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...

A protein identifiable as calmodulin has been isolated from oat (Avena sativa, var Garry) tissues. This protein is relatively heat stable, binds to hydrophobic gels, and phenothiazines in a calcium-dependent fashion, and binds to antibody to rat testes calmodulin. Based on its migration on sodium dodecyl sulfate-polyacrylamide gels, ultraviolet absorption spectrum, and amino acid composition, oat calmodulin is essentially identical to calmodulin isolated from other higher plants. Radioimmunoassays indicate that calmodulin is associated with isolated oat protoplasts, mitochondria, etioplasts, and nuclei and also appears to be a component of oat cell wall fractions.Much evidence has accumulated recently to support the hypothesis that Ca 2 plays a major role in mediating the adaptations of plants to certain environmental changes (12,22). As in animals, at least some Ca2-imediated responses in plants are controlled by Ca2-binding regulatory proteins. Among these, the most studied and best characterized is calmodulin (1).We have published reports suggesting a possible role for calmodulin in mediating phytochrome and gravitropic responses in Avena saliva (oats) (2, 21). As part of our ongoing research on this question, we have isolated and characterized calmodulin from oats and have estimated its content, both in intact tissue and in isolated subcellular fractions, by radioimmunoassay. Here we report the results of these experiments.MATERIALS AND METHODS Plant Material. Except where indicated, the starting material for all extractions was taken from the coleoptiles and primary leaves of 3-to 4-d-old dark-grown oat (A vena sativa, var. Garry) seedlings, harvested 5 to 7.5 mm above the seed. The oats were grown on water-saturated vermiculite at 27C.Calmodulin Isolation. Two extraction methods were employed.'Supported by grants from the National Aeronautics and Space Administration (NSG 7480), The National Science Foundation (PCM 81-03429), and The Robert A. Welch Foundation (F 858) to S. J. R.

Vitamin C breaks DNA only in the presence of oxygen. Superoxide dismutase has no effect on the reaction but catalase suppresses it. Superoxide also gives rise to breaks in DNA suppressible by both superoxide dismutase and catalase. The hydroxyl radical seems to be the agent responsible for strand cleavage itself.

The metabolism of polysaccharides by pea stem segments treated with and without auxin was investigated using a centrifugation technique for removing solution from the free space of the cell wail. Glucose is the predominant sugar in both the ethanol-soluble and ethanol-insoluble fracdons of the cell wall solution extracted with water. In the water-soluble, ethanol-insoluble polysaccharides, arabinose, xylose, galactose, and glucose make up 9.5, 23.8, 23.9, and 39.9%, respectively, of the neutral sugars, while rhamnose, fucose, and mannose are present at concentrations between 0.5 and 2.0%.Auxin treatment enhances the levels of xylose and glucose in ethanolinsoluble polysaccharides relative to controls, and this difference can be detected within 30 minutes of auxin treatment. Cellulose-binding experiments show that the enhanced levels of xylose and glucose are in a polymer having the cellulose-binding properties of xyloglucan. 3H-glucose labeling experiments confirm the auxin-enhanced metabolism of the xyloglucan fraction; however, increased labeling of arabinose is also observed in auxintreated sections. Auxin treatment also causes a marked increase in the level of uronic acids centrifuged from pea internode sections. Thus, after 3 hours of incubation in indoleacetic acid, the level of uronic acids in the ethanol-insoluble polysaccharides which can be recovered by centrifugation is increased 2-to 3-fold over sections incubated in water. These auxinenhanced changes in xylose, glucose, and uronic acids are correlated with enhanced rates of section growth.Incubation of excised pea internode sections in acidic buffers also enhances the rate of xyloglucan and polyuronide metabolism. This acidenhanced metabolism of xyloglucan and polyuronide is inhibited by low temperature, suggesting that it is enzyme-mediated.Extraction of the ceUl wan solution with CaC2 increases the yield of an neutral sugars. Arabinose and mannose are increased 4-and 3-fold, respectively, and xylose and glucose by about 20%, while galactose levels are 40% higher in cell wan solution extracted with CaCI2 than in that extracted with water. Although calcium increases the amoumt of neutral sugars extracted, it does not affect the auxin-induced changes in neutral sugars. Extraction of the cell wanl solution with ethyleneglycol-bis-4f-aminoethyl ether)-N,N'tetraacetic acid enhances the yield of uronic acids and also increases the difference due to auxin treatment.The cell wall has become the focus of research into the mechanism whereby plant hormones stimulate cell expansion. Heyn (7)

The effect of ethylene on cel wail metabolism in sections excised from etiolated pea stems was studied. Ethylene causes an inhibition of elongation and a pronounced radial expansion of pea internodes as shown by an increase in the fesh weight of excised, 1-cm sections. Cell wail metabolism was studied using centrifugation to remove the cell wall solution from sections. The principal neutral sugars in the cell wail solution extracted with H20 are arabinose, xylose, galactose, and glucose. Both xylose and glucose decline relative to controls in air within 1 hour of exposure to ethylene. Arabinose to do so by altering the orientation of the cellulose microfibrils in the cell wall (2, 6, 7, 17). Apelbaum and Burg (2) showed that the birefringence of pea stem cell walls was changed following treatment with ethylene, and this altered birefringence pattern was associated with the disappearance of cortical microtubules. Ethylene does not promote radial expansion by stimulating cell division; rather, in sub-apical tissue of etiolated peas, ethylene inhibits cell division (21). Although ethylene has been shown to alter the orientation of cellulose microfibrils, little is known of changes which occur in the noncellulosic polymers of the radially expanding pea stem cell wall. The effects of ethylene on growth contrast with those of auxin since auxin stimulates elongation of intact or excised pea internodes (6, 7, 10). In auxin-treated tissue, cell elongation is correlated with an increase in cell wall plasticity (9) and with the enhanced metabolism of a water-soluble xyloglucan fragment (12,13,22,23) and a pectic polymer (23) in the cell wall. There have been numerous attempts to correlate changes in cell wall polymers with ethylene-induced growth; however, most studies have emphasized changes in the hydroxyproline-rich proteins of the cell wall. Sadava and Chrispeels (19) found a strong positive correlation between the deposition of hydroxyproline-rich protein in the cell wall of excised pea segments and the cessation of cell elongation induced by Ethrel treatment. Similarly, in intact peas, Ridge and Osborne (18) and Nee et al. (16) correlated an increase in hydroxyproline in the cell wall with ethylene treatment.Because auxin and ethylene produce qualitatively different modes of cell expansion, we undertook an investigation of the metabolism of the cell wall using a centrifugation technique to displace components from the free space of the tissue. This technique was chosen because our previous experiments (22,23) showed a strong correlation between changes which could be monitored in the solution centrifuged from the cell wall and auxininduced elongation.MATERIALS AND METHODS Seeds of Pisum sativum L. cv. Alaska were grown in vermiculite in darkness at 24 to 26 C. After 7 days, the stems were cut at the level of the vermiculite and either placed directly in ice water (time zero) or 300 stems were placed in each oftwo 400-ml beakers containing 100 ml H20. For ethylene treatment, the beakers containing the stems...