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. crescita delle piante <ol><li> Superficie sterilizzare i semi. <ol><li> Sterilizzare semi (fino a 30 mg) con il metodo a umido oa secco. Ad esempio, per il metodo di sterilizzazione a secco, aggiungere 1 ml glaciale HCl 37% in 50 ml di candeggina, in un bicchiere di vetro in un essiccatore chiuso per almeno 4 ore e fino a tutta la notte. Eseguire questo passaggio in una cappa chimica. </li></ol></li><li> Diffondere i semi sterili su piastre di agar con impianto di 0,8% a 0.5x Murashige &amp; Skoog (MS). Avvolgere l...

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

Qui, presentiamo un metodo per determinare l&#39;accumulo di cellulosa cristallina nei diversi tessuti che compongono le radici di Arabidopsis, mantenendo informazioni anatomiche. Come tale, fornisce un ulteriore passo verso la comprensione processi di crescita nelle piante con una risoluzione cellulare. Questo metodo può essere applicato anche per lo studio di specie vegetali aggiuntivi e organi. Un certo numero di punti deve essere considerato quando si applica il metodo. Prima a...

La parete cellulare vegetale è una struttura dinamica. cellule in crescita sono circondate da una parete cellulare primaria, la cui organizzazione permette alle cellule di espandersi. Cellule che cessano di crescere deposito una parete secondaria più rigida che migliora il supporto meccanico della pianta. Entrambe le pareti cellulari sono composti microfibrille di cellulosa annegate in una matrice di polisaccaridi di diverse strutture (ad esempio, emicellulosa e pectina) che variano di tutti i diversi stadi di sviluppo e tessuti 1,2. Cellulosa viene sintetizzato come catene di (1,4) -β-D-glucano che sono strettamente allineati a formare la microfibril di una struttura cristallina. cellulosa amorfo si riferisce alle regioni in cui le catene glucano sono meno ordinate. Il rapporto tra il cristallino ei domini amorfi è un parametro pensato per influenzare le proprietà meccaniche della parete cellulare, fornendo resistenza meccanica e caratteristico viscoelastico rispettivamente 3. Diversi metodi sono statisviluppato per rilevare e quantificare le due forme di disposizione di cellulosa, tra i quali la diffrazione a raggi X e polarizzazione incrociata / angolo magic filatura NMR allo stato solido 4. Diffrazione di raggi X può essere utilizzato per determinare la percentuale di cristallina rispetto domini cellulosa amorfi nel campione 5. Un metodo alternativo utilizza frazionamento di contenuti parete cellulare in materiale acido insolubile e acido solubile, per distinguere tra il cristallino e cellulosa amorfa o altri polimeri, rispettivamente. In questo approccio, incorporazione di glucosio marcato ([14 C] glucosio) viene utilizzato per quantificare il 6,7 cellulosa. Questi metodi richiedono grandi quantità di materiale vegetale per le analisi di organi interi, nella migliore delle ipotesi, e, di conseguenza, sono adeguatamente sensibili a variazioni tessuto-specifica nella struttura della parete cellulare. Visualizzazione microfibrille di cellulosa ad una risoluzione cellulare può essere ottenuto in studi di imaging vivi combinati con coloranti fluorescenti 8,9, che può identificare variazionil&#39;orientamento delle microfibrille di cellulosa. Tuttavia, questi coloranti non sono utilizzati per la quantificazione, non sono specifici a cristallino cellulosa e possono interferire con la normale struttura della parete cellulare 8. PolScope è una tecnica di imaging che si basa sulla capacità di cellulosa cristallina dividere fasci di luce e ritardare parte della luce 10. ritardo luce è più forte per microfibrille che giacciono perpendicolarmente alla direzione di propagazione della luce. Per microfibrille con orientamento simile, maggiore è il grado di cristallinità, maggiore è il ritardo di luce 11. Quindi, PolScope viene usato per studiare sia i livelli relativi e l&#39;orientamento delle microfibrille di cellulosa. Radici presentano crescita lineare, durante il quale le cellule originari alla nicchia di cellule staminali, sulla punta della radice, subire una serie di divisioni cellulari, prima che rapidamente si espandono 12. Le cellule che compongono la radice si espandono in un unidirezionale (anisotropico)maniera, come dettato dalle piccole ormoni molecola di segnalazione che hanno un impatto le proprietà della parete cellulare 13. Risposte differenziale ormoni, nel tempo e nello spazio, forniscono un mezzo per assicurare una crescita equilibrata organo 14. Quindi, l&#39;analisi ad alta risoluzione di struttura della parete cellulare in grado di fornire importanti informazioni necessarie per comprendere meglio la connessione tra le risposte specifiche per tipo di cellule per la crescita dell&#39;intero organo. Qui, si segnala la realizzazione di PolScope per studiare l&#39;accumulo di tessuto-specifica di cellulosa cristallina nelle radici di Arabidopsis, come osservato nelle sezioni anatomiche di alta qualità. Questo metodo ha recentemente scoperto l&#39;accumulo specifico tipo di cellule di cellulosa cristallina in risposta alla perturbazione spaziale delle attività ormonale 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.

Studiamo l&#39;impatto delle risposte specifiche per tipo di cellula brassinosteroids (BRS), usando la radice Arabidopsis come modello organo 15-17. Quando il recettore BRI1 BR si rivolge, sullo sfondo di BRI1 mutante, a un sottoinsieme di cellule epidermiche chiamate cellule non-capelli (Figura 2A, B), inibisce l&#39;espansione delle cellule unidirezionale di cellule vicine, e la crescita integrale della radice 15. analisi PolScop...

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.

Risoluzione Quantificazione alta del cristallino cellulosa accumulo in<em&gt; Arabidopsis</em&gt; Roots a Monitor tessuto-specifica delle cellule parete Modifiche

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.

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JoVE Journal

Biology

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

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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 ...

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

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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

Ad alta risoluzione Imaging e analisi dei singoli microtubuli astrali Dynamics in germogliamento lievito

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

Combinazione di sincronizzazione cellulare mitotica e microscopia confocale ad alta risoluzione per studiare il ruolo delle proteine del ciclo cellulare multifunzione durante la mitosi

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 ...