我々は編集支援のためのセービンラッセルに感謝しています。この作品は、ローレンス·バークレーとの間の契約DE-AC02-05CH11231を通じてエネルギー省合同バイオエナジー研究所（http://www.jbei.org）米国エネルギー省でサポートされている、科学局、生物環境研究局の一部であった国立研究所と米国エネルギー省。

<ol>
	<li>Boerjan, W., Ralph, J., Baucher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lignin+biosynthesis.">Lignin biosynthesis.</a> Annual review of plant biology. 54, 519-546 (2003).</li><li>Vanholme, R., Demedts, B., Morreel, K., Ralph, J., Boerjan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lignin+biosynthesis+and+structure.">Lignin biosynthesis and structure.</a> Plant physiology. 153, 895-905 (2010).</li><li>Humphreys, J. M., Chapple, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rewriting+the+lignin+roadmap.">Rewriting the lignin roadmap.</a> Current opinion in plant biology. 5, 224-229 (2002).</li><li>Adler, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lignin+chemistry&#8212;past,+present+and+future.">Lignin chemistry&#8212;past, present and future.</a> Wood Sci. Technol. 11, 169-218 (1977).</li><li>Brien, T. P., Feder, N., McCully, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polychromatic+Staining+of+Plant+Cell+Walls+by+Toluidine+Blue.">Polychromatic Staining of Plant Cell Walls by Toluidine Blue.</a> 59, 368-373 (1964).</li><li>Mori, B., Bellani, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+staining+for+cellulosic+and+modified+plant+cell+walls.">Differential staining for cellulosic and modified plant cell walls.</a> Biotechnic &amp; histochemistry: official publication of the Biological Stain Commission. 71, 71-72 (1996).</li><li>Maeda, H., Ishida, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+of+binding+of+hexopyranosyl+polysaccharides+with+fluorescent+brightener.">Specificity of binding of hexopyranosyl polysaccharides with fluorescent brightener.</a> Journal of biochemistry. 62, 276-278 (1967).</li><li>Wood, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+in+the+interaction+of+direct+dyes+with+polysaccharides.">Specificity in the interaction of direct dyes with polysaccharides.</a> Carbohydrate Research. 85, 271-287 (1980).</li><li>Hughes, J., McCully, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+an+optical+brightener+in+the+study+of+plant+structure.">The use of an optical brightener in the study of plant structure.</a> Stain technology. 50, 319-329 (1975).</li><li>Verbelen, J. P., Kerstens, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarization+confocal+microscopy+and+congo+red+fluorescence:+a+simple+and+rapid+method+to+determine+the+mean+cellulose+fibril+orientation+in+plants.">Polarization confocal microscopy and congo red fluorescence: a simple and rapid method to determine the mean cellulose fibril orientation in plants.</a> Journal of microscopy. 198, 101-107 (2000).</li><li>Anderson, C. T., Carroll, A., Akhmetova, L., Somerville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+cellulose+reorientation+during+cell+wall+expansion+in+Arabidopsis+roots.">Real-time imaging of cellulose reorientation during cell wall expansion in Arabidopsis roots.</a> Plant physiology. 152, 787-796 (2010).</li><li>Liljegren, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phloroglucinol+Stain+for+Lignin.">Phloroglucinol Stain for Lignin.