An Examination of Centrifugation as a Method of Extracting an Extracellular Solution from Peas, and Its Use for the Study of Indoleacetic Acid-induced Growth

Abstract

MATERIALS AND METHODSA technique of centrifuging pea epicotyl sections which extracts watersoluble cell wall polysaccharides with less than 1.5% cytoplasmic contamination as revealed by malate dehydrogenase activity determinations was developed. Tests Reactions occurring in the cell wall are often difficult to study because of the problems in separating the solution that bathes the cell wall from the complex solution within the cell. Any process which ruptures the plasmalemma leads to a mixing of intracellular and extracellular components, making it difficult to determine whether compounds such as enzymes and water-soluble polysaccharides were localized in the cell wall or the cytoplasm.Abeles et al.(1) devised a method of centrifuging pea stem sections to remove the extracellular solution. They found that pea stem sections could be centrifuged at 3,000g, and a solution was removed which contained cellulase, an enzyme believed to be present in the cell wall. Using a similar technique, Stafford and Bravinder-Bree (13) localized a peroxidase isozyme within the cell wall of sorghum, but some cytoplasmic isozymes were also detected. Ferrari and Amison (6) refined this technique further and found that centrifuging pea stem sections at 500g released cellulase. However, some of the cells must have been broken because a cytoplasmic enzyme, malate dehydrogenase, was also found in the solution spun from the sections.The present study describes a method of centrifuging soluble substances from the cell walls of plant stem sections and evaluates the damage to the cells and contamination of the cell wall solution by the cytosol. ' Present address: Department of Botany, University of California, Berkeley, Calif. 94720.General Procedure. Seeds of Pisum sativum var. Alaska were grown in Vermiculite for 7 days in darkness. Under fluorescent room lights and using a specially designed harvesting rack, 1.2-cm sections were excised approximately 1 cm below the apical hook. All subsequent operations were performed in the light. These sections were packed vertically in the barrel of a 20-ml plastic syringe (20 mm i.d.) cut off at the 6-ml mark to form a small tube (Fig. 1). The apical end of the sections rested against a 0.32-cm porous polyethylene disk in the bottom of the tube (Bel-Art Products, Pequanock, N.J.). Once packed, the sections were maintained in a vertical position, retaining the original apical end above the basal end, except during centrifugation. Each tube contained 95 to 105 sections weighing approximately 3 g. Among tubes packed for each experiment, the number of sections varied by less than four per tube and the weight varied by less than 0.1 g/tube. The packed tubes were placed in distilled H20 covering the bottom I to 3 mm of tissue. One h after excising the sections, the tubes were connected to a circulating pump and 20 mm Kphosphate adjusted to pH 6.0 with HCI was passed through the tubes at 200 ml/min -tube for 30 min at 26 C to remove cytoplasmic contamination from cut surfaces. The total volume of...

Compartmentation of Solutes and Water in Developing Sugarcane Stalk Tissue

“…In the current study, the apoplast solution was collected at 480g, well below the force reported to induce leakage. Analyses of immature internode tissues in other species of Saccharum that accumulate sugars have produced iJa values greater than -0.5 MPa, indicating that the high apoplastic solute concentrations were not due to systemic errors in the centrifugation (4,14), while fully hydrated sugarcane stalk tissue yielded sufficient solution for repeated analyses without the addition of water. Contamination from the phloem during centrifugation would not be expected to constitute a large error, since the phloem has been estimated to comprise less than 0.5% of the volume in mature stalk tissue (9).…”

Effect of powdery mildew infection on concentrations of apoplastic sugars in pea leaves

“…The basic principle is that the whole of the apoplast is filled with water or buffer, usually by vacuum infiltration, followed by connection of the intercellular washing fluid (IWF) by centrifugation at speeds sufficiently low so as to not significantly disrupt the symplast. This method has been used to look at cell wall enzymes and compounds involved in cell growth (Terry & Bonner, 1980) as well as a means of identifying the appearance of proteins and other compounds as a response to infection by pathogens (Rohringer & Martens, 1987). Earlier Klement (1965) studied IWF from tomato leaves for its potential as a substrate for phytobacterial pathogens.…”

Soluble and Bound Apoplastic Activity for Peroxidase, β-d-Glucosidase, Malate Dehydrogenase, and Nonspecific Arylesterase, in Barley (Hordeum vulgare L.) and Oat (Avena sativa L.) Primary Leaves

“…Soluble or weakly bound enzymes have been extracted from the apoplast by vacuum infiltration followed by gentle centrifugation (21,(23)(24)(25). This technique works well with stems which require minimal support during centrifugation, but when used with cereals which have thin and relatively fragile leaves it yields small volumes of solution with low protein content (21).…”

“…By criteria developed in several laboratories (6,9,11,15,17,18,21,25): (a) enzymes washed from tissues by solutions not damaging the plasmalemma are soluble in the apoplast or weakly bound to the cell wall; (b) soluble cytoplasmic enzymes can be obtained by disrupting cells and pelleting organelles and broken cell walls; (c) ionically bound enzymes may be extracted from washed wall pellets by salt solutions of high ionic strength; and (d) enzyme activity remaining in the salt-washed wall pellet is covalently bound or entrapped into the wall. We have applied these criteria in our attempt to localize activity for four enzymes which may have roles in regulating apoplastic phenolic metabolism in young primary leaves of oats and barley.…”