We thank Dr. M. Rosenberg for her advice and help with anatomical sectioning. We also thank D. Eisler for his technical assistance. This research was supported by grants from FP7-PEOPLE-IRG-2008, Binational Agriculture research and Development (BARD; IS-4246-09), and Israel Science Foundation (ISF; 592/13).

1.植物成長<ol><li>表面の種子を滅菌します。 <ol><li>湿式または乾式法により（30ミリグラムまで）の種子を滅菌します。例として、乾燥滅菌法のために、少なくとも4時間、一晩まで、閉じたデシケーター中でガラスビーカーに、50ミリリットル漂白剤に1ミリリットルの氷のHCl 37％を追加します。化学フード内で、この手順を実行します。 </li></ol></li><li> 0.5×ムラシゲ＆スクーグ（MS）培地中の0.8％植物寒天を含むプレート上で無菌種子を広げます。パラフィルムまたは多孔質テープでプレートを包み、アルミホイルでカバーしています。 </li><li>均一な発芽を促進するために4日 - 1 4℃でプレートを保管してください。 </li><li> シロイヌナズナの苗を成長させるのに適した成長室にプレートを転送します。寒天培地の上に根の成長を維持し、侵入を防止するために、垂直方向に板を置きます。 </li><li> 7日発芽（DAG）の後固定液するために苗木を転送します。 </li></ol> 2.固定<ol><li> 50ミリリットルリチャードソンSolutioを準備蒸留水で1％のホウ砂（緩衝剤）、1％メチレンブルーおよび1％アズール（組織学的染料）の等容量を組み合わせることにより、n個。使用前に0.22μmのPVDFフィルターユニットを駆動シリンジで濾過。 </li><li> 0.05 Mナトリウムカコジル酸（緩衝剤）中の1.25％グルタルアルデヒド：固定液の50ミリリットルを準備します。 注：これらの物質は毒性があり、化学フード内で処理する必要があります。 </li><li>最終的な固定液を得るために、固定溶液にリチャードソン溶液100μlを（2.1を参照）（2.2を参照）を追加します。 </li><li>マーク対応するサンプル名で6ウェルプレートの各ウェル。 </li><li>苗木、一度転送は、完全に液体で覆われるように、ウェルに（2.3を参照）3ミリリットル、最終的な固定液を - 2を配置します。 </li><li>慎重に各ウェルに10苗まで、鉗子を使用して、転送します。パラフィルムを使用してプレートをシール。 4℃で、暗闇の中で、少なくとも一晩、一週間まで保管してください。 </li></ol> 3.脱水<ol> &lt;LI&gt;苗を脱水します。ここに記載されているように、エタノールの濃度の増加を含む、新たな6ウェルプレートのウェル間にそれらを転送する：10％エタノールを15分間、 15分間、30％エタノール。 15分間、50％エタノール。 15分間70％エタノール。 15分間85％のエタノール。 15分間、95％エタノール。一晩、最低でも4°C、95％エタノール。慎重にプラスチックピンセットで好ましく、その子葉、によってそれらを把握することにより、苗を処理します。 </li></ol> 4.潜入<ol><li>浸透媒体を準備します。小さなガラス瓶で50ミリリットルの基本的な樹脂液キットでhistoresinキット活性化因子の1袋の内容物を混合。 20分間磁石をかき混ぜます。浸透媒体は、数週間、4℃で維持することができます。 </li><li>マーク・各対応するサンプル名で新しい6ウェルプレートのウェルと2でいっぱい - 浸潤培地の3ミリリットル。 </li><li>ピンセットを用いて浸透培地に95％エタノール溶液から苗木を移します。