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[59] Vacuoles and spherosomes
572
SUBCELLULAR FRACTIONS DERIVED FROM PLANT TISSUE
[59]
[59] V a c u o l e s a n d S p h e r o s o m e s
By PHILIPPE MATILE and ANDRES WIEMKEN Vacuoles Plant vacuoles cannot be isolated according to a universal scheme of cell fractionation. This circumstance is chiefly due to the wide variation of physical properties of these organelles (size, shape, density, etc.). In addition, the mechanically resistant cell walls require drastic procedures of tissue homogenization which inevitably result in the destruction of large vacuoles. The disintegration of parenchymatous tissues using conventional techniques will always result in bursting and fragmentation of these organelles. The soluble substances contained in these vacuoles are, therefore, mainly present in the soluble fraction of extracts. On the other hand, meristematic cells may contain small vacuoles that are not destroyed upon grinding or blending of the tissue. It appears that the preparation of vacuoles depends largely on the selection of suitable objects. One of these is represented by spheroplasts I whose fragility permits a gentle lysis and the release of even large vacuoles. Isolation of Vacuoles from Spheroplasts The procedure involves the following steps: (i) preparation of osmotically stabilized spheroplasts by enzymatic digestion of the cell walls, (ii) osmotic lysis of spheroplasts and release of intact vacuoles, and (iii) isolation and purification of vacuoles, la The following method has been successfully used in several laboratories. Spheroplasts. A growing population of Saccharomyces cerevisiae is collected by centrifugation. After washing with distilled HzO, the cells are suspended in a medium containing buffered osmoticum (0.6 M sorbitol, 10 mM citrate buffer, pH 6.8) supplemented with 0.14 M cysteamineHCI (approx. 1 × 109 cells/ml). The suspension is indubated for 20 minutes at 27 °. The cells are now sedimented and washed once with the above osmoticum (no supplement). The subsequent incubation in the presence of snail gut enzyme (Helicase, lyophilizdd product of l'Industrie Biologique Fran~aise S.A. Gennevilliers, Seine, France; Glusulase, or corresponding preparations) is carried out under the following conditions: 1 ml of Helicase solution (30 mg of enzyme p~r milliliter of 1The term spheroplast has now been generally adopted in cases wh~re the question whether the wall has been completely eliminated or not is irrelevant. lap. Matile and A. Wiemken, Arch. Mikrobiol. 56, 148 (1967).
[59]
VACUOLES AND SPHEROSOMES
573
osmoticum) is used for suspending 1 g of packed cells; incubation at 30 ° in an oscillating water bath. Within 45-90 minutes the cells are converted into protoplasts (controls in phase contrast microscope; note the spherical shape of spheroplasts) which are now cooled down to 0 °, the subsequent working temperature. Separation of spheroplasts from the Helicase solution is achieved by centrifugation in a swing-out rotor (20 minutes, 1000 g) through a gradient ranging from 0.6 M sucrose to 0.6 M sorbitol. The sedimented protoplasts are washed twice with a solution of 0.7 M sorbitol. Note that the successful conversion of yeast cells into protoplasts is possible only in growing cells. Resting cells (e.g., commercially available cakes of bakers' yeast) must be incubated in an appropriate culture medium for a few hours prior to the preparation of protoplasts. Liberation of Vacuoles. Osmotic lysis is induced by placing the packed protoplasts in a medium containing 0.1 M mannitol or sorbitol, and 8% (w/v) Ficoll (Pharmacia, Uppsala, Sweden). Resuspension is carried out by gently stirring and swirling with a glass rod. Microscopical controls are necessary in order to check the completeness of lysis and release of vacuoles upon gentle agitation of the suspension. It is important that vacuoles are freed as completely as possible from adhering cytoplasm. This can be achieved by moving the suspension repeatedly through a Pasteur pipette. Isolation and Purification of Vacuoles. The product of lysis is placed in centrifuge tubes and carefully overlayered with about equal volumes of 7.50% and 7.0% (w/v) Ficoll dissolved in 0.1 M mannitol or sorbitol in 10 mM citrate buffer, pH 6.8. After centrifugation in a swing-out rotor (15 minutes, 8500 g) the vacuoles have formed a white layer on top of this system from where they are carefully removed. Turbidity in the Ficoll layers indicates the presence of small vacuoles and lipid granules which have not flotated to the top under the conditions used. The sediment contains, among other products of lysis, vacuoles that have not been completely liberated from the protoplasts. If the preparation of flotated vacuoles is contaminated with lipid granules, a further purification may be achieved by sedimenting the vacuoles (30 minutes, 2000 g) after having slowly diluted the Ficoll with 1.5 volumes of 0.6 M sorbitol in 10 mM citrate buffer, pH 6.8. Critique. Osmotic lysis seems to change the permeability properties of the tonoplast. The above procedure, therefore, is not suitable for investigating the micromolecular contents of vacuoles. To study the low molecular weight contents osmotic lysis can be substituted by metabolic lysis of yeast protoplasts in the presence of glucose and chelating agents. ~,3 In 2K. J. Indge, J. Gen. Microbiol. 51, 433 (1968). ~K. J. Indge, J. Gen. Microbiol. 51, 441 (1968).
