Hydrolases in Vacuoles from Castor Bean

Vacuoles were prepared from endosperm tissue of 4-day-old castor bean seedlings (Ricinus communis var. Hale) and purified on a stepped sucrose gradient. It was shown by assays of marker enzymes that there was only trace contamination of the final preparation by other organeiles (mitochondria, glyoxysomes, nuclei, spherosomes, and plastids) and by cytoplasmic components. Hydrolytic enzymes (acid protease, carboxypeptidase, phosphodiesterase, RNAase, phytase and /8-glucosidase) were present in the isolated vacuoles in amounts indicating a primarily vacuolar localization in vivo. The vacuoles also contained storage protein and high concentrations of sucrose. The over-all results indicate that the vacuoles from castor bean endosperm are the site of hydrolysis of the constituents of the protein bodies and are a temporary storage compartment for the sucrose produced from fat and protein reserves. During the early growth of the castor bean seedling there is a massive production ofenzymes and organelles concerned with the conversion of fat to sucrose in the endosperm (3). The amino acid precursors required for the synthesis ofenzymes and other protein constituents arise from the hydrolysis of protein bodies present in the endosperm. Thus, in a single tissue active synthesis of specific proteins occurs concomitantly with massive protein breakdown. This raises the question of how the catalytically important protein components are protected from attack by the enzymes bringing about protein hydrolysis in the same cells. The most obvious possibility is that hydrolysis of storage protein occurs in a segregated compartment, the vacuole. In fact, early in germination small vacuoles are formed by the dissolution of the water-soluble protein matrix surrounding the protein crystalloid of the protein bodies and these coalesce to give a large central vacuole containing crystalloid remnants (2, 22). The concept of the vacuole as a discrete site of hydrolytic activity in higher plant cells is an old one and has received strong support from Matile (13) particularly from experiments with younger tissues. Recently, methods have been developed for the isolation of vacuoles in quantities sufficient to allow a direct test of their storage and lytic function (4, 5, 7, 9, 26, 27). Somewhat surprisingly, in what the authors emphasize is the first detailed examination ofthis question in mature cells (from petals ofHippeastrum) assays for various acid hydrolytic enzymes revealed that they were not present in the vacuole but in the cytosol (5). However, in an earlier brief report Matile (12) had shown that the vacuoles that arise from protein bodies in germinating pea seeds contain an acid protease and other hydrolytic enzymes. The vacuoles in the endosperm tissue of young castor bean seedlings have a similar origin. The present paper describes a detailed investigation of vacuoles isolated in high purity from this tissue and it is shown clearly that they have both a lytic and storage function. 'Supported by National Science Foundation Grant PCM 75-23566. 2 M. N. received a travel grant from The Japan Society for Promotion of Science and a grant from Matsunaga Foundation (Tokyo). MATERIALS AND METHODS Plant Material. Seeds of castor bean (Ricinus communis var. Hale) were soaked in running tap water for I day and germinated in moist Vermiculite at 30 C. Preparation of Protoplasts. Fourteen endosperm halves, removed from 4-day-old seedlings, were used for preparation of protoplasts as described previously (15). The protoplasts (about 4 x 10) were washed twice with 0.7 M mannitol. To obtain the protoplast extract, 0.5 ml of homogenizing medium (150 mM Tricine-KOH [pH 7.51, 15% sucrose, 0.1 mm EDTA) was added and the protoplasts were ruptured by sonication for 10 sec at 0 C. Appropriate amounts of this homogenate (5-200 ,ul) were used for the assays described below. Preparation of Vacuoles. Sliced tissue from 14 endosperm halves was incubated for a total of 5 hr in the enzyme solution (0.5% Macerozyme R-10 and 2% cellulose "Onozuka" R-10 in 0.7 M mannitol) and the enzyme solution was removed (15). Seven ml 0.7 M mannitol was added to the slices and the flask was briefly swirled by hand. This treatment released vacuoles and other components from the slices. The mannitol solution was collected by filtration through nylon bolting cloth (35 mesh) and the process was repeated using fresh mannitol solution until 40 ml of filtrate had been collected. Microscopic examination showed the presence of protoplasts and other components in this crude