苗は府であることを確認しますLLY媒体によって覆われます。 </li><li>植物組織に最大の浸透を確実にするために、少なくとも4日間、4℃で保管してください。 </li></ol> 5.ブロックの準備<ol><li>金型を埋め込むの各ウェルの中に小さなラベル、サンプル名と鉛筆でマークされたが、置きます。 </li><li> 15 mlの浸潤培地（4.1を参照）1mLの硬化剤を添加することにより培地を埋め込む準備します。重合の問題を回避するために、15ミリリットル未満のボリュームを準備しようとしないでください。 </li><li>メディアを埋め込む200μlで金型内に各ウェルの半分を記入し、金型の形状に切断オーバーヘッドフィルム片で覆いますが、それぞれの側に大きなサイズに1ミリメートル。重合を可能にするために、少なくとも2時間室温でインキュベートします。 5時間を超えないようにしてください。 </li><li>培地を埋め込む追加のバッチを調製し（5.2を参照）、根の先端が金型内に配置されている間、重合を回避するために、氷上に保ちます。 </li><li>メスを用いて解剖スコープの下で各根端をカットし、非常に慎重に位置iトン：垂直方向の横断面または水平方向に縦断面のため、約2時 - 金型外周から3ミリメートル。オーバーヘッドフィルムで解決し、カバーを遮断して型を完全に埋めます。それが重合された後、ブロッキング溶液がわずかに縮小することに注意してください。 </li><li>最低でも、2時間室温で保管してください。 </li><li>ブロックを乾燥させるため、乾燥シリカゲルと箱に入れて、最低でも、室温で一晩おきます。 </li></ol> 6.セクショニング<ol><li>ナイフメーカーでガラス棒からガラスナイフを準備します。組織切片用にガラスナイフを使用してください。正方形にガラス棒をカットした後、ダイヤモンドスコアリングツールを使用して、2つのナイフを生産するために斜めの正方形をカットします。 </li><li>セクショニング機を使用して3ミクロン幅のスライス、 - 2へのセクションをブロックします。スライドガラスの上に置いてスライス、蒸留水の液滴の上に、低熱に設定したヒートブロック上にスライドを配置 - 水が蒸発するまで、（50〜60℃）。 </li></ol><ol><li>元の溶液の0.2％に（2.1を参照してください）​​リチャードソン液を希釈し、スライスをカバーするために、それの1ミリリットルを使用します。 5秒間ヒートブロック上に置き、次いで蒸留水で洗浄します。 </li><li>ホットプレート上でスライドを乾燥させます。解剖顕微鏡下で観察し、顕微鏡下で彼らの後に識別を容易にするために、関心のスライスの位置を示すために、マーカーでスライドの背面をマーク。 </li><li>マウンティング培地100μlを添加して、カバースリップでカバーしています。 </li></ol> 8.画像取得<ol><li>リター情報と偏光子/干渉光学フィルタ（ 図1 AD）を得るのに適しPolscopeシステムを装備した光学顕微鏡下でルートセクションを含む覆われたスライドを配置します。 </li><li>サンプル全体の明確な可視化を可能にする選択倍率（ 例えば 、本研究では40X）と顕微鏡の焦点を合わせますブロック部が、無根元セクションが含まれている空のフィールド上の電子。無傷できれいな地域を探してみてください。背景を設定するには、この領域を使用します。 </li><li>イメージング・システムおよびソフトウェアのパラメータを設定します。設定範囲を17 nmまで。 （8.2を参照）、「自動露出」を適用し、空のフィールドから背景画像を得ます。一つの背景画像は、実験ごとに使用されます。 </li></ol>セルロースミクロフィブリル配向の9 Polscope分析<ol><li>縦断面の分析。 <ol><li>顕微鏡下で長手方向部分を含むスライドを置き、それのリター画像を得ます。関心の根元部に顕微鏡の焦点を合わせます。新しいファイル名を指定します。 </li><li> 「ライブAbrio &quot;窓からabrioの画像（位相差情報と画像）をキャプチャします。 