I. Isolation of Cell Walls

II. Cellulose Determination; Total Sugar and Uronic Acids

III. Fractionation of Cell Wall Polysaccharides

IV. Methyl Esterification of Uronic Acids

V. Monosaccharide Composition

VI. Linkage (methylation) Analysis

VII. Fourier Transform Infrared (FTIR)

VIII. Protocol for the screening of the UniformMu maize population with near infrared reflectance spectroscopy


I. Isolation of Cell Walls

The major goal is to conveniently isolate the cell wall components in their entirety from all cytoplasmic components. To do this, the key is to ensure that all cells are broken and then membranes and adhering basic pI proteins are dislodged.

Homogenization of fresh tissues in glass-glass grinders or ‘Polytron’ is usually incomplete owing to the inherent toughness of the walls. Freezing the tissues or cells in liquid nitrogen improves cell breakage by growth of ice crystals ruptures individual cells. Still, cells in liquid culture can ‘escape’ the ice induced breakage. Nitrogen gas decompression, a further sophistication of the French press, can be used with small cell cluster to achieve complete cell breakage (Carpita 1985).

Owing to their greater density, cell walls may be isolated after homogenization by simple settling or low-speed centrifugation. This is a preferred technique if amounts of tissue are small. However, a significant contamination with starch grains often results from use of sedimentation. As an alternative, cell walls can be easily retained by filtration on nylon mesh of 47-µm square (‘Nitex cloth’; Tetko, Industries, Briarcliff, NY), whereas starch grains easily pass through.

A secondary concern in cell wall isolation is to not lose some of the less tenaciously held components, such as acidic and neutral pI proteins and water-soluble pectins. Basic pI proteins typically strongly adhere to the acidic wall matrix. Thus, cell wall isolation techniques vary depending on whether the carbohydrates or the proteins/active enzymes are the target material.

Carpita, N. C. 1985. Tensile strength of living plant cells. Plant Physiol. 79, 484-488.

 

A. Isolation of carbohydrate components.

In these applications, the soluble/enzymic proteins of the wall are removed, leaving mostly the pectins, cross-linking glycans and cellulose. Cross-linked structural proteins, such as HRGPs, PRPs, and GRPs are retained. AGPs, which are quite water soluble are always lost regardless of isolation technique.

1. Phenol : acetic acid : water (2:1:2; v/v/v) at ambient temperature

Homogenization of frozen tissue in this mixture effectively extracts all soluble protein, including the wall proteins, and the strong acid prevents extraction of water-soluble pectins. The extract can be pelleted by centrifugation, resuspended in water, and passed through Nitex cloth for collection of the walls. This is a very effective and simple method, but caution should be exercised because of the use of phenol. See Fry et al. (1988) for more information on this classic method.

2. An alternative to the potentially dangerous phenol:acetic acid:water method we use routinely is simply homogenize the frozen tissues at ambient temperature in 50 mM Tris-HCl, pH 7.2 containing 1% SDS, in a scintered glass-glass grinder (Duall; Thomas Glass). The suspension are heated to 70°C for 30 min to ensure inactivation and extraction of potentially wall-modifying enzymes. After cooling, the suspension is filtered through a 2.5-cm diameter 37-mm nylon cloth disk positioned in a Millipore-type manifold, and then washed sequentially with plenty of water, ethanol (to remove the SDS), acetone, and then back into water.

B. Isolation of enzyme-active walls

Whereas the principal of the first methods is to clear the carbohydrate components of protein, the following method is to prepare clean cell walls but have them retain their neutral and basic proteins/enzymes in a native and active state. The cell wall milieu is mildly acidic and buffered between pH 4.5-5.5, depending on growth status.

Walls are isolated by filtration after homogenization in freshly-prepared ice-cold 50 mM Na acetate or Na MES, pH 5.5, containing 50 mM NaCl, and 30 mM ascorbate. The salt helps prevent loose association of cytoplasmic proteins, and the ascorbate inactivates polyphenol oxidases (usually without affecting enzyme activity). The walls are washed thoroughly with ice-cold 100 mM NaCl, water, acetone (-2°C), and back into water or 10 mM Na acetate, pH 5.5. Aliquots of the wall material can be used directly in enzyme assays, but additional procedures will be necessary to extract the basic pI proteins. Because the cell wall is essentially an affinity matrix, wall protein enrichment can easily be 100-fold by first isolating cell walls.

Proteins vary in their tenacity of binding to the cell wall and are typically extracted sequentially with 200 mM CaCl2 in the acetate buffer, followed by 3 M LiCl in acetate buffer. This sometimes takes a considerable volume of extractant, and the proteins can be conveniently concentrated to 1/100 volume by filling dialysis tubing with the extracts and laying them on beds of PEG 8000, followed by dialysis against the final desired buffer. Any extraneous material that precipates can be removed by centrifugation.

Another way to extract cell wall proteins/enzymes is to suspend the living cells or vacuum-infiltrated tissues in 200 mM CaCl2 in acetate buffer. This methods is reasonably effective because of enhanced purity, but is of lower efficiency. Likewise, proteins have also been centrifuged at 1000 x g from vacuum-infiltrated tissues without significant disruption of cell integrity and contamination with cytoplasmic proteins (Terry et al. )


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