglycolic acid (TGA) method is a highly reliable method of estimating the total lignin content in the plant biomass. This method estimates the lignin content by forming thioethers with the benzyl alcohol groups of lignin, which are soluble in alkaline conditions and insoluble in acidic conditions. The total lignin content is estimated using a standard curve generated from commercial bamboo lignin.

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid TGA

Here, we present a modified TGA method for estimation of lignin content in herbaceous plant biomass. This method estimates the lignin content by forming specific thioether bonds with lignin and presents an advantage over the Klason method, as it requires a relatively small sample for lignin content estimation.

チオグリコール酸(TGA)を用いた植物バイオマスリグニン含量の推定

Lignin is a natural polymer that is the second most abundant polymer on Earth after cellulose. Lignin is mainly deposited in plant secondary cell walls ...

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

生物学

Quantitative 31P NMR Analysis of Lignins and Tannins

The development of sustainable biorefinery products is confronted, among others, with the challenge of lignin and tannin valorization. These abundant, renewable aromatic biopolymers have not been widely exploited due to their inherent structural complexity and high degrees of variability and species diversity. The lack of a defined primary structure for these polyphenols is further compounded with complex chemical alterations induced during processing, eventually imparting a large variety of structural features of extreme significance for any further utilization efforts.
Consequently, a protocol for the rapid, simple, and unequivocal identification and quantification of the various functional groups present in natural polyphenols, is a fundamental prerequisite for understanding and accordingly tailor their reactivity and eventual utility.
Quantitative 31P NMR offers the opportunity to rapidly and reliably identify unsubstituted, o-mono substituted, and o-disubstituted phenols, aliphatic OHs, and carboxylic acid moieties in lignins and tannins with broad application potential.
The methodology consists of an in situ quantitative lignin or tannin labeling procedure using a suitable 31P containing probe, followed by the acquisition of a quantitative 31P NMR spectrum in the presence of an internal standard. The high natural abundance of the 31P nucleus allows for small amounts of the sample (~30 mg) and short NMR acquisition times (~30-120 min) with well-resolved 31P signals that are highly dependent on the surrounding chemical environment of the labeled OH groups.

Quantitative 31P NMR Analysis of Lignins and Tannins

31P NMR is a powerful tool for the structural elucidation of polyphenols. This fast, easy, precise, quantitative, and highly reproducible analytical procedure, that allows for the quantification and differentiation of the different types of hydroxy, phenolic, and carboxylic groups in lignins and tannins has now become a routine analytical tool.

リグニンとタンニンの定量 31P NMR分析

The development of sustainable biorefinery products is confronted, among others, with the challenge of lignin and tannin valorization. These abundant, ...

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 ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

の組織化学的染色<em&gt;シロイヌナズナ</em&gt;二次細胞壁の要素

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester of glycerol and modified fatty acids that constitutes the framework of the cuticle that covers aerial plant surfaces. Suberin is a related lipid polyester that is deposited on the cell walls of certain tissues, including the root endodermis and the periderm of tubers, tree bark and roots. These lipid polymers are highly variable in composition among plant species, and often differ among tissues within a single species. Here, we describe a detailed protocol to study the monomer composition of cutin in Arabidopsis thaliana leaves by sodium methoxide (NaOMe)-catalyzed depolymerisation, derivatization, and subsequent gas chromatography-mass spectrometry (GC/MS) analysis. This method can be used to investigate the monomers of insoluble polyesters isolated from whole delipidated plant tissues bearing either cutin or suberin. The method can by applied not only to characterize the composition of lipid polymers in species not previously analyzed, but also as an analytical tool in forward and reverse genetic approaches to assess candidate gene function.

Lipid polyesters constitute the structural components of two cell wall modifications, the plant cuticle and suberin-containing diffusion barriers. In this video, we describe a method to depolymerize cutin from whole delipidated leaves. The method can be applied to investigating mutants compromised in either cutin or suberin biosynthesis.

植物キューティクル脂質ポリエステルモノマーの単離と組成分析

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester ...

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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ラマンイメージングと多変量解析を組み合わせて、植物細胞壁のリグニン、セルロース、およびヘミセルロースを視覚化する

のリグニンダウンレギュレーション

化学のをクリックして、植物の木質化のダイナミクスを可視化する: 二重分類は至福である!

チオグリコール酸(TGA)を用いた植物バイオマスリグニン含量の推定

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

リグニンとタンニンの定量 ³¹P NMR分析

ラベルフリーその場でイメージング

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

の組織化学的染色

植物キューティクル脂質ポリエステルモノマーの単離と組成分析