</a> Cold Spring Harbor Protocols. , (2010).</li><li>Nakano, J., Meshitsuka, G., lin, S., Dence, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Detection of Lignin Methods in Lignin Chemistry. , (1992).</li><li>Sibout, R., 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=CINNAMYL+ALCOHOL+DEHYDROGENASE-C+and+-D+are+the+primary+genes+involved+in+lignin+biosynthesis+in+the+floral+stem+of+Arabidopsis.">CINNAMYL ALCOHOL DEHYDROGENASE-C and -D are the primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis.</a> The Plant cell. 17, 2059-2076 (2005).</li><li>Teather, R. M., Wood, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Congo+red-polysaccharide+interactions+in+enumeration+and+characterization+of+cellulolytic+bacteria+from+the+bovine+rumen.">Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen.</a> Applied and environmental microbiology. 43, 777-780 (1982).</li><li>Yeung, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+beginner's+guide+to+the+study+of+plant+structure.">A beginner's guide to the study of plant structure.</a> Proceedings of the 19th Workshop/Conference of the Association for Biology Laboratory Education (ABLE. 19, 365-36 (1998).</li><li>Turner, S. R., Somerville, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collapsed+xylem+phenotype+of+Arabidopsis+identifies+mutants+deficient+in+cellulose+deposition+in+the+secondary+cell+wall).">Collapsed xylem phenotype of Arabidopsis identifies mutants deficient in cellulose deposition in the secondary cell wall).</a> The Plant Cell Online. 9, 689-701 (1997).</li><li>Iiyama, K., Pant, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+the+M&#228;ule+colour+reaction.+Introduction+of+methylated+syringyl+nuclei+into+softwood+lignin.">The mechanism of the M&#228;ule colour reaction. Introduction of methylated syringyl nuclei into softwood lignin.</a> Wood Sci. Technol. 22, 167-175 (1988).</li><li>Yang, F., 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=Engineering+secondary+cell+wall+deposition+in+plants.">Engineering secondary cell wall deposition in plants.</a> Plant biotechnology journal. 11, 325-335 (2013).</li><li>Zhong, R., Ripperger, A., Ye, Z. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+deposition+of+lignin+in+the+pith+of+stems+of+two+Arabidopsis+mutants.">Ectopic deposition of lignin in the pith of stems of two Arabidopsis mutants.</a> Plant physiology. 123, 59-70 (2000).</li><li>Chapple, C. C., Vogt, T., Ellis, B. E., Somerville, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Arabidopsis+mutant+defective+in+the+general+phenylpropanoid+pathway.">An Arabidopsis mutant defective in the general phenylpropanoid pathway.</a> The Plant cell. 4, 1413-1424 (1992).</li><li>Fagard, 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=PROCUSTE1+Encodes+a+Cellulose+Synthase+Required+for+Normal+Cell+Elongation+Specifically+in+Roots+and+Dark-Grown+Hypocotyls+of+Arabidopsis.">PROCUSTE1 Encodes a Cellulose Synthase Required for Normal Cell Elongation Specifically in Roots and Dark-Grown Hypocotyls of Arabidopsis.</a> The Plant Cell Online. 12, 2409-2423 (2000).</li></ol>