図2Cに示すように、セルの周囲に沿って実行して染色されたセルロースによって識別することができ、細胞壁を含み、セクションをキャプチャするようにしてください。</li></ol></li><li>画像解析。 <ol><li>画像スタック内の関連する画像をダブルクリックします。 「表示設定」タブで「オリエンテーション擬似カラー」を選択します。 </li><li>セルロースミクロフィブリル角度を推定するには、カラーホイールの場合と細胞壁の対応する陰に一致します。 注： 図2Cにおけるカラーホイールは、ミクロフィブリルの長軸に沿った方向を示しているミクロフィブリルに沿って存在する光の遅相軸を反映しています。セルロースミクロフィブリル角度、色、セルの長軸により示される角度です。たとえば、インセットで伸長細胞は、細胞の長軸に対して約90°傾いたシアン色を示します。 </li><li>細胞壁の平均ミクロフィブリル傾角を定量化するために、ソフトウェアのツールバーから「燃性および方位角スタンプ」ツールを使用します。ツール、ポイントを選択し、一方で得られた画像中の関心領域をクリックしてください。 注：このウィル度の角度（方位）（ 図2C）の表示におけるリットル結果。 </li><li>プラグインメニューから代替オープンアクセス偏光画像解析ソフトウェアでは、ランチ「ポル・アナライザ」プラグイン。新しい開いたウィンドウで、「複屈折」タブを開いてを選択し、背景ファイルと画像ファイルを保存します。 </li><li>画像ウィンドウでは、「仕様」を選択します。そして、「ROIのピクセルサイズを「（この研究のために5ピクセルを使用）を調整。 &quot;仕様&quot;でウィンドウを選択し、「結果表内の値を示して」。 </li><li>画像ウィンドウ内の関心領域をマークするために、多角形ツールを使用してください。セルロースミクロフィブリルの配向を得るために、「オリエンテーションライン」を選択します。 注：これは度の角度（方位）の表示になります。 注：2つの方法のいずれかにより算出された平均角度は、その後、スライドに沿って根元部の位置に正規化する必要があります。からのずれを修正するにはスライドに沿って全体の根元部の水平位置、（セクションへと根の成長軸、 図2Cに沿って垂直つまり、）その長軸に沿ってセルに隣接するセル壁の平均ミクロフィブリル傾角を定量化します。根元部が正確スライドに沿って配置されている場合、この角度は180°です。ミクロフィブリル角度を計算するために、180°から隣接するセル壁の平均測定ミクロフィブリル角度を引きます。スライド上に平らに細胞壁の平均測定ミクロフィブリル角度になった違いを追加します。 </li></ol></li></ol>結晶セルロース蓄積の10 Polscope分析<ol><li>横（クロス）セクションを使用してください。彼らのセルロースミクロフィブリルは（9で決定されるように）同様の角度で整列しているときに、結晶セルロース蓄積が細胞のみの間で比較することができることに注意してください。 </li><li>画像スタック内の関連する画像をダブルクリックします。 P」を選択表示設定」タブ」の「seudo色。 </li><li>燃性を測定しました。ズームアップ画像に。 「リージョン」を選択し、関心のあるすべての領域をマークします。すべての値を「クリップボードにコピー」「測定」タブに移動します。 <ol><li> Excelにデータを転送し、（リターダンスの領域を意味する）必要な情報を抽出します。データを正規化します。 注：スライス間の厚さの違いを考慮するために、スライド内の特定の細胞壁のエッジに対する相対値を表します。標準化するために、同じ画像内の異なる細胞壁の平均リターダンスによって目的の細胞壁に対応する平均燃性を分けます。例えば、外側の表皮細胞壁は、私たちの研究では、内側皮質細胞壁に正規化しました。 </li></ol></li><li>サンプルあたり3根を最小限に抑えて、ルートごとに少なくとも3つのセクションのための測定を繰り返します。 </li></ol>

