Name: glycan profiling of plant cell wall polymers using microarrays
Uploaded: 2012-12-17T13:00:00
Description: Watch this Scientific Journal Video about Glycan Profiling of Plant Cell Wall Polymers using Microarrays at JoVE.com

IEM would like to acknowledge the Danish Research Council (FTP and FNU) for funding. ERL acknowledges the support of an ARC DP grant. AB acknowledges the support of the ARC Centre of Excellence in Plant Cell Walls grant.

1. Tissue Collection &amp; Preparation
<ol>
	<li>Collect 100 mg fresh weight of plant tissues (a minimum of 10 mg dry weight) in at least triplicate for each tissue of interest. The following steps describe the preparation of cell wall material from vegetative tissues. In the case of storage tissues, un-wanted starch is enzymatically removed before proceeding with the extraction of cell wall polymers as previously described13.</li>
	<li>Homogenize the samples to a fine powder with liquid nitrogen using a Qia...

<ol>
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G., Orfila, C., Limberg, G., 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=Modulation+of+the+degree+and+pattern+of+methyl-esterification+of+pectic+homogalacturonan+in+plant+cell+walls.+implications+for+pectin+methyl+esterase+action,+matrix+properties,+and+cell+adhesion.">Modulation of the degree and pattern of methyl-esterification of pectic homogalacturonan in plant cell walls. implications for pectin methyl esterase action, matrix properties, and cell adhesion.</a> J. Biol. Chem. 276, 19404-19413 (2001).</li><li>Doblin, M. 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J., 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=High-throughput+mapping+of+cell-wall+polymers+within+and+between+plants+using+novel+microarrays.">High-throughput mapping of cell-wall polymers within and between plants using novel microarrays.</a> The Plant J. 50, 1118-1128 (2007).</li><li>S&#248;rensen, I., Willats, W. G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+and+characterization+of+plant+cell+walls+using+carbohydrate+microarrays.">Screening and characterization of plant cell walls using carbohydrate microarrays.</a> Methods Mol. Biol. 715, 115-121 (2011).</li><li>Moller, I. E., Licht, D. e. F. i. n. e., Harholt, H. H., J, , 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=The+dynamics+of+plant+cell-wall+polysaccharide+decomposition+in+leaf-cutting+ant+fungus+gardens.">The dynamics of plant cell-wall polysaccharide decomposition in leaf-cutting ant fungus gardens.</a> PLoS One. 6 (3), e17506 (2011).</li><li>Pettolino, F. A., Walsh, C., Fincher, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+procedures+for+the+determination+of+polysaccharide+composition+of+plant+cell+walls.">Chemical procedures for the determination of polysaccharide composition of plant cell walls.</a> Nature Protocols. , (2012).</li><li>Clausen, M. H., Willats, W. G. T., Knox, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+methyl+hexagalacturonate+hapten+inhibitors+of+anti-homogalacturonan+monoclonal+antibodies+LM7,+JIM5+and+JIM7.">Synthetic methyl hexagalacturonate hapten inhibitors of anti-homogalacturonan monoclonal antibodies LM7, JIM5 and JIM7.</a> Carbohydrate Res. 338, 1797-1800 (2003).</li><li>Verhertbruggen, Y., Marcus, S. E., Haeger, A., 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=Developmental+complexity+of+arabinan+polysaccharides+and+their+processing+in+plant+cell+walls.">Developmental complexity of arabinan polysaccharides and their processing in plant cell walls.</a> Plant J. 59, 413-425 (2009).