Subcellular Fractionation of Plant Tissues
The successful isolation of intact, functional organelles from plant tissue is fraught with difficulties. Leaves are often covered in waxy cuticles, and frequently contain silica (as in grasses) and toxic components, such as phenolics, proteolytic enzymes, and high concentrations of acids and salts in the vacuole. In addition, all higher plants have one major barrier in common, the presence of a rigid, cellulose cell wall, which has to be broken in order to release the organelles. Mechanical isolation methods, i.e., where leaf material is macerated in a mechanical homogenizer, are likely to succeed only for a limited number of species, e.g., pea and spinach, where the leaves do not contain large amounts of tough, thickened tissue. Otherwise, the prolonged homogenization required to release significant numbers of organelles from tough leaves, e.g., of grasses, such as wheat or barley, results in most of them being broken and inactive. For this reason, the only viable method for obtaining good-quality organelles from species such as these is to isolate them from protoplasts. Although chloroplasts can be successfully isolated from protoplasts, the yield of mitochondria is so small that, unless very large-scale protoplast preparations are employed, the technique is generally inappropriate for mitochondrial isolation. This means that there are many plant species from whose leaves it has proven impossible to isolate good-quality mitochondria using existing techniques.
KeywordsSucrose Starch EDTA Cysteine Respiration
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