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The authors have no conflicts of interest.

解剖学的情報を維持しながら、ここでは、 シロイヌナズナの根を構成する異なる組織における結晶セルロースの蓄積を決意するための方法を提示します。このように、それは、細胞の解像度で、植物の成長過程を理解することに向けた追加のステップを提供します。この方法はまた、付加的な植物種および器官の研究に適用することができます。 この方法を適用?...

植物細胞壁は動的な構造です。増殖している細胞は、一次細胞壁、細胞を拡大することを可能にする組織で囲まれています。堆積物を植物の機械的支持を強化し、より剛性の二次壁を成長させることやめる細胞。両方の細胞壁は、異なる発達段階や組織1,2間で異なる別の構造（例えば 、ヘミセルロースおよびペクチン）の多糖類のマトリックス中に埋め込 ​​まれたセルロースミクロフィブリルで構成されています。セルロースはしっかり結晶構造のミクロフィブリルを形成するように整列されている（1,4）-β-D-グルカンの鎖として合成されます。アモルファスセルロースはグルカン鎖が少ない順序付けられている領域を意味します。結晶と非晶質領域との間の比が3それぞれ、機械的強度及び粘弾性特性を提供することにより、細胞壁の機械的特性に影響を与えると考えられて一つのパラメータです。いくつかの方法がありましたX線回折及び交差分極/固体NMR 4を回転マジック角を検出し、それらのうち、セルロース配置の二つの形態を定量化するために開発されました。 X線回折は、試料5中の非晶質セルロースドメインに対する結晶の割合を決定するために用いることができます。別の方法は、それぞれ、結晶質および非晶質セルロースまたは他のポリマーとの間で区別するために、酸不溶および酸可溶性物質に細胞壁コンテンツの分画を使用しています。このアプローチでは、標識グルコース（[14 C]グルコース）の組み込みは、セルロース6,7を定量化するために使用されます。これらの方法は、せいぜい、全臓器分析のための植物材料を大量に必要とし、従って、細胞壁構造における組織特異的な変化に対して不十分敏感です。セルラー解像度でセルロースミクロフィブリルの可視化の変化を同定することができる蛍光色素8,9、と組み合わせるライブイメージング研究で達成することができますセルロースミクロフィブリルの向き。これらの色素を定量化するために使用されていないが、それらは結晶セルロースに特異的ではなく、細胞壁8の正常な構造を妨害し得ます。 Polscopeは、光ビームを分割し、光10の一部を遅延させる結晶セルロースの能力に依存イメージング技術です。光の位相差は、光の伝播方向に垂直に横たわるミクロフィブリルのために最強です。同様の配向のミクロフィブリルのために、光のリターダンス11より大きい、結晶化度が高いです。したがって、polscope、セルロースミクロフィブリルの相対的なレベルと方向の両方を研究するために使用されます。 彼らは急速に12を展開する前に、根は、細胞分裂のシリーズを受け、根の先端に、細胞は、幹細胞ニッチに由来その間線形成長を示します。ルートを含む細胞は、単方向（異方性）に拡大します方法、セル壁13の特性に影響を与える小分子シグナル伝達ホルモンによって指示されます。ホルモンに対する微分応答は、時間と空間に、バランスのとれた臓器成長14を確保する手段を提供します。したがって、細胞壁構造の高分解能の分析は、より良い全体の臓器の成長に細胞型特異的応答との間の接続を理解するために必要な重要な情報を提供することができます。ここで、我々は高品質の解剖学のセクションで観察されるように、 シロイヌナズナの根に結晶セルロースの組織特異的な蓄積を研究するpolscopeの実装を報告しています。この方法は、最近、ホルモン活性15の空間的な摂動に応答して、結晶セルロースの細胞型特異的蓄積を明らかにしました。

<table>
	<tbody>
		<tr>
			<td>Murashige &#38; Skoog (MS)</td>
			<td>Duchefa</td>
			<td>M0221</td>
			<td></td>
		</tr>
		<tr>
			<td>Borax</td>
			<td>LOBA CHEMIE&#160;</td>
			<td>6038</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene blue</td>
			<td>Sigma</td>
			<td>M-9140</td>
			<td></td>
		</tr>
		<tr>
			<td>Azure</td>
			<td>Sigma</td>
			<td>861065</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Driven 0.22mm PVDF Filter&#160;</td>
			<td>MILLEX-GV</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>EMS</td>
			<td>16220</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Cacodylate</td>
			<td>Sigma</td>
			<td>C-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica kit historesin&#160;</td>
			<td>Leica</td>
			<td>7022 18 500</td>
			<td></td>
		</tr>
		<tr>
			<td>embedding molds</td>
			<td>Agar Scientific</td>
			<td>AGG3530</td>
			<td></td>
		</tr>
		<tr>
			<td>film 100&#181; P.P.C.&#160;</td>
			<td>www.Jolybar.com</td>
			<td></td>
			<td>overhead film</td>
		</tr>
		<tr>
			<td>Knivemaker</td>
			<td>LKB</td>
			<td>7800</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultratome III</td>
			<td>LKB</td>
			<td>8800</td>
			<td>Ultratome</td>
		</tr>
		<tr>
			<td>Shandon immumount&#160;</td>
			<td>Thermo</td>
			<td>9990402</td>
			<td>mounting medium</td>
		</tr>
		<tr>
			<td>light microscope</td>
			<td>Nikon</td>
			<td>Eclipse 80i</td>
			<td></td>
		</tr>
		<tr>
			<td>Abrio imaging system</td>
			<td>CRI</td>
			<td>Abrio imaging system</td>
			<td></td>
		</tr>
		<tr>
			<td>Abrio V2.2 software</td>
			<td>CRI</td>
			<td>Abrio V2.2 software</td>
			<td></td>
		</tr>
		<tr>
			<td>Open access polarized light image analysis software &#160;</td>
			<td>OPS</td>
			<td>OpenPolScope</td>
			<td><a href="http://www.openpolscope.org/">http://www.openpolscope.org/</a></td>
		</tr>
	</tbody>
</table>

