Estamos agradecidos a Sabin Russell para asistencia en la edición. Este trabajo formó parte de la Joint BioEnergy Institute DOE (http://www.jbei.org) apoyado por el Departamento de Energía de los EE.UU., Oficina de Ciencia, Oficina de Investigación Biológica y Ambiental, a través del contrato DE-AC02-05CH11231 entre Lawrence Berkeley Laboratorio Nacional y el Departamento de Energía de EE.UU..

<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.

Secciones de tallo de A. thaliana se utilizan ampliamente para estudiar la organización de las células en la pared celular secundaria y para examinar cualitativamente las diferencias entre el tipo salvaje y las plantas transgénicas. Las técnicas utilizadas para el corte de las muestras son de corte directo de las manos; o cuando las muestras están incrustados en agarosa o fijador, seccionamiento se puede hacer con un vibratome o microtomo. En contraste con el corte manual, los dos últimos permiten reducir...

La pared celular vegetal tiene una gran cantidad de información dentro de sus diversos componentes: lignina, celulosa, hemicelulosa (xilano, glucuronoxilano, xiloglucano, arabinoxilano, vinculación mixta de glucano, o glucomanano), y pectina. Técnicas histológicas proporcionan importantes pistas visuales en el estudio de las diferencias dentro de las paredes celulares secundarias en los niveles de organización y celulares. Diversas técnicas histológicas fueron desarrollados y se pueden encontrar en la literatura, pero estas técnicas puede ser difícil y requiere mucho tiempo para los principiantes debido a protocolos muy detalladas con instrucciones visuales simples son rara vez disponible. El objetivo de este estudio es proporcionar pautas sencillas para las técnicas de tinción histológica para obtener imágenes de alta calidad. Seccionar el tejido del tallo es el primer paso en la visualización de las paredes celulares y formas celulares. Aunque las secciones cortadas a mano son baratos y requieren menos tiempo para prepararse, el uso de un vibratome ofrece consistencia yproduce imágenes de alta calidad. El uso de un vibratomo permite producir mejor calidad de los datos mediante la generación de incluso secciones con el mismo espesor, lo que ayuda a producir imágenes nítidas y reduce significativamente el riesgo de producir diferencias inexactas entre las muestras que se causada simplemente por una mala preparación de la muestra. El uso de resinas para fijar especímenes frescos puede ser un desafío para los principiantes y todavía puede llevar mucho tiempo, incluso para los expertos en el análisis que debe hacerse rápidamente. Además, se hace imposible para medir cualquier actividad biológica con la muestra cuando se ha incorporado en una resina. Una técnica simple que emplea agarosa y un molde casero es útil para la incrustación de tejidos del tallo, y también se puede utilizar en otras aplicaciones que requieren la disección de los tejidos blandos. En comparación con la incrustación de especímenes en la resina, este método tiene la ventaja de mantener los tejidos vivos y la reducción de la manipulación de la muestra. Seccionamiento tejidos a través de un vibratomo es altamente preciso y genera homogensecciones unidades organizativas, que, dependiendo del objetivo del estudio, a continuación, se pueden utilizar con varias técnicas de tinción diferentes. Los métodos más sencillos para visualizar la lignina y otros compuestos aromáticos emplean radiación ultravioleta (UV). La excitación de las moléculas de base de compuestos aromáticos por la luz UV es una técnica antigua, pero sigue siendo uno de los métodos más rápidos para la visualización de la lignina. Sin embargo, la visualización UV en realidad no es ideal para la detección de la lignina, porque la luz UV excitará otros compuestos aromáticos. La lignina se compone principalmente de tres componentes básicos, los monolignoles (alcoholes hidroxicinamil: alcohol coniferil, alcohol sinapílico y alcohol p-cumaril) 1-3. Mancha Phloroglucinol puede proporcionar pistas sobre la magnitud de cinnamaldehydes presentes en el xilema, fibras y tejidos traqueales 4. Floroglucinol es un buen indicador de cinnamaldehydes generales y puede diferenciar entre cinnamaldehydes y otros compuestos aromáticos. Los monómeros de alcohol sinapílico se pueden detectary diferenciados por el uso de Maule mancha. Azul de toluidina O es un colorante policromático y por lo tanto tiene la capacidad de manchar diferentes elementos de la pared celular en diferentes colores 5,6. El principal uso de azul de toluidina O es detectar pectina y 5,6 lignina. La ventaja de la utilización de azul de toluidina O es que muchos elementos de la pared celular pueden ser visualizados en un solo paso. Tanto calcoflúor blanco y rojo congo son fáciles de trabajar con y se pueden utilizar para visualizar celulosa. Calcofluor manchas blancas de celulosa, callosa, y otros β-glucanos, no sustituidos o sustituidos débilmente 6-9, mientras que el congo manchas rojas directamente a β-glucanos (1 → 4) y, en particular y celulosa 10,11. El objetivo de este estudio es proporcionar pautas sencillas para el uso de las técnicas de tinción antes mencionados para obtener imágenes de alta calidad a partir de A. thaliana tallos.