</li><li>Heimburg-Molinaro, J., Song, X., Smith, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UNIT+12.10+Preparation+and+Analysis+of+Glycan+Microarray.">UNIT 12.10 Preparation and Analysis of Glycan Microarray.</a> Current Protocols in Protein Science. , (2011).</li><li>McCartney, L., Blake, A., Flint, J., 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=Differential+recognition+of+plant+cell+walls+by+microbial+xylan-specific+carbohydrate-binding+modules.">Differential recognition of plant cell walls by microbial xylan-specific carbohydrate-binding modules.</a> PNAS. 103, 4765-4770 (2006).</li><li>Ca&#241;o-Delgado, A. I., Metzlaff, K., Bevan, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+eli1+mutation+reveals+a+link+between+cell+expansion+and+secondary+cell+wall+formation+in+Arabidopsis+thaliana.">The eli1 mutation reveals a link between cell expansion and secondary cell wall formation in Arabidopsis thaliana.</a> Development. 127, 3395-3405 (2000).</li><li>Manabe, Y., Nafisi, M., Verhertbruggen, 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=Loss-of-Function+Mutation+of+REDUCED+WALL+ACETYLATION2+in+Arabidopsis+Leads+to+Reduced+Cell+Wall+Acetylation+and+Increased+Resistance+to+Botrytis+cinerea.">Loss-of-Function Mutation of REDUCED WALL ACETYLATION2 in Arabidopsis Leads to Reduced Cell Wall Acetylation and Increased Resistance to Botrytis cinerea.</a> Plant Physiology. 155, 1068-1078 (2011).</li><li>Updegraff, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semimicro+determination+of+cellulose+in+biological+materials.">Semimicro determination of cellulose in biological materials.</a> Anal. Biochem. 32, 420-424 (1969).</li><li>21Moller, I., Marcus, S. E., Haeger, A., 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=High-throughput+screening+of+monoclonal+antibodies+against+plant+cell+wall+glycans+by+hierarchial+clustering+of+their+carbohydrate+microarray+binding+profiles.">High-throughput screening of monoclonal antibodies against plant cell wall glycans by hierarchial clustering of their carbohydrate microarray binding profiles.</a> Glycoconjugate Journal. 25, 37-48 (2007).</li><li>S&#248;rensen, I., Pettolino, F. A., Wilson, S. 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=Mixed+linkage+(1&#8594;3),(1&#8594;4)-&#946;-D-glucan+is+not+unique+to+the+Poales+and+is+an+abundant+component+of+Equisetum+arvense+cell+walls.">Mixed linkage (1&#8594;3),(1&#8594;4)-&#946;-D-glucan is not unique to the Poales and is an abundant component of Equisetum arvense cell walls.</a> Plant J. 54 (13), 510-521 (2008).</li><li>Domozych, D. S., S&#248;rensen, I., Willats, W. G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+distribution+of+cell+wall+polymers+during+antheridium+development+and+spermatogenesis+in+the+Charophycean+green+alga,+Chara.">The distribution of cell wall polymers during antheridium development and spermatogenesis in the Charophycean green alga, Chara.</a> 2104, 1045-1056 (2009).</li><li>Singh, B., Avci, U., Eichler Inwood, S. 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+specialized+outer+layer+of+the+primary+cell+wall+joins+elongating+cotton+fibers+into+tissue-like+bundles.">A specialized outer layer of the primary cell wall joins elongating cotton fibers into tissue-like bundles.</a> Plant Physiol. 150, 684-699 (2009).</li><li>&#216;bro, J., S&#248;rensen, I., Derkx, P., 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=High-throughput+screening+of+Erwinia+chrysanthemi+pectin+methylesterase+variants+using+carbohydrate+microarrays.">High-throughput screening of Erwinia chrysanthemi pectin methylesterase variants using carbohydrate microarrays.</a> Proteomics. 9, 1861-1868 (2009).</li></ol>