vacuole fraction (see Fig. IB). Twenty ml of the filtrate was layered on 10 ml of 40%1o (w/w) sucrose-containing 0.1 mm EDTA in a 50-ml tube. The tubes were centrifuged at 300g for 5 min. Only vacuoles were sedimented; protoplasts and other cell components remained in the 0.7 M mannitol layer. To obtain the vacuolar extract the purified vacuole fraction containing about 7 x 105 vacuoles was mixed with 0.5 ml of homogenizing medium (as above) and ruptured by sonication for 10 sec at 0 C. Samples of this homogenate were used directly for the enzyme and other assays described below. For the hydrolytic enzymes 10 to 100 ,ul (equivalent to about 104-105 vacuoles) were used. Larger amounts, up to 500 Al, were used for the other assays. When vacuoles were prepared for sucrose analysis, 40%o (w/w) sorbitol containing 0.1 mM EDTA was used in place of40o (w/w) sucrose containing 0.1 mM EDTA, and sucrose was omitted from the homogenizing medium. Enzyme and Chemical Assays. All hydrolytic enzyme assays were essentially those ofTully (22). The extracts were not dialyzed and rates of reaction were corrected for zero time blank values. Acid protease was assayed with bovine hemoglobin as substrate. The reaction mixture contained the following components in 1 ml: K-malate (pH 3.5), 100 ,umol; bovine hemoglobin, 10 mg. After incubating for 30 min at 37 C, the reaction was stopped by adding I ml of 10%o trichloroacetic acid. The solutions were centrifuged and the supernatant solutions were assayed for amino-N by the ninhydrin method (21) using glycine as a standard. Carboxypeptidase was assayed by hydrolysis of Z-Phe-Ala.3 The reaction mixture contained the following components in I ml: K-acetate 'Abbreviations: bis-PNPP: bis-(p-nitrophenyl phosphate); PNP: p-nitrophenol; PNPP: p-nitrophenyl phosphate; PNP-13-D-Glu: p-nitrophenylf,-D-glucoside; Z-Phe-Ala: carbobenzoxy phenylalanylalanine. 44 www.plantphysiol.org on July 22, 2017 Published by Downloaded from Copyright © 1978 American Society of Plant Biologists. All rights reserved. VACUOLES FROM CASTOR BEAN ENDOSPERM (pH 4.5), 100,mol; Z-Phe-Ala, 1 ,Amol. The reactions were stopped by adding 1 ml of 10%o trichloroacetic acid after incubation at 37 C for 30 min and the alanine assayed with ninhydrin. Acid phosphatase was assayed with PNPP as substrate. The reaction mixture contained the following components in 0.5 ml: K-acetate (pH 5), 50,umol; PNPP, 2.5,umol. After incubating for 5 min at 37 C, the reaction was stopped and the free PNP color developed by adding 2.5 ml of 0.2 N NaOH. The A at 400 nm was measured, and PNP concentration determined from a standard curve. Phosphodiesterase was assayed by hydrolysis of bis-PNPP. The reaction mixture contained the following components in 0.5 ml: Kmalate (pH 5), 50 ,umol; bis-PNPP, 2.9 ,umol. After incubation for 10 min at 37 C the reaction was stopped with 2.5 ml 0.2 N NaOH. Phosphodiesterase activity was calculated by measuring the A at 400 nm and correcting for PNPPase activity. RNA was assayed by hydrolysis of Torula RNA following the method of Pitt and Combes (18). The reaction mixture contained the following components in 0.2 ml: K-acetate (pH 5), 20 ,umol; RNA, 0.1 ,ug. After incubation for 30 min at 37 C the reaction was stopped by the addition of 200 ,ul of 4% HC104 plus 0.25% uranyl-Mg-acetate, and the mixture was chilled on ice for 30 min. After adding 2.6 ml of H20, the insoluble protein and RNA were removed by centrifugation, and the A at 260 nm was measured. Phytase was assayed by the hydrolysis of Na-phytate. The reaction mixture contained the following components in 0.5 ml: K-malate (pH 5), 50 ,umol; Na-phytate, 5 ,umol. After incubation for 30 min at 37 C, the reaction was stopped with 0.5 ml of 10%o trichloroacetic acid. The Pi released was measured by the method of Fiske and Subbarow (6). ,B-Glucosidase was assayed by hydrolysis of PNP-,/-D-Glu. The reaction mixture contained the following components in 0.5 ml: K-acetate (pH 5), 50 ,umol; PNP-fl-D-Glu, 1.25 umol. After I hr of incubation at 37 C, the reaction was stopped and PNP determined as for acid phosphatase. Other enzyme and chemical assay methods were those described in the literature as follows: acid lipase (14), alkaline lipase (14), catalase (11), fumarase (19), NADH-Cyt c reductase (8), NADHmalate dehydrogenase (1), RuP2 carboxylase (16), triose-P isomerase (17), and DNA (7). Sucrose was measured by the resorcinol method (20) after remQval of glucose and fructose (24). Protein was assayed by the Lowry method (10) after precipitation and washing in trichloroacetic acid (10%, w/v).