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.

私たちは、モデルの臓器15-17としてシロイヌナズナの根を使用して、ブラシノステロイド（のBR）への細胞型特異的応答の影響を研究しています。 BR受容体BRI1を対象としているときに、BRI1変異体のバックグラウンドで、非有毛細胞（ 図2A、B）と呼ばれる表皮細胞のサブセットには、隣接セルの一方向性の細胞増殖を阻害し、そして?...

Watch this Scientific Journal Video about High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications at JoVE.com

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-resolution-quantification-crystalline-cellulose-accumulation

Research

JoVE Journal

Biology

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

8.1K ビュー.  Technion-Israel Institute of Technology. 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.

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

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

成長円錐の高分解能イメージングのためのアメフラシから神経細胞の培養

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

生物学

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

日立F - 7000蛍光分光光度計とPicoGreen色素を使用してdsDNAの定量化

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

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 ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

の長期、高分解能共焦点タイムラプスイメージング<em&gt;シロイヌナズナ子葉</em発芽過程&gt;表皮

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Using Flatbed Scanners to Collect High-resolution Time-lapsed Images of the Arabidopsis Root Gravitropic Response

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories can face is the development and attainment of these methods. Observation of phenotypes in a process of interest is a typical objective of research labs studying gene function and this is often achieved through image capture. A particular process that is amenable to observation using imaging approaches is the corrective growth of a seedling root that has been displaced from alignment with the gravity vector. Imaging platforms used to measure the root gravitropic response can be expensive, relatively low in throughput, and/or labor intensive. These issues have been addressed by developing a high-throughput image capture method using inexpensive, yet high-resolution, flatbed scanners. Using this method, images can be captured every few minutes at 4,800 dpi. The current setup enables collection of 216 individual responses per day. The image data collected is of ample quality for image analysis applications.

This protocol describes a process for rapid collection of images of Arabidopsis seedlings responding to a gravity stimulus using commercially-available flatbed scanners. The method allows for inexpensive, high-volume capture of high-resolution images amenable for downstream analysis algorithms.

シロイヌナズナ根重力屈性反応の高分解能時間経過画像を収集するためにフラットベッドスキャナを使用して

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories ...

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 ...

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. A detailed analysis of IRF3 activation is essential to understand how pathogens induce or evade the innate antiviral response. Distinct activated forms of IRF3 can be distinguished based on their phosphorylation and monomer vs dimer status. In vivo discrimination between the different activated species of IRF3 can be achieved through the separation of IRF3 phosphorylated forms based on their mobility shifts on SDS-PAGE. Additionally, the levels of IRF3 monomer and dimer can be monitored using non-denaturing electrophoresis. Here, we detail a procedure to reach the highest resolution to gain the most information regarding IRF3 activation status. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple total and phosphospecific antibodies. This experimental strategy constitutes an affordable and sensitive approach to acquire all the necessary information for a complete analysis of the phosphorylation-mediated activation of IRF3.

Here we describe a procedure allowing a detailed analysis of the phosphorylation-dependent activation of the IRF3 transcription factor. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple phosphospecific antibodies.

IRF3のリン酸化依存性活性化を監視するために、高解像度の方法

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. ...

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 ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

透過処理と無傷の細胞でミトコンドリア機能を評価するために高解像度呼吸計測

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

VirusMapperと超解像顕微鏡のためのオープンソースの単一粒子分析

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

結晶セルロース蓄積中の高解像度の定量化

要約

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