IEMは資金調達のためにデンマークの研究評議会（FTPとFNU）を承認したいと思います。 ERLは、ARCのDP助成金の支援を認めている。 ABは、植物の細胞壁の助成金で優秀のARCセンターの支援を認めている。

1。組織収集＆準備<ol><li>関心のある各組織には、少なくとも三重に植物組織100 mgの生体重（10 mgの乾燥重量の最小値）を収集します。次の手順では、植物組織からの細胞壁物質の調製を記載している。貯蔵組織の場合には、非指名澱粉を酵素的に前述の13のような細胞壁ポリマーの抽出を続行する前に削除されます。 </li><li> 24チューブアダプタセットと3 mmのタングステンカーバイドビー​​ズ（30ヘルツ、2×30秒）で、キアゲンTissueLyserをIIを用いて液体窒素で微粉末にサンプルをホモジナイズする。唯一のいくつかのサンプルが処理されている場合、あるいは、乳鉢と乳棒を使用しています。 </li><li> 10ミリリットルプラスチックのコニカルチューブにホモジネートを転送します。 </li><li>室温で10ミリリットル80パーセントv / vエタノールでホモジネートを洗浄し、2分間激しくボルテックスすることによって細胞壁材料を準備します。 </li><li> 3500 xgでサンプルを遠心分離し、上清を捨てる。上澄みが透明になるまで70％エタノールが特にクロロフィルを含む組織のために、または少なくとも3回洗いを繰り返します。 </li><li> 100％アセトンで最終洗浄を行い、一晩空気乾燥する際には、アルコール性残留物（AIR）を含有するペレットを残す。 </li><li>ふるい0.4ミリメートル2メッシュを使用したAIRサンプルは罰金、均質な粉末を達成し、いつかホモジネートに残るほど、貧弱グラウンド粒子を除去する。 </li><li>マイクロチューブ、サンプルの混合を助けるために3ミリメートルのガラスビーズを含んだそれぞれに10mgのAIRサンプルを計量する。 </li></ol> 2。細胞壁糖鎖の抽出<ol><li>ペクチン、およびペクチンに関連付けられたポリマーは、各試料に50μMのCDTA液（pH7.5）500μlを加えてサンプルから抽出されます。 </li><li>簡潔にボルテックスチューブは溶媒が試料物質と接触していることを確認してから、8 Hzで3時間TissueLyserを用いて混合します。 </li><li>慎重に、12,000 xgでサンプルを遠心R4℃で上清と店を残したまま削除</li><li> 13000×gで残っている溶剤、ボルテックス、遠心を薄めて除去するために脱イオン水1ml中のペレットを洗浄します。ペレットを乱さずに、この手順を繰り返して、次のステップに進む前に、チューブから液体をすべて削除します。 </li><li> CDTA抽出のために2.4 - 架橋結合型糖鎖を順次ステップ2.1で説明したのと同じ手順を使用して、0.1％（w / v）の水素化ホウ素ナトリウムと4M NaOHを500μlの残りの細胞壁ペレットから抽出されます。 NaBH 4をそれによって多糖類の剥離を防止するベースアルコールに多糖類の還元末端にアルデヒド基（またはケト基）を低減するために追加されます。 </li><li>遠心分離後、上清を再び除去され、4℃で保存し、ペレットは、次の抽出に進む前に、脱イオン水で2回洗浄した。 </li><li>オプションの手順として、セルロースなどの残留性ポリマーは、500μlのcadoxen（31％v / vの1,2 - diamiで抽出する2.4 - 手順2.1で説明したのと同じ手順を使用して、0.78 MのCDOとnoethane）。代わりに、残りのペレットで絶対セルロース含有量は、酢酸/硝酸アッセイ（説明を参照してください）​​を使用して決定することができます。 </li></ol> 3。印刷マイクロアレイ<ol><li>任意の粒子状物質を除去するためにxgの13000で抽出された細胞壁ポリマーを含有する上清を遠心分離します。 </li><li>ウェルポリプロピレン384サンプルは、組織の種類と抽出の種類に応じて配置され、あらかじめデザインされたカスタムレイアウトを使用したマイクロタイタープレートに各サンプル50μlをロードします。 </li><li>脱イオン水で0,5、および25×シリアル希釈系列の細胞壁ポリマー試料を希釈します。 </li><li>そのようなピンの高さなどのパラメータは、収集および滞留時間、および洗浄工程は、マイクロアレイを制御するソフトウェアで設定されます。 </li><li>印刷室の湿度は、サンプルの蒸発を防ぐために60％に制御されます。 </li><li>印刷ジョブがLabNEXTソフト制を使用して開始されreとマイクロアレイのレイアウトに対応したプログラム。 </li><li>ロボットは機械で平板に取り付けられた20×20cmのニトロセルロース膜上にサンプルプレートから溶液をプリントする毛管チャネルピンを使用しています。アレイ上の各スポットは、溶液15 NLが含まれており、三重に印刷されています。 </li><li>印刷ジョブが完了した後、同一のマイクロアレイは、個々の配列への膜とカットで隣同士に印刷されます。 </li><li>各実験において、配列は多かれ少なかれサンプル、希釈または複製に対応するために修正することができます。 </li></ol> 4。糖鎖マイクロアレイのプロービング<ol><li>印刷後、非特異的結合を低減するために2時間、室温で生理食塩水（MPBS）をリン酸緩衝に溶解し5％w / vの脱脂粉乳で個々のマイクロアレイをブロック。 </li><li> MPBSで2時間の細胞壁糖鎖エピトープに特異的なモノクローナル抗体を用いたプローブのマイクロアレイ。モノクローナルantibの過半数細胞壁糖鎖に対するodiesは、次の3つの会社から市販されており、Biosupplies（ <a href="http://www.biosupplies.com.au" target="_blank">www.biosupplies.com.au</a> ）、Carbosourceサービス（ <a href="http://www.carbosource.net" target="_blank">www.carbosource.net</a> ）とPlantProbes（ <a href="http://www.plantprobes.net" target="_blank">www.plantprobes.net</a> ）。 </li><li>ネガティブコントロール、のみMPBS無一次抗体と共にインキュベートマイクロアレイを含む。 </li><li>マイクロアレイのリン酸塩で3回洗浄すると、非特異的な結合を除去するために5分間緩衝生理食塩水（PBS）。 </li><li> 2時間MPBSにおける西洋ワサビペルオキシダーゼ（HRP）に結合させた二次抗体を用いてマイクロアレイを探る。細胞壁糖鎖に対する最もモノクローナル抗体は抗マウスまたは抗ラット二次抗体を必要とします。 </li><li>非特異的結合を除去するために5分間3回PBS緩衝液での洗浄手順を繰り返します。 </li><li>発色（3,3 - ジアミノベンジジン）またはchemilを使用してマイクロアレイを開発uminecent（ルミノール）基板。 </li></ol> 5。定量化<ol><li>現像後、高解像度（1,200 dpiの）デスクトップスキャナを使用して、個々のマイクロアレイをスキャンして、負の、16ビットのTIFFファイル（ 図3）のように画像を保存します。 </li><li>自動グリッドツールを搭載画像処理ソフトウェア（LabNEXT）Xplore使用して、各スポットの積分強度を計算します。インテグラルスポット強度は、各スポットを囲むグリッド領域内のピクセルの合計から導出されます。 </li><li>各マイクロアレイのグリッドデータをtxtファイルとしてエクスポートされ、手動で解析するためのExcelスプレッドシートにインポートすることができます。オンラインツール（ <a href="http://arraytemplates.charliehart.com.au/" target="_blank">http://microarray.plantcell.unimelb.edu.au/は</a> ）自動的に翻訳して、個々のtxtファイルからのデータを処理するために開発されました。 </li><li>インテグラルスポット強度は &#39;平均スポットを得るために、印刷複製し、希釈液で平均化されている各サンプルの強度&quot;の値（ 図2）。代わりに、アレイ上のちょうど1希釈値に対応するスポット信号は、各サンプルの相対的な糖鎖エピトープの存在量を定量化するために使用されています。 </li><li>異なるサンプル間の相対的な平均スポット強度は、Excelまたはオンラインヒートマッパーツール（で条件付き書式を使用してヒートマップ（ 図4）として提示されている<a href="http://bar.utoronto.ca/welcome.htm" target="_blank">http://bar.utoronto.ca/welcome.htm</a> ）。各抗体の種類のデータを100に修正され、5％のカットオフ値は、バックグラウンド信号と誤検出を​​除去するために課せられています。 </li></ol>