CoMPP is a fast and sensitive method for profiling the glycan composition of hundreds plant-derived samples in a matter of days. This method complements the already available bacterial or mammalian glycan array platforms for high-throughput screening of carbohydrate interactions with glycan-binding proteins such as lectins, receptors, and antibodies16. With a large diversity of probes available for detecting cell wall glycans, it is possible to gain detailed information about glycan epitopes belonging to all m...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>3 mm Tungsten Carbide beads</td>
			<td>Qiagen</td>
			<td>69997</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collection microtubes (1.2 mm)</td>
			<td>Qiagen</td>
			<td>19560</td>
			<td>1.5 ml microfuge tubes can also be used</td>
		</tr>
		<tr>
			<td>Qiagen TissueLyser II</td>
			<td>Qiagen</td>
			<td>85300</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>3 mm glass beads</td>
			<td>Sigma Aldrich</td>
			<td>Z143928</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CDTA</td>
			<td>Sigma Aldrich</td>
			<td>34588</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cadmium oxide</td>
			<td>Sigma Aldrich</td>
			<td>202894</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1,2-diaminoethane</td>
			<td>Sigma Aldrich</td>
			<td>03550</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane (0.22 &mu;m pore size)</td>
			<td>GE-water &amp; process technologies</td>
			<td>EP2HY00010</td>
			<td>different pore sized membranes are suitable for different pin types</td>
		</tr>
		<tr>
			<td>Xact II microarrayer robot</td>
			<td>Labnext</td>
			<td>001A</td>
			<td>the Xact II robot was fitted with a custom 20 x 20 cm ceramic plate to which the nitrocellulose membrane is attached</td>
		</tr>
		<tr>
			<td>Xtend RM microarray pins</td>
			<td>Labnext</td>
			<td>0037-350</td>
			<td>pins must be suitable for spotting on membranes</td>
		</tr>
		<tr>
			<td>384 well microtiter plates (polypropylene)</td>
			<td>Greiner</td>
			<td>781207</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-glycan monoclonal antibodies</td>
			<td>Plant Probes/ 
			CarboSource/Biosupplies</td>
			<td>&nbsp;</td>
			<td>Websites; PlantProbes (<a href="http://www.plantprobes.net" target="_blank">www.plantprobes.net</a>), Carbosource (<a href="http://www.carbosource.net" target="_blank">www.carbosource.net</a>) and Biosupplies (<a href="http://www.biosupplies.com.au" target="_blank">www.biosupplies.com.au</a>).</td>
		</tr>
		<tr>
			<td>Anti-Rat IgG (whole molecule) - Peroxidase antibody produced in goat.</td>
			<td>Sigma</td>
			<td>A9037</td>
			<td>the type of secondary antibodies depends on the primary antibody used (e.g. raised in rat, mouse, goat etc).</td>
		</tr>
		<tr>
			<td>SIGMAFAST 3,3'-Diaminobenzidine tablets</td>
			<td>Sigma</td>
			<td>D4293</td>
			<td>the type of developing reagent depends on the secondary antibodies used and the detection method (colourmetric, or chemiluminecent).</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico Chemiluminescent Substrate</td>
			<td>Thermoscientific</td>
			<td>34080</td>
			<td>see above</td>
		</tr>
		<tr>
			<td>Xplore Image Processing Software</td>
			<td>LabNext</td>
			<td>008</td>
			<td>many software types with automatic gridding tools are available to measure pixel value of microarray spots.</td>
		</tr>
		<tr>
			<td>Plant polysaccharides</td>
			<td>Sigma/Megazyme</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

glycan profiling of plant cell wall polymers using 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 raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

The relative abundance of glycans in six tissue types (anther filaments, pollen, ovaries, petals, sepals and stigma) from Nicotiana alata flowers was determined using CoMPP. Figure 3A shows a representative microarray that has been probed with mAb JIM5 specific for partially (low) methylesterified homogalacturonan (HG), an epitope which occurs on pectic polysaccharides14. The JIM5 epitope is detected in CDTA extracts of all flower tissues however is highest in pollen and lowest in stigmatic tissue (<s...

Watch this Scientific Journal Video about Glycan Profiling of Plant Cell Wall Polymers using Microarrays at JoVE.com

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

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

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