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. Plant Growth
<ol>
	<li>Surface sterilize seeds.
	<ol>
		<li>Sterilize seeds (up to 30 mg) by wet or dry method. As an example, for dry sterilization method, add 1 ml glacial HCl 37% in 50 ml bleach, in a glass beaker in a closed desiccator for at least 4 hr and up to overnight. Perform this step in a chemical hood.</li>
	</ol>
	</li>
	<li>Spread the sterile seeds on plates with 0.8% plant agar in 0.5x Murashige &#38; Skoog (MS) medium. Wrap plates with Parafilm or porous tape and cover with aluminum foil.</li>
	<li>...

<ol>
	<li>Cosgrove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+the+plant+cell+wall.">Growth of the plant cell wall.</a> Nat Rev Mol Cell Biol. 6, 850-861 (2005).</li><li>Wolf, S., Hematy, K., Hofte, H. <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+and+cell+wall+signaling+in+plants.">Growth control and cell wall signaling in plants.</a> Annu Rev Plant Biol. 63, 381-407 (2012).</li><li>Mazeau, K., Heux, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Dynamics+Simulations+of+Bulk+Native+Crystalline+and+Amorphous+Structures+of+Cellulose.">Molecular Dynamics Simulations of Bulk Native Crystalline and Amorphous Structures of Cellulose.</a> J. Phys. Chem. B. 107, 2394-2403 (2003).</li><li>Zhao, H., 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=Studying+cellulose+fiber+structure+by+SEM,+XRD,+NMR+and+acid+hydrolysis.">Studying cellulose fiber structure by SEM, XRD, NMR and acid hydrolysis.</a> Carbohydr Polym. 68, 235-241 (2007).</li><li>Fujita, 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=Cortical+microtubules+optimize+cell-wall+crystallinity+to+drive+unidirectional+growth+in+Arabidopsis.">Cortical microtubules optimize cell-wall crystallinity to drive unidirectional growth in Arabidopsis.</a> Plant J. 66, 915-928 (2011).</li><li>Peng, 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=Fractional+separation+and+structural+features+of+hemicelluloses+from+sweet+sorghum+leaves.">Fractional separation and structural features of hemicelluloses from sweet sorghum leaves.</a> Bioresources. 7, (2012).</li><li>Xu, S. L., Rahman, A., Baskin, T. I., Kieber, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+leucine-rich+repeat+receptor+kinases+mediate+signaling,+linking+cell+wall+biosynthesis+and+ACC+synthase+in+Arabidopsis.">Two leucine-rich repeat receptor kinases mediate signaling, linking cell wall biosynthesis and ACC synthase in Arabidopsis.</a> Plant Cell. 20, 3065-3079 (2008).</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 Physiol. 152, 787-796 (2010).</li><li>Baskin, T. I., Beemster, G. T., Judy-March, J. E., Marga, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disorganization+of+cortical+microtubules+stimulates+tangential+expansion+and+reduces+the+uniformity+of+cellulose+microfibril+alignment+among+cells+in+the+root+of+Arabidopsis.">Disorganization of cortical microtubules stimulates tangential expansion and reduces the uniformity of cellulose microfibril alignment among cells in the root of Arabidopsis.</a> Plant Physiol. 135, 2279-2290 (2004).</li><li>Iyer, K. R. K., Neelakantan, P., Radhakrishnan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Birefringence+of+native+cellulosic+fibers.+I.+Untreated+cotton+and+ramie.">Birefringence of native cellulosic fibers. I. Untreated cotton and ramie.</a> J. Polym. Sci. A-2: Polymer Physics. 6, 1747-1758 (1968).</li><li>Abraham, Y., Elbaum, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+microfibril+angle+in+secondary+cell+walls+at+subcellular+resolution+by+means+of+polarized+light+microscopy.">Quantification of microfibril angle in secondary cell walls at subcellular resolution by means of polarized light microscopy.</a> New Phytol. 197, 1012-1019 (2013).</li><li>Petricka, J. J., Winter, C. M., Benfey, P. 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>

Here, we present a method for determination of the accumulation of crystalline cellulose in the different tissues composing the Arabidopsis roots, while maintaining anatomical information. As such, it provides an additional step towards understanding growth processes in plants at a cellular resolution. This method can be also applied for the study of additional plant species and organs.
A number of points must be considered when applying the method. First, crystalline cellulose accumu...

