この作品は、ドイツ科学財団（DFG）とドイツ連邦政府と州政府のエクセレンスイニシアチブによって資金を供給された。

1。植物材料の架橋<ol><li> 50 mlのプラスチックチューブで収穫シロイヌナズナの葉の1〜2グラム（6〜10cmのロゼットの直径）と架橋のバッファと40mlのためにいっぱいに。 </li><li>葉は右側のバッファの表面上に合わせたフィルタースポンジを詰めることによって、例えば、浸漬滞在を確認してください。その後、デシケーター中にチューブを置きます。 </li><li>真空は、10分間浸潤。各チューブに架橋を停止するには2Mグリシンの2.5mLを追加し、そしてグリシンの均等分散を保証するためにチューブを反転させる。 </li><li>真空は、別の5分間に潜入し、ふるいに葉を収穫しながら液を捨てる。 </li><li>洗浄は、ガラスのビーカーの水の1L、徹底的にペーパータオルで乾燥した葉で二回葉。 </li><li>新鮮なプラスチック製のチューブで葉を収集し、液体窒素中で凍結する。店は-80葉° Cまでのクロマチンの単離。 </li></ol> 2。クロマチンの単離<ol><li>葉は、使用するまで液体窒素中に保管されたモルタルと雌しべで粉砕した。 </li><li>を50 mLプラスティックチューブに粉を移し、30mLを抽出バッファー＃1で一時停止。 </li><li>オーバーヘッドシェーカー上で4℃で15分間インキュベートする。 </li><li>新鮮な50 mLのプラスチックチューブにミラクロスの4つの層を介してろ液は、懸濁液。 </li><li> 4℃で2800 × gで20分のためにスピン℃に</li><li>上清を除去し、1mLの抽出バッファー＃2のペイントブラシでペレットを懸濁する。まず、ペイントブラシでペレットを中断し、バッファの残りを追加し、バッファ＃2の小さなボリュームを追加します。 </li><li> 4℃で12,000 xgで10分間1.5mlのマイクロチューブとスピンして懸濁液を移す℃に</li><li>一方、1.5mlの抽出バッファー＃3を含む液2ml、マイクロチューブを用意する。 </li><li>紡糸管（ステップ2.7）から上清を除去し、抽出緩衝液第3位の300μLでペレットを懸濁する。最初のバッファの小さなボリュームを追加する＃3、ペイントブラシでペレットを中断し、残りのバッファを追加します。 </li><li>ステップ2.8で作成した1.5​​ mLの抽出バッファー＃3の上に慎重にピペット中断ペレットを（ステップ2.9）。二つの相が混在しないことを確認してください。 4℃で16000 × gで1時間のためにスピン℃に</li><li>上清を除去し、超音波処理のバッファの300μLでペイントブラシでクロマチンペレットを中断。 </li><li>約400bpのDNAの大きさに、sonotrodeと、または超音波浴中でクロマチンを超音波処理してください。超音波処理するときに、クロマチンは° Cは熱に敏感な架橋を保護するために約30以上に加熱されることはありませんことを確認してください。それは別の超音波デバイスの間に著しく変化するため、超音波処理の期間は、実験的に決定する必要があります。ガイダンスについては、二つの異なるデバイスの設定が用意されています： </li></ol>設定は25％の電力で0.6mL微量遠心管でBandelinブランドsonotrodeを公開クロマチ​​ンと超音波処理のための5倍〜20バースト、周期= 50。氷浴では20 番目のバーストの後に、編集作業中に、涼しいサンプル。 DiagenodeのBioruptor超音波洗浄機と超音波処理の場合は、水と氷で風呂を記入し、&quot;高&quot;に設定して1.5mlのマイクロチューブに30秒間十倍を超音波で分解する。毎30秒後、別の30秒間クールなサンプル。 <ol start="13"><li>スピンは4で16,000 gで5分間サンプルを超音波処理℃で溶解していない物質を沈殿させる。上清を直接免疫沈降に使用することができます。また、サンプルは、衝撃、液体窒素で凍結し、-80℃で保存できますさらに使用するまでのC。 </li></ol> 3。免疫沈降<ol><li> 40μlのタンパク質 - 抗体 - 結合バッファーのアガロースおよび1.8mLを含む2 mLのマイクロ遠心チューブを準備します。クロマチンの調製（ステップ2.13）の200μLを追加。 </li><li>オーバーヘッドシェーカーで1時間のためのチューブをインキュベートします。 </li><li>プロテインA -アガロースビーズを破棄し、免疫沈降のために上清を使用してください。 </li><li>クロマチンの濃度を（&#39;input&#39;）を決定するために40μlの-アリコートを削除します。 </li><li> 30μlのプロテ​​インA -アガロースおよび400μL音波処理クロマチンの懸濁液に特異的な試薬と設備の下の表に示されている修正特異的抗体の量を追加します。 </li><li> 4時オーバーヘッドシェーカー上で2.5時間または一晩インキュベート℃に</li><li> 440 xgで2分間ビーズをスピンダウン</li><li>各洗浄バッファーの900μL（下のリストを参照）ビーズペレットを洗浄します。各バッファの場合は、4℃でオーバーヘッドシェーカーで10分間バッファーでビーズをインキュベートして、440 xgで2分間スピンし上清を捨て、そして次のバッファを追加します。 <ol><li>低塩の洗浄バッファー</li><li>高塩分洗浄バッファー</li><li> LiClを洗浄バッファー</li><li> TE洗浄バッファー</li></ol></li><li>最終洗浄後、完全に残っているバッファを削除します。 </li></ol> 4。ヒストンとDNAの脱架橋、および沈殿したDNAの精製<ol><li>アガロースをペレットと40μlの&quot;入力&quot;のサンプル手順3.4、ボルテックスミキサーで5分のミックス、スピンのサンプルから、とincuに脱架橋バッファー100μLを加える℃で一晩65℃で脱灰する。 </li><li>市販のDNAやPCR精製キットでDNAを精製。 </li><li>通常は最後のカラム溶出液の1:5希釈の5μlのは、実行するのに十分であり、従来の遺伝子座特異的リアルタイム定量RT - PCR（RT - QPCR）。 </li></ol> 5。バッファの表<table border="1"><tbody><tr><td colspan="2"> 架橋バッファ </td></tr><tr><td>酪酸ナトリウム</td><td> 10mMの</td></tr><tr><td>スクロース</td><td> 400mmの</td></tr><tr><td>トリス- HCl、pH8.0の</td><td> 10mMの</td></tr><tr><td> β-メルカプトエタノール</td><td> 5mMの</td></tr><tr><td>ホルムアルデヒド</td><td> 3％（v / v）の</td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> 抽出緩衝液第1位 </td></tr><tr><td>酪酸ナトリウム</td><td> 10mMの</td></tr><tr><td>スクロース</td><td> 400mmの</td></tr><tr><td>トリス- HCl、pH8.0の</td><td> 10mMの</td></tr><tr><td> β-メルカプトエタノール</td><td> 5mMの</td></tr><tr><td>完全なプロテアーゼ阻害剤</td><td> 1X </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> 抽出バッファー＃2 </td></tr><tr><td>酪酸ナトリウム</td><td> 10mMの</td></tr><tr><td>スクロース</td><td> 250mmの</td></tr><tr><td>トリス- HCl、pH8.0の</td><td> 10mMの</td></tr><tr><td> β-メルカプトエタノール</td><td> 5mMの</td></tr><tr><td> MgCl 2の </td><td> 10mMの</td></tr><tr><td>トリトンX - 100 </td><td> 1％（w / v）の</td></tr><tr><td>完全なプロテアーゼ阻害剤</td><td> 1X </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> 抽出緩衝液第3位 </td></tr><tr><td>酪酸ナトリウム</td><td> 10mMの</td></tr><tr><td>スクロース</td><td> 1.7M </td></tr><tr><td>トリス- HCl、pH8.0の</td><td> 10mMの</td></tr><tr><td> β-メルカプトエタノール</td><td> 5mMの</td></tr><tr><td> MgCl 2の </td><td> 2mMの</td></tr><tr><td>トリトンX - 100 </td><td> 0.15％（w / v）の</td></tr><tr><td>完全なプロテアーゼ阻害剤</td><td> 1X </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> 超音波処理のバッファ </td></tr><tr><td>トリス- HCl、pH8.0の</td><td> 25mMの</td></tr><tr><td> EDTA - NaOHで、pH8.0の</td><td> 5mMの</td></tr><tr><td> SDS </td><td> 0.5％（w / v）の</td></tr><tr><td>完全なプロテアーゼ阻害剤</td><td> 1X </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> 抗体結合バッファー </td></tr><tr><td>トリス- HCl、pH8.0の</td><td> 50mmの</td></tr><tr><td> EDTA - NaOHで、pH8.0の</td><td> 1mMの</td></tr><tr><td> NaClの</td><td> 150MM </td></tr><tr><td>トリトンX - 100 </td><td> 0.1％（w / v）の</td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> 低塩の洗浄バッファー </td></tr><tr><td> NaClの</td><td> 150MM </td></tr><tr><td> SDS </td><td> 0.1％（w / v）の</td></tr><tr><td>トリトンX - 100 </td><td> 1％（w / v）の</td></tr><tr><td> EDTA - NaOHで、pH8.0の</td><td> 2mMの</td></tr><tr><td>トリス- HCl、pH8.0の</td><td> 20mMの</td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> 高塩分洗浄バッファー </td></tr><tr><td> NaClの</td><td> 500mmの</td></tr><tr><td> SDS </td><td> 0.1％（w / v）の</td></tr><tr><td>トリトンX - 100 </td><td> 1％（w / v）の</td></tr><tr><td> EDTA - NaOHで、pH8.0の</td><td> 2mMの</td></tr><tr><td>トリス- HCl、pH8.0の</td><td> 20mMの</td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> LiClを洗浄バッファー </td></tr><tr><td>リチウム塩化</td><td> 250mmの</td></tr><tr><td>ノニデット- P40 </td><td> 1％（v / v）の</td></tr><tr><td>デソキシコール酸ナトリウム</td><td> 1％（w / v）の</td></tr><tr><td> EDTA - NaOHで、pH8.0の</td><td> 1mMの</td></tr><tr><td>トリス- HCl、pH8.0の</td><td> 20mMの</td></tr></tbody></table><table border="1"><tbody><tr><td> TE洗浄バッファー </td></tr><tr><td> EDTA - NaOHで、pH8.0の</td><td> 10mMの</td></tr><tr><td>トリス- HCl、pH8.0の</td><td> 1mMの</td></tr></tbody></table><table border="1"><tbody><tr><td> Decrosslinkingバッファ </td></tr><tr><td>トリス- HCl、pH 6.8の</td><td> 62.5mM </td></tr><tr><td> NaClの</td><td> 200mmの</td></tr><tr><td> SDS </td><td> 2％（w / v）の</td></tr><tr><td> DTT </td><td> 10mMの</td></tr></tbody></table> 6。代表的な結果： βをエンコードするシロイヌナズナPR2防御遺伝子の発現抗菌活性を持つ1,3 -グルカナーゼ12は 、ベンゾチアジアゾール（BTH）、植物ホルモンであるサリチル酸3の合成アナログで誘導した。 図1は、PR2の発現の活性化は、ヒストンのリジン残基のアセチル化の増加に関連付けられている実証PR2プロモーター上のH3とH4。ヌクレオソーム密度が遺伝子の活性化は13減少するため、ヒストンアセチル化の値は、ヒストン3の不変領域に対する抗体を用いて得られた信号のために標準化した。 <img src="/files/ftp_upload/3096/3096fig1.jpg" alt="図1"/> 図1 BTHは、シロイヌナズナのPR2プロモーターのヒストンアセチル化を誘導する 。植物は100μMBTHまたは水和剤のコントロールで処理した。 72時間後、葉を採取し、クロマチンを単離した。アセチル化 - 特異的抗体（図に示される）で沈殿後に得られた信号は、ヒストンH3の不変ドメインに対する抗体で沈殿させることにより得られる信号に対して標準化した。沈殿したクロマチンは、RT -定量PCRにより定量した。データは、BTHの治療がない場合のヒストン修飾のレベルの標準化されています。

