Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Summary

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.

Abstract

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 simultaneously^9,10,11.

Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 & 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.

Protocol

1. Tissue Collection & Preparation

1. 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.
2. Homogenize the samples to a fine powder with liquid nitrogen using a Qiagen TissueLyser II, with 24 tube adapter sets and 3 mm tungsten carbide beads (30 Hz, 2 x 30 sec). Alternatively if only a few samples are being processed....

Results

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 (

Discussion

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.......

Materials

List of materials used in this article

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