The plant cell wall is a dynamic structure. Growing cells are surrounded by a primary cell wall, the organization of which allows cells to expand. Cells that cease to grow deposit a more rigid secondary wall that enhances the mechanical support of the plant. Both cell walls are composed of cellulose microfibrils embedded in a matrix of polysaccharides of different structures (e.g., hemicellulose and pectin) that vary across the different developmental stage and tissues1,2. Cellulose is synthesized as chains of (1,4)-&#946;-D-glucan that are tightly aligned to form the microfibril of a crystalline structure. Amorphous cellulose refers to the regions where the glucan chains are less ordered. The ratio between the crystalline and the amorphous domains is one parameter thought to affect the mechanical properties of the cell wall, by providing mechanical strength and viscoelastic characteristic, respectively3. Several methods have been developed to detect and quantify the two forms of cellulose arrangement, among them X-ray diffraction and cross polarization/magic angle spinning solid-state NMR4. X-ray diffraction can be used to determine the proportion of crystalline versus amorphous cellulose domains in the sample5. An alternative method uses fractionation of cell wall content into acid-insoluble and acid-soluble material, to distinguish between the crystalline and amorphous cellulose or other polymers, respectively. In this approach, incorporation of labeled glucose ([14C]Glucose) is used to quantify the cellulose6,7. These methods require large volumes of plant material for whole organ analyses, at best, and hence, are inadequately sensitive to tissue-specific variation in cell wall structure. Visualization of cellulose microfibrils at a cellular resolution can be achieved in live imaging studies combined with fluorescent dyes8,9, that can identify changes in the orientation of the cellulose microfibrils. However, these dyes are not used for quantification, they are not specific to crystalline cellulose and may interfere with the normal structure of the cell wall8. Polscope is an imaging technique that relies on the ability of crystalline cellulose to split light beams and retard part of the light10. Light retardation is strongest for microfibrils that lie perpendicularly to the direction of light propagation. For microfibrils with similar orientation, the higher the degree of crystallinity, the larger the light retardance11. Hence, polscope is used to study both the relative levels and orientation of the cellulose microfibrils.
Roots exhibit linear growth, during which cells originating at the stem cell niche, at the tip of the root, undergo a series of cell divisions, before they rapidly expand12. The cells comprising the root expand in a unidirectional (anisotropic) manner, as dictated by small molecule signaling hormones that impact the properties of the cell wall13. Differential responses to hormones, in time and space, provide a means of ensuring balanced organ growth14. Hence, high resolution analysis of cell wall structure can provide important information necessary to better understand the connection between cell type-specific responses to whole organ growth. Here, we report the implementation of polscope to study tissue-specific accumulation of crystalline cellulose in Arabidopsis roots, as observed in high quality anatomical sections. This method recently uncovered cell type-specific accumulation of crystalline cellulose in response to spatial perturbation of hormonal activity15.

<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.

We study the impact of cell type-specific responses to brassinosteroids (BRs), using the Arabidopsis root as a model organ15-17. When the BR receptor BRI1 is targeted, in the background of bri1 mutant, to a subset of epidermal cells called non-hair cells (Figure 2A,B), it inhibits unidirectional cell expansion of neighboring cells, and whole-root growth15. Polscope analysis was performed to reveal the mechanism underlying this inhib...

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.

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 ...

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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Biology

8.0K vistas.  Technion-Israel Institute of Technology. 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 ste...