Name: high resolution quantification of crystalline cellulose accumulation in arabidopsis roots to monitor tissue-specific cell wall modifications
Uploaded: 2016-05-10T13:00:00
Description: 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

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?...

<ol>
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E., Marga, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disorganization+of+cortical+microtubules+stimulates+tangential+expansion+and+reduces+the+uniformity+of+cellulose+microfibril+alignment+among+cells+in+the+root+of+Arabidopsis.">Disorganization of cortical microtubules stimulates tangential expansion and reduces the uniformity of cellulose microfibril alignment among cells in the root of Arabidopsis.</a> Plant Physiol. 135, 2279-2290 (2004).</li><li>Iyer, K. R. K., Neelakantan, P., Radhakrishnan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Birefringence+of+native+cellulosic+fibers.+I.+Untreated+cotton+and+ramie.">Birefringence of native cellulosic fibers. I. Untreated cotton and ramie.</a> J. Polym. Sci. 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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Arabidopsis+root+development.">Control of Arabidopsis root development.</a> Annu Rev Plant Biol. 63, 563-590 (2012).</li><li>Vanstraelen, M., Benkova, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormonal+Interactions+in+the+Regulation+of+Plant+Development.">Hormonal Interactions in the Regulation of Plant Development.</a> Annu Rev Cell Dev Biol. , (2012).</li><li>Singh, A. P., Savaldi-Goldstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+control:+brassinosteroid+activity+gets+context.">Growth control: brassinosteroid activity gets context.</a> J Exp Bot. 66, 1123-1132 (2015).</li><li>Fridman, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root+growth+is+modulated+by+differential+hormonal+sensitivity+in+neighboring+cells.">Root growth is modulated by differential hormonal sensitivity in neighboring cells.</a> Genes Dev. 28, 912-920 (2014).</li><li>Vragovic, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translatome+analyses+capture+of+opposing+tissue-specific+brassinosteroid+signals+orchestrating+root+meristem+differentiation.">Translatome analyses capture of opposing tissue-specific brassinosteroid signals orchestrating root meristem differentiation.</a> Proc Natl Acad Sci U S A. 112, 923-928 (2015).</li><li>Hacham, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brassinosteroid+perception+in+the+epidermis+controls+root+meristem+size.">Brassinosteroid perception in the epidermis controls root meristem size.</a> Development. 138, 839-848 (2011).</li><li>Gu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+cellulose+synthase-associated+protein+required+for+cellulose+biosynthesis.">Identification of a cellulose synthase-associated protein required for cellulose biosynthesis.</a> Proc Natl Acad Sci U S A. 107, 12866-12871 (2010).</li><li>Fendrych, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmed+cell+death+controlled+by+ANAC033/SOMBRERO+determines+root+cap+organ+size+in+Arabidopsis.">Programmed cell death controlled by ANAC033/SOMBRERO determines root cap organ size in Arabidopsis.</a> Curr Biol. 24, 931-940 (2014).</li></ol>

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 ...

