Proc. Natl. Acad. Sci. USA Vol. 77, No. 1, pp. 428-432, January 1980 Cell Biology Protein bodies of mung bean cotyledons as autophagic organelles (acid hydrolase/autophagy/lysosome) WILLEM VAN DER WILDEN, ELIOT M. HERMAN, AND MAARTEN J. CHRISPEELS Department of Biology, C-016, University of California, San Diego, La Jolla, California 92093 Communicated by Warren L. Butler, October 19, 1979 ABSTRACT We present evidence that protein bodies con- during seedling growth, while the membrane surrounding the stitute the principal lytic compartment in storage parenchyma protein body remained intact (i). Recently, biochemical evi- cells of mung bean cotyledons and propose that they play a role dence has been obtained which confirms that the hydrolysis of in cellular autophagy. We developed a method to isolate protein the bodies (6-8). No ev- bodies by incubating tissue slices with cell wall-degrading en- reserve protein occurs within protein zymes and fractionating the cellular organelles on a Ficoll idence has yet been presented for the degradation within the gradient. About 75-80% of the protein bodies present in the protein bodies of macromolecules other than the stored reserves, protoplasts were recovered intact in a band at the 5/25% Ficoll and this function, postulated by Matile (5), is as yet undocu- interface. This band contained a similar proportion of the cel- mented (1). lular a-mannosidase, N-acetyl-glucosaminidase, ribonuclease, The central vacuole of the plant cells contains many acid acid phosphatase, phosphodiesterase, and phospholipase D. hydrolases (9-12), and there is now considerable evidence that P-Amylase was present in the cells but not in the protein bodies. it plays a role in autophagy and the breakdown of cellular or- Ultrastructural observations showed that on the 3rd day of Nishi- seedling growth protein bodies contain small vesicles (0.3-1.0 ganelles and macromolecules (for a review see ref. 13). #m) with a cytoplasmic content (ribosomes, membrane vesicles, mura and Beevers (11) have shown that vacuoles derived from mitochondria). Later in seedling growth these vesicles appeared the fusion of "empty" protein bodies also contain numerous empty. We believe that these are autophagic vesicles resulting hydrolases. There is, however, little information about the en- from invaginations of the protein body membrane and that their zyme content of the protein-filled protein bodies of ripening cytoplasmic contents are digested by the acid hydrolases'present or germinating seeds. Presented here is evidence that such in the protein bodies. protein bodies contain many acid hydrolases and constitute the principal lytic compartment in the storage parenchyma cells. The protein reserves of many seeds are stored in special tissues We suggest on the basis of morphological observation that and contained in protein bodies, large (2-10,m in diameter) protein bodies play a role in cellular autophagy during seed spherical organelles bounded by a single membrane. Although germination. most of the content of the protein bodies can be accounted for by the reserve proteins, these organelles also contain phytin, MATERIALS AND METHODS lectins, and certain acid hydrolases (for a recent review see ref. Seeds of mung bean [Vigna radiata (Linnaeus) Wilczek] ob- 1). In cotyledons of leguminous seeds, formation of protein tained from a local dealer were sterilized in 10% commercial bodies occurs during seed ripening. Reserve proteins are syn- bleach and germinated in the dark in moist vermiculite as de- thesized on rough endoplasmic reticulum (2) and accumulate scribed (14). first in the central vacuole and later in protein bodies formed Isolation of Protoplasts and Protein Bodies. Forty cotyle- de novo (for a review see ref. 3). During seedling growth the dons were cut in 1-mm slices and incubated in a solution of 1% reserve proteins are catabolized within the protein bodies. Macerase (Calbiochem), 1% Cellulysin (Calbiochem), and 0.1% When the protein bodies appear "empty," their limiting Polycillin in mannitol medium [0.6 M mannitol/10 mM sodium membranes fuse to form a central vacuole (4). These observa- 2-(N-morpholino)ethanesulfonate buffer at pH 5.5]. After tions indicate a clear ontogenetic relationship between protein 10-12 hr of incubation at 25°C with gentle shaking the mac- bodies and vacuoles in legume cotyledons and suggest that erated tissue was washed six times with mannitol medium protein bodies may be considered as storage-protein-filled containing 0.1 mM EDTA to remove the digestive enzymes. vacuoles. The macerated tissue was resuspended in this medium to free Matile (5) proposed more than 10 years ago that protein protoplasts from the tissue slices and was carefully strained bodies have three functions: (i) they contain reserves, especially through nylon cloth (pore width approximately 500 Am) to protein and phytin; (ii) upon germination they become hy- remove large tissue fragments. The protoplasts were allowed drolytic compartments in