The authors have no financial conflicts to disclose.

シロイヌナズナステム部が広く二次細胞壁中の細胞の組織化を研究し、定性的に、野生型およびトランスジェニック植物の間の差を調べるために使用される。標本を区画するために一般に使用される技術は、直接手で切っている。標本はアガロースまたは固定液に埋め込まれたとき、または、切片をビブラトームまたはミクロトームで行うことができます。手の切断とは対照的に、最...

リグニン、セルロース、ヘミセルロース（キシラン、グルクロノキシラン、キシログルカン、アラビノキシラン、混合連鎖グルカン、またはグルコマンナン）、およびペクチン：植物細胞壁は、その様々な構成要素内の情報の過多を保持している。組織学的技術は、組織および細胞レベルでの二次細胞壁の中の違いを研究する上で重要な視覚的な手がかりを提供する。様々な組織学的技術が開発されており、文献に記載されていますが、これまでに利用可能な場合、単純な視覚的な指示に非常に詳細なプロトコルはめったにないため、これらの技術は、初心者のために挑戦し、時間がかかる場合があります。本研究の目的は、高品質の画像を得るために組織学的染色技術のための簡単​​なガイドラインを提供することである。 茎組織を区画すると、細胞壁と細胞形状の可視化の最初のステップです。ハンドカットのセクションでは、安価であり、準備する時間がかからないが、ビブラトームを使用すると、一貫性を提供し、高品質の画像が得られる。ビブラトームを使用すると、シャープな画像を生成するのに役立ち、著しく単純悪い試料調製に起因するサンプル間の不正確な差を生成するリスクを低減し、同じ厚さであってもセクションを生成することにより、より良好なデータ品質を製造することができる。新鮮な標本を修正するために樹脂を使用すると、初心者のための挑戦することができますし、分析を迅速に行う必要があるときもまだ専門家のために、時間がかかる場合があります。また、それは、樹脂中に埋め込まれたときに試料に任意の生物学的活性を測定することができなくなる。アガロース及び自家製の金型を用いたOne簡単な手法は、幹組織を包埋するのに役立ち、それはまた、軟組織の切開を必要とする他の用途に利用することができる。樹脂中に包埋標本を比較すると、この方法は、生きている組織を維持し、サンプル操作を低減するという利点を有する。ビブラトームを経由して組織を切片化することは、非常に正確であるとhomogenを生成研究の目的に応じて、組織単位（OU）のセクションでは、次に、いくつかの異なる染色技術と共に使用することができる。 リグニンおよび他の芳香族化合物を可視化するための最も簡単な方法は、紫外線（UV）光を使用する。 UV光による芳香族系分子の励起は古い技術であるが、それはまだリグニンの可視化のための最速の方法の一つです。 UV光は、他の芳香族化合物を励起するので、UV可視化は、実際にはリグニンを検出するための理想的ではない。 1-3：リグニンは、主に以下の3つのビルディングブロック、モノリグノール（コニフェリルアルコール、シナピルアルコール、およびp-クマリルアルコールhydroxycinnamylアルコール）で構成されている。フロログルシノール染色は木部に存在シンナムアルデヒド、繊維、および気管組織4の範囲で手がかりを提供することができます。フロログルシノールは、一般的なシンナムアルデヒドの良好な指標であり、シンナムアルデヒド、その他の芳香族化合物を区別することができます。シナピルアルコールモノマーは検出することができるとマウレ染色を使用して分化した。トルイジンブルーO多色性染料であり、したがって異なる色5,6における細胞壁の異なる要素を染色する能力を有する。トルイジンブルーOの主な用途は、ペクチンとリグニン5,6を検出することである。トルイジンブルーOを使用する利点は、細胞壁の多くの要素は、単一のステップで可視化することができることである。カルコフロールホワイトおよびコンゴーレッドの両方を作成すると処理が容易であり、セルロースを可視化するために使用することができる。 ·ホワイト汚れセルロース、カロース、および他の非置換または弱置換されたβ-グルカン6-9、コンゴレッド汚れが直接、特に10,11-β（1→4） -グルカンとセルロースにするのに対し。この研究の目的は、A.より高品質の画像を得るために、前述の染色技術を使用するための簡単なガイドラインを提供することであるシロイヌナズナの茎 。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>&#160;Agarose</td>
			<td>EMD</td>
			<td>MERC2125</td>
			<td>CAS Number: 9012-36-6</td>
		</tr>
		<tr>
			<td>Phloroglucinol</td>
			<td>Sigma</td>
			<td>P 3502</td>
			<td>1,3,5-trihydroxybenzene [CAS Number: 108-73-6]</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>EMD</td>
			<td>HX0603-75</td>
			<td>CAS Number: 7647-01-0</td>
		</tr>
		<tr>
			<td>Ammonium hydroxide</td>
			<td>EMD</td>
			<td>AX1303-6</td>
			<td>CAS Number: 1336-21-6</td>
		</tr>
		<tr>
			<td>Toulidine Blue O</td>
			<td>Sigma</td>
			<td>T3260</td>
			<td>Blutene chloride, Tolonium Chloride [CAS Number 92-31-9]&#160;</td>
		</tr>
		<tr>
			<td>Potassium permanganate</td>
			<td>Sigma</td>
			<td>223468</td>
			<td>CAS Number 7722-64-7&#160;</td>
		</tr>
		<tr>
			<td>Ethanol 190 proof</td>
			<td>KOPTEC</td>
			<td>V1401</td>