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特別な利害関係は宣言されません。

CoMPPは、数日のうちに何百もの植物由来のサンプルの糖組成をプロファイリングするための迅速かつ高感度な方法である。この方法は、レクチン、受容体、抗体16と糖結合タンパク質と糖の相互作用のハイスループットスクリーニングのためにすでに利用可能な細菌または哺乳動物糖鎖アレイ·プラットフォームを補完します。細胞壁糖鎖を検出するための利用可能なプローブの大き?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td> 3 mmのタングステンカーバイドビー​​ズ</td><td>キアゲン</td><td> 69997 </td><td></td></tr><tr><td>コレクション·マイクロチューブ（1.2ミリメートル） </td><td>キアゲン</td><td> 19560 </td><td> 1.5 mlのマイクロチューブを使用することもできます</td></tr><tr><td>キアゲンTissueLyserを世</td><td>キアゲン</td><td> 85300 </td><td></td></tr><tr><td> 3ミリのガラスビーズ</td><td>シグマアルドリッチ</td><td> Z143928 </td><td></td></tr><tr><td> CDTA </td><td>シグマアルドリッチ</td><td> 34588 </td><td></td></tr><tr><td>酸化カドミウム</td><td>シグマアルドリッチ</td><td> 202894 </td><td></td></tr><tr><td> 1,2 - ジアミノエタン</td><td>シグマアルドリッチ</td><td> 03550 </td><td></td></<html> TR&gt; <tr><td>ニトロセルロース膜（孔径0.22μmのサイズ） </td><td> GEウォーター＆プロセス·テクノロジー</td><td> EP2HY00010 </td><td>異なる大きさの細孔膜は異なるピンタイプに適しています</td></tr><tr><td> XACT IIマイクロアレイロボット</td><td> Labnext </td><td> 001A </td><td>トランザクション配信IIのロボットは、ニトロセルロース膜が添付されているカスタム20×20センチメートルセラミックプレートが取り付けられていた</td></tr><tr><td> RMのマイクロアレイ上のアイコンをXTEND </td><td> Labnext </td><td> 0037-350 </td><td>ピンは、膜上スポッティングに適していなければならない</td></tr><tr><td> 384ウェルマイクロタイタープレート（ポリプロピレン） </td><td>グライナー</td><td> 781207 </td><td></td></tr><tr><td>抗糖鎖モノクローナル抗体</td><td>植物プローブ/ CarboSource / Biosupplies </td><td></td><td>ウェブサイト; PlantProbes（ <a href="http://www.plantprobes.net" target="_blank">www.plantprobes.net</a> ）、Carbosource（ww.carbosource.net &quot;ターゲット=&quot; _blank &quot;&gt; www.carbosource.net）とBiosupplies（ <a href="http://www.biosupplies.com.au" target="_blank">www.biosupplies.com.au</a> ）。 </td></tr><tr><td>抗ラットIgG（全分子） - ヤギで産ペルオキシダーゼ抗体。 </td><td>シグマ</td><td> A9037 </td><td>二次抗体の種類（ 例えば 、ラット、マウス、ヤギなどで提起された）を使用し、一次抗体に依存します。 </td></tr><tr><td> SIGMA FAST 3,3 &#39; -ジアミノベンジジン錠</td><td>シグマ</td><td> D4293 </td><td>試薬の開発のタイプは、使用される二次抗体および検出方法（colourmetric、またはchemiluminecent）に依存します。 </td></tr><tr><td> SuperSignalウェスト·ピコ化学発光基質</td><td> Thermoscientific </td><td> 34080 </td><td>上記を参照</td></tr><tr><td>画像処理ソフトXplore </td><td> LabNext </td><td> 008 </td><td>自動グリッド化ツールを使用して、多くのソフトウェアの種類がありますマイクロアレイのスポットの画素値を測定します。 </td></tr><tr><td>植物性多糖類</td><td>シグマ/ Megazyme </td><td></td><td></td></tr></tbody></table>

