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 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 & 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.
1. Tissue Collection & Preparation
2. Extraction of Cell Wall Glycans
3. Printing Microarrays
4. Probing of Glycan Microarrays
5. Quantification
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 (Figure 3A and 4A). Interestingly, the JIM5 epitope is also detected in the NaOH extracts in pollen but not in other tissues (Figure 3A).
Quantitative information about the occurrence of a glycan of interest is obtained by including a series of purified polysaccharides as internal standards on the microarray (Figure 3B). Integral spot intensity of extracts of different tissues probed with mAb JIM5 is matched to the spot signals derived from a serial dilution of homogalacturonan (25% degree of methyl esterification, DE). In ovary tissue, approximately 157 μg of homogalacturonan containing the JIM5 epitope was present in each sample, accounting for approximately 1.5% (by weight) of total cell wall residues (Figure 3C). Whereas 625 μg homogalacturonan containing the JIM5 epitope was present in pollen tissue, accounting for approximately 6.25% (by weight) of total cell wall residue.
The relative abundance of 15 glycan epitopes are summarised as a heatmap in Figure 4 where the intensity of color bars is proportional to the adjusted mean spot intensity for each sample. A pictorial representation of this data is also illustrated in Figure 5 and allows us to rapidly interpret differences in glycan epitope occurrence between the different flower tissues.
Our results indicate that individual glycan epitopes are uniquely distributed amongst N. alata flower tissues. For example, the LM13 epitope (linearised (1-5)-α-L-arabinan15 ) is most abundant in anther filaments and sepal tissues compared to other tissues (Figure 5D). This pattern is in striking contrast to that of shorter chain (1-5)-α-L-arabinan epitopes preferentially detected by mAb LM6 (Figure 5E)15. Cross-linking glycans also have distinct patterns of occurrence in N. alata flowers. The LM10 epitope (low-substituted (1-4)-β-D-xylan) is abundant in stigma tissue compared to other tissues, but is absent from ovary tissue (Figure 5G). LM15 xyloglycan epitopes are highest in petal and anther filament compared to other tissues (Figure 5H), whereas heteromannans are highest in anther filaments (Figure 5J).
Figure 1. A simplified flow diagram representing the five key steps of Comprehensive Microarray Polymer Profiling (CoMPP).
Figure 2. An illustration of the key steps of Comprehensive Microarray Polymer Profiling (CoMPP). (A) Plant tissues are collected as three replicates, homogenized and washed in 70% EtOH and acetone to make alcohol insoluble residues (AIR). (B) Cell wall glycans are sequentially extracted from AIR samples with 50 mM CDTA and 4 M NaOH. (C) Extracted cell wall glycans are printed on nitrocellulose using a microarray robot. Each sample is represented in three concentrations and as a series of four printing replicates. (D) The printed arrays are probed individually with mAbs directed to cell wall glycans epitopes. (E) The spot signals are quantified using microarray imaging software, exported as txt files and relative mean spot intensity values are automatically calculated using an online data processing tool.
Figure 3. Glycan profiling of Nicotiana alata flower tissues. (A) A representative microarray probed with monoclonal antibody (mAb) JIM5 specific for homogalacturonan (partially, low methylesterification) is indicated as a negative, 16-bit TIFF file. (B) The amount of polysaccharide containing a glycan epitope of interest can be quantified by printing a series of polysaccharide standards. (C) Integral spot intensity of sample of interest probed by mAb JIM5 is calculated and matched to a standard curve to estimate the quantity (μg) of polysaccharide containing this epitope in the sample.
Figure 4. Glycan profiling of Nicotiana alata flower tissues represented as a heatmap. The color intensity is proportional to the relative mean spot identity value (scale bar). Relative abundance of 15 glycan epitopes detected by monoclonal antibodies (listed at the top) was analyzed for six tissue types extracted with CDTA and NaOH (left hand side). The glycan epitopes fall into three broad classes of polysaccharides; pectins, cross-linking glycans and arabinogalactan proteins (AGPs). me, methyl-esterification; DP, Degree of polymerization; mAb, monoclonal antibodies; AGP, arabinogalactan-protein.
Figure 5. Glycan profiling of Nicotiana alata flower tissues represented pictorially. A-L represents the relative abundance of 12 glycan epitopes detected by mAbs in different flower tissues. The scale bar color intensity is proportional to the relative mean spot identity value derived from either CDTA or NaOH extracts or a sum of both. me, methyl-esterification; DP, Degree of polymerization; mAb, monoclonal antibodies; AGP, arabinogalactan-protein. Click here to view larger figure.
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 major classes of polysaccharides in the plant cell wall8,9. However these encompass only a small proportion of the glycan structures that have been identified8,9, and in some circumstances limit the comprehensibility of CoMPP. Nevertheless, this capability has recently been extended by the use of carbohydrate binding modules (CBMs) against plant glycans17, and also against epitopes characterized by non-glycosyl residues such as phenolics18 and acetyl residues19 which modify glycosyl residues and play an important role in glycan function.
Although CoMPP determines the relative levels of glycans across a set of samples, it is possible to quantify the epitope levels in sequential extracts by comparing the binding signal in specific samples to that of known standards (Figure 3B and C). Despite the advantages of reduced sample volumes in a microarray-based platform, the ability to accurately quantify glycan occurrence can be hindered by differences in spot morphology or saturation of spot signal. These problems (as well as false positives) are avoided by optimising the amount of cell wall material verses extractant volume, prior to analysis, and including a series of replicates and dilutions as outlined here and in mammalian glycan array methods16. Furthermore, differences in spot morphology (caused by diffusion of samples at different rates from the point of contact) can be overcome by using non-contact ink-jet microarray technology16.
CoMPP is used in combination with established procedures for the isolation and preparation of cell walls20. It can also be used in parallel with existing biochemical, enzymatic and physical methods to gain further information about glycan content in samples of interest. For instance instead of extracting cellulosic polymers, Acetic/Nitric acid assays20 can be used to quantify cellulose content (mg) remaining after sequential extraction of pectin and cross-linking glycans with CDTA and NaOH. Enzyme treatment of glycan microarrays21 can also reveal further differences in glycan epitope distribution amongst samples, or confirm the specificity of epitopes structures detected on the microarray. The determination of glycan composition by independent analytical techniques, such as those recently described13, is also particularly important for the validation of CoMPP data.
We demonstrate that glycans are distributed in an organ or tissue specific manner in N. alata flowers. This information can give insight into the biological function of glycan epitopes or glycan synthesizing or modifying enzymes present in these different cell types. CoMPP has previously provided insight into cell wall alterations that occur in relation to plant type22 during development23,24 or in response to mutation10,25. In summary, CoMPP is a valuable tool for the studying the diverse role of glycans and glycan synthesizing and modifying genes in the plant cell wall.
The authors have nothing to disclose.
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.
Name of the reagent | Company | Catalogue number | Comments (optional) |
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-diaminoethane | 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 |