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利害の衝突は宣言されません。

プロトコルにおいて最も重要なステップは、材料の研削、およびクロマチンの超音波処理、DNAとヒストン、型と葉の物質の量の架橋です。これらの問題についての詳細は、refsを参照してください。 9と10。 超音波処理と架橋： 400bp程度の断片に超音波照射時のクロマチンを妨害することが重要です。不十分な超音波処理が長いクロマチンの断片を残すのに対し、徹底的な超音波処理は、DNAとヒストンを破壊することによりクロマチンを破壊します。テストした軌跡から遠位ヒストン修飾がローカルの変更と区別できないため、これらは、メソッドの特異性に影響を与えます。超音波処理の効率が最高のDNAを単離し、アガロースゲル電気泳動により断片化したDNAの長さを決定する、超音波処理、脱架橋DNAとヒストンの前と後のサンプルから20μLクロマチンを収集することによってテストされています。クロマチンの準備がまだ断片化したDNAの可視化を妨げる可能性のある精製のこの段階でRNAを大量に含まれているため、RNアーゼは、電気泳動前のサンプルに追加する必要があります。非断片化したクロマチンからDNAが10kbp以上の場合、断片化したクロマチンからDNAは、DNAの400bp前後の最大信号強度とスミアを形成するのに対し、サンプルは、クロマチン免疫沈降のために有用です。 我々は日常的に無傷の葉におけるクロマチン架橋のために3％（v / v）のホルムアルデヒドを使用していますが、1％（v / v）のホルムアルデヒドは、ほとんどのケースで十分な場合があります。私たちの手では、低ホルムアルデヒドの濃度が短い超音波処理時間とそれに伴うPCR反応のバックグラウンドシグナルを可能にする。しかし、ホルムアルデヒドの不十分な量は、しばしば独立した実験の間でPCR信号の低再現性につながる。これは、低濃度でホルムアルデヒドと葉の不十分な浸透に起因するかもしれない。化合物が時間とともに重合する傾向があるので頻繁にホルムアルデヒドの株式を交換することを確認してください。ホルムアルデヒドのソリューションは、約一ヶ月間安定しており、この期間はほとんどメタノールの安定化によって延長されていません。それは、実験条件を設定するときに別のホルムアルデヒド濃度をテストすることをお勧めします。 植物材料の量と研削： お互いに付着葉を避けるために、バッファの大量の葉材料の少量に潜入することが重要です。葉は、しばしば、ホルムアルデヒドの不十分な浸潤の結果を突き出し。また、あまりにも多くの植物材料は、ミラクロス組織の細孔をブロックします。粉砕効率が大幅に完璧にフィットで乳鉢と乳棒を使用してによって異なります。材料を微粉末に粉砕されるまで、我々は定期的に追加の液体窒素の欠如50秒間、3回の液体窒素冷却乳棒と乳鉢を用いて挽く。

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント</th></tr><tr><td>プロテイン- A -アガロース</td><td>ロッシュ</td><td> 11 134 515 001 </td><td>抗体のクラスによって異なります。 </td></tr><tr><td>プロテイン- G -アガロース</td><td>ロッシュ</td><td> 11 243 233 001 </td><td>抗体のクラスによって異なります。 </td></tr><tr><td>抗hyperacetyl - H4 </td><td>ミリポア</td><td> 06から946 </td><td>我々は、5μLを用い</td></tr><tr><td>抗アセチル- H4K5 </td><td>ミリポア</td><td> 07から327 </td><td>我々は、5μLを用い</td></tr><tr><td>抗アセチル- H4K8 </td><td>ミリポア</td><td> 07から328 </td><td>我々は、5μLを用い</td></tr><tr><td>抗アセチル- H4K12 </td><td>ミリポア</td><td> 07から595 </td><td>我々は、5μLを用い</td></tr><tr><td>抗アセチル- H4K16 </td><td>ミリポア</td><td> 07から329 </td><td>我々は、5μLを用い</td></tr><tr><td>抗アセチル- H4K18 </td><td>ミリポア</td><td> 07から354 </td><td>我々は、1μLを用い</td></tr><tr><td>抗アセチル- H3K9 </td><td>ミリポア</td><td> 07から352 </td><td>我々は、5μLを用い</td></tr><tr><td>抗アセチル- H3K14 </td><td>ミリポア</td><td> 07から353 </td><td>我々は、1μLを用い</td></tr><tr><td>抗トリメチル- H3K4 </td><td> Diagenodeの</td><td> PAB - 003から50 </td><td>我々は、5μLを用い</td></tr><tr><td>抗ジメチル- H3K4 </td><td>ミリポア</td><td> 07から030 </td><td>我々は、5μLを用い</td></tr><tr><td>抗モノメチル- H3K4 </td><td>アブカム</td><td> ab8895 </td><td> 2,5 -10μL </td></tr><tr><td>抗H3 </td><td>アブカム</td><td> ab1791 </td><td>我々は、ヒストンの密度を確認するために1μLを用い</td></tr><tr><td> Bioruptor </td><td> Diagenodeの</td><td> UCD - 200に</td><td></td></tr><tr><td> Sonotrode MS72 </td><td> Bandelin </td><td></td><td></td></tr><tr><td>ミラクロス</td><td>カルビオケム</td><td> 475855 </td><td></td></tr><tr><td>完全なプロテアーゼ阻害剤</td><td>ロッシュ</td><td> 11836145001 </td><td></td></tr></tbody></table>