glycan profiling of plant cell wall polymers using microarrays

植物の細胞壁は、植物の生理と発展に重要な役割を果たしており、人間社会のために原材料を提供異種糖鎖の複雑なマトリックス（ 例えば、木材、紙、繊維、バイオ燃料産業）1,2です。しかし、これらの成分の生合成と機能を理解することは困難なままである。 細胞壁糖鎖は、化学とその構築ブロック、グリコシル残基の複雑さに起因するコンフォメーションが多様である。これらのフォームの複数の位置に結合およびリング構造が異なり、異性体またはアノマー配置されているだけでなく、無糖残基の配列で置換されている。グリカンの組成物は、単一細胞壁3の異なる細胞および/ ​​または組織型あるいはサブドメインに変化する。さらに、それらの組成物はまた、開発1時、または環境手がかり4に対応して変更されます。 EXで2000遺伝子の目的税は、植物の細胞壁は、異種の糖鎖の複雑なマトリックスシロイヌナズナ 5で細胞壁糖鎖の生合成と修飾に関与することが予測された多数持っています。しかし、生合成遺伝子の比較的少ないが、機能的に4,5特徴付けられてきた。遺伝子はしばしば差別的細胞型6の間に、多くの場合、低レベルで発現しているので、逆遺伝学的ア ​​プローチが困難である。また、変異体の研究は、多くの場合、適切な細胞壁機能は7に維持されていることを確認するために、遺伝子の冗長性または代償機構によって妨げられている。したがって、新たなアプローチが急速に糖鎖構造の多様な範囲を特徴づけるために理解と細胞壁の生合成と修飾に機能ゲノミクスアプローチを容易にするために必要とされる。 モノクローナル抗体（mAb）8,9は、植物の糖鎖構造と分布を決定するための重要なツールとして浮上している。これらは、dist認識ペクチン、キシログルカン、キシラン、マンナン、グルカンやアラビノガラクタンを含む植物細胞壁糖鎖の主要なクラス内に存在INCTエピトープ。最近、それらの使用は植物や組織の種類を同時に9,10,11の広い範囲で糖鎖の相対量を決定するために、大規模なスクリーニング実験にも拡張されました。 ここでは、複数のサンプル（100秒）が減少した試薬及びサンプル量で小型化されたマイクロアレイプラットフォームを用いてスクリーニングすることができる包括的なマイクロアレイポリマープロファイリング（CoMPP）（ 図1および2）10,11と呼ばれるマイクロアレイベースグリカンスクリーニング法を提案する。マイクロアレイ上のスポット信号は正式に糖鎖エピトープの発生に関する半定量的データを与えるために定量することができる。このアプローチは、複雑な生物システム12の糖鎖変化を追跡し、細胞壁組成のグローバルな概観を提供することに適している場合は特に事前知識Ofはこれができません。

ニコチアナダイジョの花から6種類の組織における糖鎖の相対的な存在量（葯フィラメント、花粉、卵巣、花弁、萼片と偏見）がCoMPPを用いて決定した。図3Aは、部分的に（低）メチルエステル化homogalacturonan（HG）、ペクチン多糖類14日に発生したエピトープに対して特異的mAb JIM5でプロービングされた代表的なマイクロアレイを示しています。 JIM5エピトープは、しかし、柱頭?...

Watch this Scientific Journal Video about Glycan Profiling of Plant Cell Wall Polymers using Microarrays at JoVE.com

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

と呼ばれる手法<strong&gt; C</strong&gt; omprehensive<strong&gt; M</strong&gt; icroarray<strong&gt; P</strong植物細胞壁糖鎖の特性評価のための&gt; olymerプロファイリング（CoMPP）について説明する。この方法は、生物学的な文脈の広い範囲で発生したグリカンのスクリーニングを可能にする小型のマイクロアレイ分析プラットフォームで定義された糖鎖エピトープに対するモノクローナル抗体の特異性を兼ね備えています。

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 ...

glycan-profiling-of-plant-cell-wall-polymers-using-microarrays

Research

JoVE Journal

Biology

14.5K ビュー.  University of Melbourne. と呼ばれる手法 C omprehensive M icroarray P olymerプロファイリング（CoMPP）について説明する。この方法は、生物学的な文脈の広い範囲で発生したグリカンのスクリーニングを可能にする小型のマイクロアレイ分析プラットフォームで定義された糖鎖エピトープに対するモノ...

ビデオ: Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Bacterial Gene Expression Analysis Using Microarrays

マイクロアレイを用いた細菌の遺伝子発現解析

生物学

Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The plant hormone auxin rapidly stimulates cell elongation in young stems and similar tissues at least in part by an acid-growth mechanism 2, 3. Auxin activates a H+ pump in the plasma membrane, causing acidification of the cell wall solution. Wall acidification activates expansins, which are endogenous cell wall-loosening proteins 4, causing the cell wall to yield to the wall tensions created by cell turgor pressure. As a result, the cell begins to enlarge rapidly. This 'acid growth' phenomenon is readily measured in isolated (nonliving) cell wall specimens. The ability of cell walls to undergo acid-induced extension is not simply the result of the structural arrangement of the cell wall polysaccharides (e.g. pectins), but depends on the activity of expansins 5. Expansins do not have any known enzymatic activity and the only way to assay for expansin activity is to measure their induction of cell wall extension. This video report details the sources and preparation techniques for obtaining suitable wall materials for expansin assays and goes on to show acid-induced extension and expansin-induced extension of wall samples prepared from growing cucumber hypocotyls.
 To obtain suitable cell wall samples, cucumber seedlings are grown in the dark, the hypocotyls are cut and frozen at -80 &deg;C. Frozen hypocotyls are abraded, flattened, and then clamped at constant tension in a special cuvette for extensometer measurements. To measure acid-induced extension, the walls are initially buffered at neutral pH, resulting in low activity of expansins that are components of the native cell walls. Upon buffer exchange to acidic pH, expansins are activated and the cell walls extend rapidly. We also demonstrate expansin activity in a reconstitution assay. For this part, we use a brief heat treatment to denature the native expansins in the cell wall samples. These inactivated cell walls do not extend even in acidic buffer, but addition of expansins to the cell walls rapidly restores their ability to extend.

Measuring Plant Cell Wall Extension Creep Induced by Acidic pH and by Alpha-Expansin

We demonstrate the use of a constant-force extensometer to measure long-term extension (creep) of plant cell wall specimens induced by acidic buffers and expansin protein.

酸性pHでとα-エクスパンにより誘起される植物細胞壁の延長（クリープ）を測定

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

でFACSアシスト細胞型特異的発現プロファイリングを使用して組織と器官特異フィトクロム応答を調査シロイヌナズナ</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both prokaryotic and eukaryotic cells lead to translational regulation which is a major cellular mechanism of stress response. Familiar examples are the stringent response in E. coli and the Gcn2 stress response pathway in yeast ([2-6]). Recent work in E. coli and S. cerevisiae have shown that tRNA charging patterns are highly dynamic and depends on the type of stress experienced by cells [1, 6, 7]. The highly dynamic, variable nature of tRNA charging makes it essential to determine changes in tRNA charging levels at the genomic scale, in order to fully elucidate cellular response to environmental variations. In this review we present a method for simultaneously measuring the relative charging levels of all tRNAs in S. cerevisiae . While the protocol presented here is for yeast, this protocol has been successfully applied for determining relative charging levels in a wide variety of organisms including E. coli and human cell cultures[7, 8].

Genome-wide Analysis of Aminoacylation Charging Levels of tRNA Using Microarrays

We describe a method for microarray analysis to determine relative aminoacylation levels of all tRNAs from S. cerivisiae.

マイクロアレイを用いたtRNAのアミノアシル化（充電）レベルのゲノムワイド解析

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both ...

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

細胞外多糖のオリゴマスプロファイリング（オリンプ）

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

同位体タグ質量分析法を用いたチオールレドックスプロテオームのプロファイリング

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

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 ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

結晶セルロース蓄積中の高解像度の定量化<em&gt;シロイヌナズナ</em&gt;ルーツは、組織特異的な細胞壁の変更を監視します

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

個々の細胞タイプの転写プロファイリングのためのツールと​​してのレーザ支援顕微解剖（LAM）

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

An Easy Method for Plant Polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

工場ポリソームプロファイリングのための簡単​​な方法

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...