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			<td>&#160;Agarose</td>
			<td>EMD</td>
			<td>MERC2125</td>
			<td>CAS Number: 9012-36-6</td>
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		<tr>
			<td>Phloroglucinol</td>
			<td>Sigma</td>
			<td>P 3502</td>
			<td>1,3,5-trihydroxybenzene [CAS Number: 108-73-6]</td>
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		<tr>
			<td>Hydrochloric Acid</td>
			<td>EMD</td>
			<td>HX0603-75</td>
			<td>CAS Number: 7647-01-0</td>
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			<td>Ammonium hydroxide</td>
			<td>EMD</td>
			<td>AX1303-6</td>
			<td>CAS Number: 1336-21-6</td>
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			<td>Toulidine Blue O</td>
			<td>Sigma</td>
			<td>T3260</td>
			<td>Blutene chloride, Tolonium Chloride [CAS Number 92-31-9]&#160;</td>
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			<td>Potassium permanganate</td>
			<td>Sigma</td>
			<td>223468</td>
			<td>CAS Number 7722-64-7&#160;</td>
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			<td>Ethanol 190 proof</td>
			<td>KOPTEC</td>
			<td>V1401</td>
			<td>CAS Number: 64-17-5</td>
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			<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>
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			<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>
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			<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>
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			<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>
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			<td>VWR</td>
			<td>16466-044</td>
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			<td>Axygen Scientific</td>
			<td>MCT-060-C</td>
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			<td>Mitt</td>
			<td>Bel-Art</td>
			<td>380000000</td>
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			<td>10033</td>
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			<td>Microwave</td>
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			<td>NN-SD762S</td>
			<td>PELCO Pro CA 44 Instant tissue adhesive&#160;</td>
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			<td>Camera with CCD chip with no mechanical shutter&#160;</td>
			<td>Hamamatsu</td>
			<td>C4742-95</td>
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			<td>High speed color camera&#160;&#160;&#160;</td>
			<td>QImaging</td>
			<td>MicroPublisher 5.0 RTV</td>
			<td></td>
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			<td>Camera software&#160;</td>
			<td>&#160;Molecular Devices</td>
			<td>MetaMorph version 7.7.0.0</td>
			<td></td>
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			<td>Imagining anaylsis&#160;</td>
			<td>Adobe&#160;</td>
			<td>Photoshop CS4</td>
			<td></td>
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			<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>
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			<td>Frosted Micro Slides, 1mm</td>
			<td>VWR</td>
			<td>48312-003</td>
			<td>75 x 25 mm- 1mm</td>
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			<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>
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			<td>Parafilm M</td>
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			<td>BRNDPM998</td>
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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.

Stem inclusión y corte: El uso del molde de plástico hecha en casa para incrustar los tallos en 7% de agarosa demostrado ser rápido y fácil (Figura 1). Las dos partes (A y B; Figura 1) del sistema vial incrustado hacen que sea sencillo para liberar fácilmente el vástago incrustado en agarosa, tal como la agarosa no se pegue a las partes de los viales que son inertes, mantener el sistema limpio. Los viales se pueden reutilizar varias veces por año. La conveniencia de almace...

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.

La tinción histoquímica de<em&gt; Arabidopsis thaliana</em&gt; Celda Secundaria Elements Pared

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

40.1K vistas.  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.

Video: 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.

El aislamiento de protoplastos a partir de tejidos de 14 días de edad de las plántulas Arabidopsis thaliana</em

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

Investigación

Biología

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.

Floral dip transformación de la Arabidopsis thaliana Para el Examen de PTSO2:: β-glucuronidasa Reporter Expresión Génica

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.

Aislamiento eficaz y rápida de los embriones en etapa temprana de<em&gt; Arabidopsis thaliana</em&gt; Semillas

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.

Metabólicas etiquetado y de membrana de fraccionamiento para Comparativo Proteómica Análisis de<em&gt; Arabidopsis thaliana</em&gt; Culturas suspensión celular

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.

Alta resolución Cuantificación de celulosa cristalina acumulación en<em&gt; Arabidopsis</em&gt; Las raíces para monitorear la célula específica de tejido de pared Modificaciones

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.

Análisis del comportamiento de crecimiento de Arabidopsis thaliana en diferentes calidades de luz

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

Investigación espacio-temporal de la dinámica de ERL1 mediante imágenes confocales

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.

Autor destacado: Métodos basados en imágenes para estudiar los eventos de tráfico de membrana en células de linaje estomática  

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.

Métodos basados en imágenes para estudiar los eventos de tráfico de membrana en células de linaje estomática

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

Un método mejorado para medir los perfiles de hidratación del polen en Arabidopsis thaliana

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.

Autor destacado: Un bioensayo de hidratación de polen in vivo de alta resolución, de un solo grano para Arabidopsis thaliana  

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.

Un bioensayo de hidratación de polen in vivo de grano único de alta resolución para Arabidopsis thaliana

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