detection of histone modifications in plant leaves

クロマチン構造は、真核生物における遺伝子発現の調節に重要である。このプロセスでは、ヒストンH3およびH4のアミノ末端尾部でのクロマチンリモデリング、DNAメチル化、および共有結合修飾は、本質的な役割1〜2果たす。 H3およびH4ヒストン修飾は、リジンやアルギニンのメチル化、リジンのアセチル化、および1-2のセリン残基のリン酸化が含まれています。これらの修飾は遺伝子の活性化、抑制、または適切な信号（微生物体関連分子パターン、光、ホルモン、等）3-7の認識の後の式のより迅速かつ堅牢な活性化をサポートしている遺伝子のプライミング状態のいずれかで関連付けられています。 ここでは、選択された植物遺伝子上の特定のクロマチン修飾の信頼性と感度に検出する手法を提案する。技術は、特異的修飾抗体9,10、ヒストン- DNA複合体の脱架橋、とクロマチン、クロマチン免疫沈降（ChIP）の抽出及び超音波処理、ホルムアルデヒド8,9付き（修正）ヒストンとDNAの架橋に基づいており、遺伝子特異的リアルタイム定量PCR。アプローチは、トウモロコシ5,11およびシロイヌナズナ3の全身性免疫におけるC 4光合成に関連する特定のヒストン修飾を検出するために役立っています。

Watch this Scientific Journal Video about Detection of Histone Modifications in Plant Leaves at JoVE.com

Detection of Histone Modifications in Plant Leaves

特定の植物遺伝子上にヒストン修飾を検出するために信頼性の高い有用なアプローチが説明されています。アプローチは、クロマチン免疫沈降（ChIP）とリアルタイム定量PCRを兼ね備えています。それは、多様な生理学的プロセスにおいて役割を持つ特定の遺伝子のヒストン修飾の検出を可能にする。

植物の葉におけるヒストン修飾の検出

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

detection-of-histone-modifications-in-plant-leaves

Research

JoVE Journal

Biology

24.8K ビュー.  RWTH Aachen University. 特定の植物遺伝子上にヒストン修飾を検出するために信頼性の高い有用なアプローチが説明されています。アプローチは、クロマチン免疫沈降（ChIP）とリアルタイム定量PCRを兼ね備えています。それは、多様な生理学的プロセスにおいて役割を持つ特定の遺伝子のヒストン修飾の検出を可能にする。

ビデオ: Detection of Histone Modifications in Plant Leaves

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

ELISAによるネイティブ無傷ヌクレオソーム上の翻訳後修飾の検出

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

生物学

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

ゲートウェイ互換二分子蛍光相補（BiFC）システムを使用してプラントにおけるタンパク質相互作用の検出

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

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 ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

植物タンパク質複合体の翻訳後修飾の同定

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

植物の葉からのアポプラスト液の抽出のための浸透·遠心分離技術の使用<em&gt;インゲンマメ</em&gt;例として

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

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 ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

トマト毛状根の発生によるDNAの修飾のハイスループットCRISPRベクター構築と特性

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

ヒストンの抽出からデータ解析まで：ボトムアップ質量分析法を用いてヒストンの翻訳後修飾の解析のための完全なワークフロー

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

ヘリアンサス・アンヌウスにおける大規模な熱画像スクリーニングによる植物蒸散の新規レギュレーターの同定

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid tissues due to unnaturally rapid cell replication. This is particularly challenging when modeling mature chromatin, as fast replicating cells are frequently involved in DNA replication and have a heterogeneous polyploid population. Presented below is a workflow for modeling, treating, and analyzing quiescent chromatin modifications using a three-dimensional (3D) cell culture system. Using this protocol, hepatocellular carcinoma cell lines are grown as reproducible 3D spheroids in an incubator providing active nutrient diffusion and low shearing forces. Treatment with sodium butyrate and sodium succinate induced an increase in histone acetylation and succinylation, respectively. Increases in levels of histone acetylation and succinylation are associated with a more open chromatin state. Spheroids are then collected for isolation of cell nuclei, from which histone proteins are extracted for the analysis of their post-translational modifications. Histone analysis is performed via liquid chromatography coupled online with tandem mass spectrometry, followed by an in-house computational pipeline. Finally, examples of data representation to investigate the frequency and occurrence of combinatorial histone marks are shown.

This protocol outlines how a three-dimensional cell culture system can be used to model, treat, and analyze chromatin modifications in a near-physiological state.

肝組織の3D細胞培養モデルにおけるヒストン翻訳後修飾のグローバルレベルの定量化

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid ...