Isolation of Plant Cell Walls and Fractionation of Cell Wall Polysaccharides

Abstract

Plant foods are an important component of the diet of humans. Since the cell wall makes up a large component of the dry weight of plant tissues, and because in a normal diet most of the dietary fiber comes from plant cell walls, it follows that there is a need to know the composition of the cell walls. Moreover, the cell wall contributes (along with turgor pressure and the cell contents) to the texture of plant foods, and since humans consider the texture of foods an important sensory attribute, again, understanding wall composition is important. While for some plant species the cell wall composition has been studied, the actual three-dimensional organization of the polysaccharides in the cell wall has not been fully elucidated. Moreover, it appears that while some aspects are constant for all walls (i.e., the presence of similar types of polysaccharides), the relative proportion and fine structure of the polysaccharides may be different, and thus the way they are organized in the wall may be different. Determination of the composition of polysaccharides in cell walls is commonly done by chemical analytical methods, but prior to determining the composition or structure of the polysaccharides in cell walls, the walls must first be isolated from the plant tissues and separated from the intracellular material. The first step in isolating cell walls is to examine the plant material microscopically (see Basic Protocol 1) to determine the types of cells in the tissues and to detect the presence of starch. If starch is present in large amounts, it must be actively removed or else there may be more starch than cell walls, and the carbohydrate analyses will be meaningless. Some plants contain large amounts of starch in which almost every cell is packed full of granules (e.g., potato), while others contain small amounts of starch in which some of the cells contain granules (e.g., apples and unripe tomatoes), and still other plant tissues may contain very little or no starch (e.g., onions and pineapple). Even among the same plants, all material must be checked because there will be differences among varieties, stages of ripeness, and parts of the plants used.

Down-regulation of POLYGALACTURONASE1 alters firmness, tensile strength and water loss in apple (Malus x domestica) fruit

“…Cell walls were prepared and extracted as described in Melton and Smith [64]. Apple cortex was snap frozen in liquid N 2 , ground to a fine powder and buffer-saturated phenol (UltraPure ™ , Invitrogen) added.…”

Pectins influence microfibril aggregation in celery cell walls: An atomic force microscopy study

“…Isolated CWM was sequentially extracted to first remove the pectins and then the hemicelluloses (Table 1) (Melton and Smith, 2005). The filtrate was dialysed against distilled water (3Â), collected by filtration and then re-suspended in 25 mL of 50 mM HEPES buffer (pH 6.5) by stirring for 2 h at room temperature.…”

“…Plant cell walls are often studied by selectively fractionating the polysaccharide groups of which they are comprised, using sequential extractions, so that each fraction can be studied in more detail (Melton and Smith, 2005). Briefly, highly methyl esterified pectins are extracted with boiling water or cold HEPES buffer, whereas less-methylated pectins, held in the wall with calcium bridges, are solubilised with calcium chelating agents such cyclohexanetrans-1,2-diaminetetraacetic acid (CDTA).…”

Analysis of β-Galactosidase During Fruit Development and Ripening in Two Different Texture Types of Apple Cultivars

“…The cell wall extraction procedure used was a modified method from Melton and Smith (2001) . Briefly, cell wall material (CWM) was extracted using Tris-phenol buffer and dimethyl sulfoxide (DMSO) from the frozen flesh (3.0 g).…”