which the hydrolysis of the reserves to settle and the supernatant was tucked off. The protoplast occurs; (iii) the protein bodies are the cellular compartments sediment was resuspended in mannitol medium containing 0.5 in which nonstorage macromolecules are broken down by acid mM EDTA and pressed through a metal screen (pore width hydrolases. The first function has been amply documented for approximately 250 pm). The material that passed through the many plant species either by analyzing the contents of isolated screen contained some unbroken protoplasts and starch grains protein bodies or with cytochemical methods (1) The second (both were allowed to settle out), and all the cytoplasmic or- function was deduced from ultrastructural observations ganelles including protein bodies. The supernatant (after that the electron-dense protein matrix disappeared settling out) was loaded on a discontinuous Ficoll gradient (5% showing over 25% Ficoll in mannitol medium with 0.5 mM EDTA), which was centrifuged for 20 min at 9.0 X g (200 rpm in a The publication costs of this article were defrayed in part by page was charge payment. This article must therefore be hereby marked "ad- Sorvall RC-3 centrifuge with an HL-8 rotor). The gradient vertisement" in accordance with 18 U. S. C. §1734 solely to indicate fractionated in 0.5-ml fractions and each fraction was assayed this fact. for enzyme activity. 428 Downloaded by guest on September 26, 2021 Cell Biology: Van der Wilden et al. Proc. Natl. Acad. Sci. USA 77 (1980) 429 Enzyme Assays. a-Mannosidase and N-acetyl-/3-glucosa- RESULTS AND DISCUSSION minidase were assayed at pH 5.0 with p-nitrophenyl derivatives Isolation of Protein Bodies. Fig. 1A shows protoplasts ob- as substrates (14). Carboxypeptidase was measured at pH 5.0 tained after the overnight incubation of cotyledon slices with with N-carbobenzoxy-L-phenylalanine as substrate (14). Ri- cell wall-degrading enzymes at room temperature. The enzy- bonuclease was determined at pH 5.0 by the method of Am- matic digestion did not cause the release of the fragile proto- bellan and Hollander (15). Phospholipase D was assayed at pH plasts into the incubation medium until the partially digested 5.0 as described in our earlier paper (16), with radioactive tissue slices were lightly shaken in the morning. The protoplasts phosphatidylcholine as substrate. Acid phosphatase and phos- were washed free of the cell wall-degrading enzymes by re- phodiesterase were assayed at pH 5.0 with p-nitrophenyl de- peated (six times) washing with mannitol medium containing rivatives as substrates. Proteinase (vicilin peptidohydrolase) was measured at pH 5.0 with Azocoll as substrate (14). f3-Amylase 0.1 mM EDTA. Each time the protoplasts were allowed to settle was measured at pH 5.0 by determining the release of reducing out and the supernatant was removed. Preparations of proto- sugars from soluble starch as described by Bernfeld (17). Leu- plasts always contained free starch grains (Fig. 1A) because the cine aminopeptidase activity was determined at pH 7.0 with free starch grains and the starch-filled protoplasts settled out leucine p-nitroanilide as substrate (14). NADH-cytochrome together when the protoplasts were washed free of the cellu- c reductase was assayed as described (2). Catalase was measured lolytic enzymes. Although the commercial preparations of cell according to the procedure of Beers and Sizer (18). Protein was wall-degrading enzymes are rich in proteinase activity (12), no determined by the method of Lowry et al. (19), with bovine such proteinase activity was present in the isolated protoplasts serum albumin as a standard. (data not shown). This observation indicates that the protoplasts Electron Microscopy. Small pieces (1 mm3) of cotyledon were not contaminated by hydrolytic enzymes originating in tissue were fixed for 12 hr at 7°C in 50 mM sodium cacodylate, the preparations of cell wall-degrading enzymes. pH 7.4, containing 4% (wt/wt), formaldehyde and 2% (wt/wt) Washed protoplasts were broken mechanically and layered glutaraldehyde. The pieces of tissue were rinsed in the fixation (after settling out of the starch grains) on a discontinuous Ficoll buffer, then postfixed in 1% osmiumtetroxide for 18 hr at 7°C. gradient. Centrifugation resulted in the formation of a band The material was dehydrated in a graded acetone series, at the 5/25% Ficoll interface. Examination of this fraction with transferred to propylene oxide, and embedded in araldite. The the light microscope showed it to be a homogeneous preparation specimens were sectioned with glass knives and the sections of large round organelles. When neutral red was added to this were stained in uranyl acetate (10 mg/ml in water) and alkaline preparation these organelles accumulated the dye. Examination lead citrate (5 mg/ml). The sections were visualized with a with the electron microscope showed the fraction to be pri- JEOL 100 S or a Phillips 300 electron microscope. marily intact protein bodies (Fig.