The overall goal of this anatomical cross section preparation is to quantify relative crystal and cellulose accumulation at the cellular resolution enabling direct comparisons between the different tissues in the primary Arabidopsis Root. This method can help answer key questions in the plant physiology and development field such as how cellular composition of a given cell type impinges on a whole organs growth. After sterilizing Arabidopsis seed according to the text protocol, spread the sterile seeds on plates containing 0.8%plant agar and 0.5XM medium.With parafilm wrap the plates and cover with aluminum foil. Store the plates at 4 degrees Celsius for one to four days to promote uniform germination. Then transfer the plates to a growth chamber suitable for growing Arabidopsis seedlings orienting the plates vertically to maintain root growth on top of the agar medium Prepare Richardson's solution according to the text protocol Filter through a syringe driven 0.22 micron PVD filter unit before use.Next under a chemical hood prepare 50 milliliters of fixative by combining 38 ml of 0.5M sodium cacodylate with 2.5 ml of glutaraldehyde. Then add 100 microliters of Richardson's solution to the fixation solution to generate the final fixation solution. Use a pre-marked plate, then place 2 to 3 ml of final fixation solution into the wells so that the solution will fully cover the seedlings.Using plastic forceps carefully transfer up to 10 seedlings to each well. Use parafilm to seal the plates before storing in the dark at 4 degrees Celsius for at least one night and up to one week. To dehydrate the fixed seedlings use forceps to pick up the plants by their an transfer them to the wells of new marked six well plates containing increasing concentrations of ethanol for the times listed here.Prepare infiltration medium in a small glass bottle by mixing the contents of one bag of Historesin Activator with 50ml of basic resin liquid. Insert a magnet and stir the solution on stir plate for 20 minutes. Fill the labeled wells of a six well plate with 2 to 3 ml of infiltration medium.Then when the seedlings are fully dehydrated use forceps to transfer them from the 95%ethanol solution to the infiltration medium ensuring that they are fully covered by the medium. Store the samples at 4 degrees Celsius for at least four days to ensure maximum penetration into the plant tissues. To prepare blocks place small labels pencil marked with the sample name inside each well embedding molds.Prepare embedding medium by adding 1ml of hardener to 15ml of infiltration medium. With about 200 microliters of embedding medium fill half of each well in the mold. And use an overhead piece of film to cover the mold with an extra 1ml on each side.Incubate the molds at room temperature for at least two hours to allow partial polymerization. Next, prepare an additional batch of embedding medium and keep it on nice to avoid polymerization while root tips are being arranged in the mold. Add embedding medium into a small plate and place one seedling in a medium.Under a dissecting scope use a scalpel to cut each root tip then very carefully position it in the mold about 2 to 3 mm away from the mold periphery either vertically for transfer sections or horizontally for longitudinal sections. After arranging the tissue use embedding medium to fill the mold completely, and cover with an overhead film. Then incubate at room temperature for a minimum of two hours To dry the blocks place them in a desiccator with dry silica gel and leave at room temperature overnight.Using a knife maker equipped with a diamond scoring tool prepare glass knives from glass rods, by cutting the glass road into a square and then cutting the square diagonally to produce two knives. Verify that both knives have sharp edges. Mount a block into a holder.Then with a single edge industrial blade remove the excess block material up to one mm from the root tip to obtain a pyramid shape. Section the shaped block into 2 to 3 micron width slices and transfer the slices to a marked glass slide. Then add water droplets on the slices.It is important to prepare sections that are uniform in width to minimize variability of analyzed data. Place the glass slides on the heat blocks set to low heat until the water evaporates. To prepare slides for viewing under a polarized microscope dilute Richardson's solution to 2%of the original solution and use about 1ml to cover the slices.Then place the samples on the heat block for 5 seconds and use distilled water to wash them. After drying the slides on the hot plate observe the slices under a dissecting microscope and use a marker to mark the back of a slide to indicate the position of the slices of interest. Add 100 microliters of mounting medium before covering with the cover slip.Image and analyze according to the text protocol. Shown here is a cross section of the Arabidopsis primary root with radial organization of its constituent cells and tissues including non-hair cells, hair cells and cortex. This confocal image shows BRI1-GFP expression in non-hair cells in a BRI1 mutant background which inhibits the unidirectional expansion of neighboring cells and whole root growth.As seen here longitudinal sections of the roots obtained from wild type and from lines expressing BRI1 in non-hair cells displayed a similar orientation of cellulose microfibrils with a high variability of angles present in meristematic cells as compared to cells in the transition zone. This enables comparison of relative accumulation of crystal in cellulose in the corresponding meristematic and elongating cells of the two genetic backgrounds as show here in cross sections and reveals a correlation between BRI1 expression in non-hair cells and high local accumulation of crystal in cellulose. Generally in the videos new to this method will struggle because it is challenging to obtain well oriented roots during the embedding procedure.Therefore several blocks should be prepared for sectioning. Once mastered, a single block section can be done in 2 to 3 hours. While attempting this procedure it is important to remember that the accumulation of crystal in cellulose is comparable among cells if they have similar orientation of its cellulose microfibrils, as determined by the longitudinal sections.After watching this video you should have a good understanding of how to fix Arabidopsis seedlings and how embed them in blocks and how to then prepare longitudinal and transverse root cross sections. You should also have a good insight into the data that can be obtained from using polarized light microscopy.

Research

JoVE Journal

Biology

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

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

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

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

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

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

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

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

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

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

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 Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

出芽酵母における高解像度画像と個人アストラル微小管動態の解析

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

有糸分裂の多機能の細胞周期蛋白質の役割を研究する有糸分裂細胞同期と高分解能共焦点顕微鏡を組み合わせた

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...