			<td>CAS Number: 64-17-5</td>
		</tr>
		<tr>
			<td>Congo Red</td>
			<td>Sigma&#160;</td>
			<td>C6277</td>
			<td>Disodium 3,3&#39;-[[1,1&#39;-biphenyl]-4,4&#39;-diylbis(azo)]bis(4-aminonaphthalene 1-sulphonate) [CAS Number 573-58-0 ]</td>
		</tr>
		<tr>
			<td>Fluorescent Brightener 28/ Calcofluor White Stain</td>
			<td>Sigma</td>
			<td>F3543&#160;</td>
			<td>4,4&#39;-Bis[4-[bis(2-hydroxyethyl)amino]-6-anilino-1,3,5-triazin-2-yl]amino]stilbene-2,2&#39;-disulphonic acid [CAS Number 4404-43-7]&#160;</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>Leica Vibrating blade microtome VT1000S</td>
			<td>http://www.leicabiosystems.com/products/sectioning/vibrating-blade-microtomes/details/product/leica-vt1000-s/</td>
		</tr>
		<tr>
			<td>Razor</td>
			<td>American Safety razor company</td>
			<td>Item # 60-0139-0000&#160;</td>
			<td>Stainless Steel Double Edge Blade (Personna Super)</td>
		</tr>
		<tr>
			<td>Screw Cap Microcentrifuge Tubes (2ml)</td>
			<td>VWR</td>
			<td>16466-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes (0.6ml)</td>
			<td>Axygen Scientific</td>
			<td>MCT-060-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Mitt</td>
			<td>Bel-Art</td>
			<td>380000000</td>
			<td>SCIENCEWARE&#160; Hot Hand Protector Mitt</td>
		</tr>
		<tr>
			<td>Tissue adhesive&#160;</td>
			<td>Ted Pella Inc</td>
			<td>10033</td>
			<td>Store at 4&#176;C or 20&#176;C for 3 months or longer&#160; storage&#160;</td>
		</tr>
		<tr>
			<td>Microwave</td>
			<td>Panasonic</td>
			<td>NN-SD762S</td>
			<td>PELCO Pro CA 44 Instant tissue adhesive&#160;</td>
		</tr>
		<tr>
			<td>Camera with CCD chip with no mechanical shutter&#160;</td>
			<td>Hamamatsu</td>
			<td>C4742-95</td>
			<td></td>
		</tr>
		<tr>
			<td>High speed color camera&#160;&#160;&#160;</td>
			<td>QImaging</td>
			<td>MicroPublisher 5.0 RTV</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera software&#160;</td>
			<td>&#160;Molecular Devices</td>
			<td>MetaMorph version 7.7.0.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Imagining anaylsis&#160;</td>
			<td>Adobe&#160;</td>
			<td>Photoshop CS4</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Cover Glasses, Square, No.1</td>
			<td>VWR</td>
			<td>48366-067</td>
			<td>22 x 22 mm (7/8 x 7/8&#34;)-Cover glasses are corrosion-resistant and uniformly thick and flat. No. 1 thickness is 0.13 to 0.17mm.&#160;</td>
		</tr>
		<tr>
			<td>Frosted Micro Slides, 1mm</td>
			<td>VWR</td>
			<td>48312-003</td>
			<td>75 x 25 mm- 1mm</td>
		</tr>
		<tr>
			<td>TX2 Filter cube</td>
			<td>Leica</td>
			<td>11513851/11513885</td>
			<td>Filter used for Congo red analysis with a band-pass of 560/40.</td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Alcan packaging</td>
			<td>BRNDPM998</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

