Invertases in Oat Seedlings SEPARATION , PROPERTIES , AND CHANGES IN ACTIVITIES IN SEEDLING SEGMENTS

The soluble invertase activty in etiolated Avena seedlngs was highest at the apex of the coleoptile and much lower in the primary leaf, mesocotyl, and root. The activity in all parts of the seeding consisted of two invertases (I and II) which were separated by chromatography on diethylaminoethylcellulose. Both enzymes appeared to be acid invertases, but they dHffered in molecular size, pH optimum, and the kinetic parameters Km and V,, of their action on sucrose, ra tnose, and stachyose. Invertase II had low stability at pH 3.5 and below, and exhibited high sensitivity to Hge', with complete inhibition by 2 micromolar HgCl2. Segments of coleoptiles incubated in water lost about two-thirds of the total invertase activty after 16 hours. The loss of activity was due primarily to a decrease in the level of invertase IL The loss of invertase was decreased by indoleacetic acid, 2,4dichlorophenoxyacetic acid, and a-naphthaleneacetic acid but not by finaphthaleneacetic acid and p-chlorophenoxyisobutyric acid. Conditions that inhibited auxin-induced growth of the segments (20 miimlar CaC12 and 200 milimolar mannitol) also blocked the auxin effect on invertase loss. Invertase activity is present in many plant tissues including seedlings of beans (4), lentils (15), peas (9), and other species. The results of several studies have suggested that this enzyme is essential for growth by making sugars available for cell expansion (1, 8, 15). Plant invertases are classified as acid or alkaline invertases on the basis of their pH optima (1). Both types of enzymes often occur in the same tissue; the acid invertase is associated with the cell wall whereas the alkaline invertase is located in the cytoplasm (1). We found that acid invertase extracted with 0.5 M NaCl from oat seedlings could be separated into two peaks of activity by chromatography on DEAE-cellulose. This paper describes the procedure for that separation and some of the properties of the invertases. Also, segments of oat seedlings were incubated in water and different aqueous solutions, and changes in invertase activity were examined. MATERIALS AND METHODS Seedlings of Avena sativa cv. Victory were grown in moist Vermiculite in stainless steel trays in the dark at 22 C. The seedlings were harvested after 5 days when approximately 4 cm tall. For the preparation of crude invertase, 100 g of whole seedlings was blended with 400 ml cold 0.5 M NaCl in a VirTis homogenizer.' All subsequent steps were conducted at about 4 C. The 'Mention of trademark of proprietary product does not constitute a guarantee or warranty of product by the United States Department of Agriculture, and does not imply its approval to the exclusion of other products that may be suitable. homogenate was stirred for 0.5 h, then centrifuged at 8,000g for 20 min. The protein in the supernatant solution was precipitated with ammonium sulfate at 75% of saturation and collected after centrifugation. It was dissolved in 35 ml of 0.15 M NaCl and dialyzed overnight against 4 liters of 0.15 M NaCl. The reaction mixture for the invertase assay consisted of 0.1 ml enzyme solution, 0.2 ml 0.1 M sodium acetate (pH 5), 0.1 ml 0.15 M NaCl, and 0.1 ml 0.73 M sucrose. The enzyme solution was diluted with 0.15 M NaCl to produce approximately l-,umol reducing groups. The blank prepared for each sample was like the assay mixture but was heated before the addition of sucrose. The sample and blank tubes were incubated for 30 min at 30 C, and the reaction was terminated by the addition of 0.5 ml 0.5 M dibasic Na-phosphate followed by immersion of the test tubes in boiling water for 3 min. A 0.5-ml aliquot was then analyzed for reducing groups by the arsenomolybdate method of Nelson (10) standardized with glucose. A unit of invertase is defined as that amount which catalyzes the release of 1-,umol reducing groups under the conditions of the assay. The invertase activity in the sediment obtained upon centrifugation of the 0.5 M NaCl extract of the seedlings was also determined. The sediment was washed with 0.5 M NaCl and then suspended in water with a Polytron homogenizer. A 0.5-ml aliquot of the suspension was added to a reaction mixture consisting of 0.5 ml 0.2 M acetate (pH 4.5), 0.5 ml 0.15 M NaCl, and 0.5 ml 0.73 M sucrose. After 30 min at 30 C, the sample was centrifuged and the supernatant analyzed for reducing groups as described above for the soluble enzyme. Pectinesterase, polygalacturonase, fi-galactosidase, and /3-glucosidase were measured as described earlier (13). The levels of these enzymes are presented for comparative purposes. Protein was measured by the biuret method. Isoelectric focusing was performed with a Desaga/Brinkmann TLE double chamber according to the manufacturers instructions. A glass plate (20 x 20 cm) was coated with a suspension of 7 g Sephadex G-75 superfine in 100 ml of 2% pH 2-10 pH isolytes (Brinkmann Instruments). The layer was dried at room temperature until fine cracks appeared along the edges. The enzyme solutions were concentrated to about 3 ml by ultrafiltration with an Amicon model 52 cell and a PM-10 membrane and dialyzed against 2% glycine (pH 6.5). Sephadex G-75 was added to the solution to produce a fairly liquid suspension. A trough was cut out of the gel layer in the middle of the plate and filled with the enzyme suspension. Focusing was conducted at 4 C at 200 v for 12 h and then at 800 v for 2 h. One-cm strips of the gel were cut parallel to the electrodes, suspended in a small volume of 0.15 M NaCl, and assayed for invertase. The studies on changes in invertase in oat seedling segments were conducted with 1-cm segments cut from seedlings approximately 4 cm tall after removing the top 3 mm. The leaves were not removed from the coleoptiles. The segments were stirred in deionized H20 for 1 h before extraction or incubation. Each experiment consisted of six samples of 10-g oat segments/200 ml 136 www.plantphysiol.org on October 31, 2017 Published by Downloaded from Copyright © 1980 American Society of Plant Biologists. All rights reserved. INVERTASES IN OAT SEEDLINGS solution, and the entire experiment was replicated several times on different days. Extracts of the segments were prepared and assayed for invertase according to the standard procedures. The standard deviation among samples of segments incubated in water was 11%, which may be taken as the error in these measurements. The results of representative experiments are presented here.