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. Crecimiento de las Plantas <ol><li> Superficie esterilizar semillas. <ol><li> Esterilizar semillas (hasta 30 mg) por el método húmedo o seco. Como ejemplo, para el método de esterilización en seco, añadir 1 ml glacial HCl 37% en 50 ml de lejía, en un vaso de vidrio en un desecador cerrado durante al menos 4 horas y hasta durante la noche. Realice este paso en una campana química. </li></ol></li><li> Esparcir las semillas estériles en placas con agar planta de 0,8% en 0.5x Murashige y Skoog (MS). Envolver la...

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A continuación, presentamos un método para la determinación de la acumulación de celulosa cristalina en los diferentes tejidos que componen las raíces de Arabidopsis, mientras que el mantenimiento de información anatómica. Como tal, proporciona un paso adicional hacia la comprensión de los procesos de crecimiento de las plantas con resolución celular. Este método también se puede aplicar para el estudio de especies y órganos de las plantas adicionales. Un número de punt...

La pared celular vegetal es una estructura dinámica. las células que crecen están rodeados por una pared celular primaria, la organización de la que permite que las células se expanden. Las células que dejan de crecer depósito una pared secundaria más rígido que mejora el soporte mecánico de la planta. Ambas paredes celulares están compuestas de microfibrillas de celulosa incrustadas en una matriz de polisacáridos de diferentes estructuras (por ejemplo, hemicelulosa y pectina) que varían en los diferentes tejidos etapa de desarrollo y 1,2. La celulosa se sintetiza como cadenas de (1,4) -β-D-glucano que están estrechamente alineadas para formar la microfibrillas de una estructura cristalina. celulosa amorfa se refiere a las regiones en las que las cadenas de glucano están menos ordenadas. La relación entre el cristalino y los dominios amorfos es un parámetro cree que afecta a las propiedades mecánicas de la pared celular, proporcionando resistencia mecánica y característica viscoelástica, respectivamente 3. Varios métodos han sidodesarrollado para detectar y cuantificar las dos formas de disposición de celulosa, entre ellos de difracción de rayos X y polarización cruzada / ángulo mágico de giro de RMN de estado sólido 4. Difracción de rayos X se puede usar para determinar la proporción de cristalino frente a dominios celulosa amorfa en la muestra 5. Un método alternativo utiliza fraccionamiento del contenido de la pared celular en el material ácido insoluble y soluble en ácido, para distinguir entre el cristalino y celulosa amorfa u otros polímeros, respectivamente. En este enfoque, la incorporación de glucosa marcada ([14 C] glucosa) se utiliza para cuantificar el 6,7 celulosa. Estos métodos requieren grandes volúmenes de material de la planta para los análisis de órganos enteros, en el mejor, y por lo tanto, son insuficientemente sensibles a la variación específica de tejido en la estructura de la pared celular. Visualización de microfibrillas de celulosa en una resolución celular se puede lograr en los estudios de imagen en vivo en combinación con colorantes fluorescentes 8,9, que puede identificar cambios enla orientación de las microfibrillas de celulosa. Sin embargo, estos tintes no son utilizados para la cuantificación, que no son específicos de celulosa cristalina y pueden interferir con la estructura normal de la pared celular 8. Polscope es una técnica que se basa en la capacidad de celulosa cristalina para dividir los rayos de luz y retardar parte de la luz 10. retraso de luz es más fuerte para microfibrillas que se encuentran perpendicularmente a la dirección de propagación de la luz. Para microfibrillas con orientación similar, cuanto mayor sea el grado de cristalinidad, mayor será el retardo de la luz 11. Por lo tanto, polscope se utiliza para estudiar tanto los niveles relativos y la orientación de las microfibrillas de celulosa. Roots exhiben crecimiento lineal, durante el cual las células se origina en el nicho de células madre, en la punta de la raíz, someterse a una serie de divisiones celulares, antes de que rápidamente se expanden 12. Las células que comprenden la raíz se expanden en un unidireccional (anisotrópica)forma, según lo dictado por pequeñas moléculas de señalización de hormonas que afectan a las propiedades de la pared celular 13. Respuestas diferenciales a las hormonas, en tiempo y espacio, proporcionan un medio para asegurar el crecimiento equilibrado de órganos 14. Por lo tanto, el análisis de alta resolución de la estructura de la pared celular puede proporcionar información importante necesaria para entender mejor la conexión entre las respuestas de tipo específico de células para el crecimiento de órganos conjunto. Aquí, se presenta la implementación de polscope para estudiar la acumulación específica de tejido de celulosa cristalina en las raíces de Arabidopsis, como se observa en las secciones anatómicas de alta calidad. Este método no cubierto recientemente específica del tipo celular acumulación de celulosa cristalina en respuesta a la perturbación espacial de la actividad hormonal 15.

<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.

Se estudia el impacto de las respuestas específicas del tipo de células a los brasinoesteroides (BRS), el uso de la raíz de Arabidopsis como modelo de órganos 15-17. Cuando el receptor BRI1 BR está dirigido, en el fondo de mutante bri1, a un subconjunto de células de la epidermis llamadas células no pelo (Figura 2A, B), que inhibe la expansión de células unidireccional de células vecinas, y el crecimiento conjunto de la raíz 1...

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.

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 ...

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

Las culturas de las células nerviosas de Aplysia para imágenes de alta resolución de los Conos de Crecimiento

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 ...

Investigación

Biología

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

La cuantificación de ADN de doble cadena con el Hitachi F-7000 espectrofotómetro de fluorescencia y Dye PicoGreen

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

Perfiles de Polímeros Glycan la pared celular vegetal utilizando microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

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

A largo plazo, de alta resolución de imagen confocal Time Lapse de<em&gt; Cotiledones Arabidopsis</em&gt; Epidermis durante la germinación

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

El uso de escáneres planos para recopilar de alta resolución de duración transcurrido Imágenes de la gravitrópica Respuesta Arabidopsis Root

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

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 ...

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

Una alta resolución Método de Seguimiento activación fosforilación dependiente de IRF3

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

De alto rendimiento CRISPR vector de la construcción y caracterización de las modificaciones del ADN mediante la generación de raíces peludas tomate

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

Imágenes de alta resolución y análisis de la dinámica de microtúbulos astrales individuales de levadura en ciernes

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

Combina la sincronización celular mitótica y microscopia Confocal de alta resolución para estudiar el papel de las proteínas multifuncional ciclo celular durante la Mitosis

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 ...