|The physical properties of Plant Cell Walls have been exploited in the production of building materials, textiles, and paper. They are an important component of our food, and are a potential source of carbohydrates for biofuels.
Presented on this page are Nature Protocols that could be used to further our understanding of plant cell walls: their development, chemical composition and structure, and the biochemistry underlying their formation.
|Whole Plant Cell Wall Characterization using Solution-state 2D-NMR
Shawn D. Mansfield, Hoon Kim, Fachuang Lu, and John Ralph
This protocol is used to determine lignin subunit composition, lignin interunit linkage distribution, and polysaccharide profiles.
Example results from poplar, pine, corn and Arabidopsis are shown.
Starting with dried, ground plant material, the protocol covers:
– Removal of non-structural components by either Soxhlet extraction or solvent extraction
– Optional removal of starch
– Milling of the structural components
– Optional acetylation
– Optional removal of trace metals
– Acquisition and processing of 2D 1H–13C HSQC spectra
|Determining the polysaccharide composition of plant cell walls
Filomena A Pettolino, Cherie Walsh, Geoffrey B Fincher & Antony Bacic
This protocol is used to analyze the monosaccharide composition of cell wall polysaccharides by GC-MS of their acetyl derivatives. Addition of a methylation step prior to hydrolysis allows the determination of the types of linkage that each monosaccharide is involved in. Supplementary Table 1 shows the analytical characteristics of most of the partially methylated and fully acetylated alditol acetates that are common to plant cell walls.
Results for Arabidopsis leaf cell walls are used an example of data analysis in the procedure.
Starting with harvested plant tissue samples, the protocol covers:
– Cell disruption by grinding
– Removal of non-structural components by solvent extraction
– Optional removal of starch by digestion with amylase
– Optional reduction step (if uronic acids are thought to be present)
– Reduction with sodium borodeuteride .
– GC-MS to determine the cell wall monosaccharide composition
– Methylation analysis to determine the monosaccharide linkage composition
|Radiometric and spectrophotometric in vitro assays of glycosyltransferases involved in plant cell wall carbohydrate biosynthesis
Christian Brown, Felicia Leijon & Vincent Bulone
This protocol covers biochemical assays that can be used to discover and characterize glycosyltransferases. These enzymes are membrane proteins with multiple transmembrane domains and are unstable outside of this matrix. It is therefore a good idea to perform exploratory assays using membrane fractions or detergent extracts derived from plant cells, and radiolabelled nucleotide sugars (e.g., UDP-[U-14C]glucose). Spectrophotometric assays can be performed as described in Box 1 and Figure 2 using highly enriched glycosyltransferases (prepared, e.g. by recombinant expression and tag-based purification procedures).
Starting from a plant cell suspension culture (example preparation and collection shown in Box 2), the radiometric assay procedure covers:
– Preparation of microsomal membranes from plant cell suspension cultures
– Preparation of detergent extracts from membrane suspensions
– Preparation of assay mixtures and assay reactions for the radiometric assay of GTs
A – Measuring the radioactivity incorporated into ethanol-insoluble carbohydrates, OR
B – Assay of GTs that form 66% (vol/vol) ethanol-soluble carbohydrates: example of a UDP-galactosyltransferase
(a) Protocol used to assay GTs that form ethanol-insoluble carbohydrates (e.g., cellulose synthase). (b) Protocol used to assay GTs that form soluble carbohydrates (e.g., nonprocessive GTs involved in the biosynthesis of oligosaccharide precursors).
|Preparation of plant cells for transmission electron microscopy to optimize immunogold labeling of carbohydrate and protein epitopes
Sarah M Wilson & Antony Bacic
This protocol is used to prepare plant material for analysis by immunogold labelling and TEM. The purpose is to look at the process of cell wall development: What is the spatial distribution of the polysaccharides? Where are the relevant synthases located? How do these variables change with time? It is therefore important that the fixing process not only preserves the fine structure of the plant cells (which is in itself more difficult thatn for animal cells), but also the antigenicity of both protein- and carbohydrate epitopes.
Procedures for two example sample types where cell wall formation would be expected to be seen are given (developing barley grain and pollen grains from ornamental tobacco).
Starting with the plant sample of interest, the protocol covers:
– Traditional chemical fixation for EM with subsequent carbohydrate epitope labeling (standard procedure using barley grain as the example)
– High-pressure freezing and freeze substitution (with resin) for fixing pollen tubes (could also be used for cultured cells)
– Block trimming
– Coating grids with formvar
– Ultrathin sectioning (with more detailed advice including transfer of the sections onto grid in Box 1)
– Antibody labeling
– Post-staining with triple lead citrate
|Imaging of plant cell walls by confocal Raman microscopy
Notburga Gierlinger,Tobias Keplinger & Michael Harrington
Raman microscopy is a non-destructive technique for obtaining spatially resolved information regarding chemical composition. In the case of cellulose microfibrils, the Raman signature comprises 15 significant bands and the Raman intensity depends on the orientation of the microfibrils relative to the direction of the laser polarization. Changes in fiber orientation of result from changes in cellulose crystallinity.
Starting with a plant sample of interest, the protocol covers:
– Preparation of sample with a a planar imaging surface and intact cell walls. It can be cut by hand, by conventional microtechnique or by cryosectioning. It can also be embedded in PEG before sectioning
– Preparation of the sections on glass slides for Raman imaging
– Setting up the Raman microscope for scanning (including calibration/optimisation using a silicon wafer)
– Acquisition of Raman images
– Pre-processing of the spectra and image generation