埋め込みと分割幹： 埋め込 ​​むための自家製のプラスチック金型の使用は、アガロース迅速かつ簡単に（ 図1）であることが判明した7％で生じる。二つの部分（AとB、 図1）組み込みバイアルシステムのは、それが簡単な、容易にクリーンなシステムを維持し、アガロースが不活性であるバイアル部分に付着しないようにアガロース中に埋め込 ​​まれた茎?...

Watch this Scientific Journal Video about Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements at JoVE.com

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 ...

histochemical-staining-arabidopsis-thaliana-secondary-cell-wall

Research

JoVE Journal

Biology

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

40.3K ビュー.  Joint Bioenergy Institute. 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.

ビデオ: Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

の14日齢の苗の組織からのプロトプラストの単離シロイヌナズナ</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

生物学

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&amp;#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

フローラルディップ変換のシロイヌナズナ調べに pTSO2：：β-グルクロニダーゼレポーター遺伝子発現

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

から初期段階の胚の効率的かつ迅速な単離<em&gt;シロイヌナズナ</em&gt;種子

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

比較プロテオーム解析のための代謝標識および膜分画<em&gt;シロイヌナズナ</em&gt;サスペンション細胞培養

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

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 ...

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light conditions, which are preferably variable in quantity and quality so that studies under different setups can be conducted. These requirements are met by climatic chambers featuring light emitting diodes (LED) lights, which can &#8211; in contrast to fluorescent lights &#8211; be set to different wavelengths. LEDs are energy conserving and emit virtually no heat even at light intensities, which often constitutes a problem with other light sources. The presented protocol provides a step-by-step guidance of how to program a climatic chamber equipped with variable LED lights as well as describing several approaches for in depth analysis of growth phenotypes. Depending on the experimental set-up various characteristics of the growing plants can be observed and analyzed. Here we describe how to determine fresh weight, leaf area, photosynthetic activity, and stomatal density. We demonstrate that in order to obtain reliable data and draw valid conclusions it is mandatory to use a sufficient number of individuals for statistical evaluation. Taking too few plants for this kind of analysis results in high statistical errors and consequently in less clear interpretations of the data.

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Here, we present a protocol for studying plant growth behavior and especially phenotypes in a reproducible manner. We show how to provide variable and at the same time stable light conditions. Proper analyses depend on sufficient sample numbers and valid statistical evaluations.

異なる光質におけるシロイヌナズナ成長挙動の解析

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light ...

共焦点イメージングによるERL1ダイナミクスの時空間的研究

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

著者スポットライト:気孔系統細胞における膜輸送イベントを研究するための画像ベースの方法  

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

気孔系譜細胞における膜輸送事象を研究するための画像ベースの手法

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...

シロイヌナズナにおける花粉の水和プロファイルを測定するための改良された方法

A High-Resolution, Single-Grain, In Vivo Pollen Hydration Bioassay for Arabidopsis thaliana

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is established between the interacting partners. Studies across a range of species have revealed that a series of molecular checkpoints regulate the pollen-stigma interaction to ensure that only compatible, generally intraspecific pollen is successful in effecting fertilization. In species that possess a 'dry stigma', such as the model plant Arabidopsis thaliana, the first post-pollination, prezygotic compatibility checkpoint is the establishment of pollen hydration. 
This phase of pollination is tightly regulated, whereby signals from the pollen grain elicit the release of water from the stigma, thus permitting pollen hydration. The ability to accurately measure and track pollen hydration over time is key to the design of experiments directed at understanding the regulation of this critical step in reproduction. Published protocols frequently utilize flowers that have been excised from the parent plant, maintained on liquid or solid media, and bulk pollinated. 
This paper describes a noninvasive, in vivo pollination bioassay that permits minute-by-minute hydration tracking of individual A. thaliana pollen grains at high resolution. The assay is highly reproducible, able to detect very subtle variations of pollen hydration profiles, and thus is suitable for the analysis of mutants that affect pathways regulating pollination. Although the protocol is lengthier than those described for bulk pollinations, the precision and reproducibility it provides, along with its in vivo nature, make it ideal for the detailed dissection of pollination phenotypes.

著者スポットライト:シロイヌナズナの高解像度、単一粒、In vivo花粉水和バイオアッセイ  

An improved method to measure pollen hydration profiles in Arabidopsis thaliana is described here. The new method offers higher resolution, is noninvasive, and is highly reproducible. The protocol represents a new tool for a finer dissection of the processes that regulate the early stages of pollination.

シロイヌナズナの高分解能単粒in in vivo花粉水和バイオアッセイ

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is ...