574
SUBCELLULAR FRACTIONS DERIVED FROM PLANT TISSUE
[59]
this case sedimentation of vacuoles or flotation in isotonic gradients of Ficoll, sorbitol, and sucrose having higher densities than those described above must be applied. Properties of Isolated Yeast Vacuoles. At 0 ° the isolated yeast vacuoles are stable for many hours. They are osmotically active, but the tonoplasts of vacuoles obtained from osmotically lysed protoplasts seem to be rather permeable with regard to micromolecules. The lysosomal nature of yeast vacuoles is demonstrated by the presence of various digestive enzymes in a concentrated form. The preparations are virtually free of enzymes localized in mitochondria or in the cytoplasmic matrix. Considerable fractions of the total amino acid pools and polyphosphates have been detected in fractions containing vacuoles prepared from metabolically lysed protoplasts. 4 Suitable Objects. Fungi which have been used for protoplast formation and subsequent liberation of vacuoles comprehend Saccharomyces cerevisiae, 1 Saccharomyces carlsbergensis, 3 Candida utilis, ~ Fusarum culm o r u m 2 A variety of fungi have been used for preparing protoplasts. 7 Macroconidia of Neurospora crassa whose cell walls had been weakened by the action of lytic enzymes can be osmotically lysed and intact vacuoles be released2 The observation of free vacuoles in preparations of higher plant protoplasts 9 suggests that the above technique may be successfully modified for the isolation of higher plant vacuoles. Isolation of Meristematic Vacuoles from Plant Tissues The subsequently described procedure for the isolation of small vacuoles (up to 2 ~m in diameter) has been worked out using maize seedlings. 1° Tissue Homogenization. Medium: 0.5 M sorbitol, 50 mM Tris.HC1 buffer pH 7.6, 1 mM EDTA. Root tips, ca. 5 mm in length, are washed in ice-cold distilled water and blotted on filter paper. Five grams of wet tips are placed in a mortar together with 5 ml of medium and 2.5 g of quartz sand (grain size 0.1-0.8 mm). The grinding is done with a minimum of pressure; reduction of the quartz to small pieces is avoided. The resulting brei is diluted with ca. 20 ml of medium and subjected to an initial cen' K . J. Indge, 1. Gen. Microbiol. 51, 447 (1968). A. Wiemken, unpublished results. 6 I. Garcia-Acha, F. Lopez-Belmonte, and J. R. Villanueva, Can. J. Microbiol. 13, 433 (1967). ~J. R. Villanueva, in "The Fungi" (G. C. Ainsworth and A. S. Sussman, eds.), Vol. 2. Academic Press, New York, 1966. 8 p. Matile, Cytobiologie 3, 324 (1971). ~E. C. Cocking, Nature (London) 187, 962 (1960). lop. Matile, Planta 79, 181 (1968).
[59]
VACUOLES AND SPHEROSOMES
575
trifugation (10 minutes, 500 g; elimination of sand, starch, cell walls, etc.). Isolation of Vacuoles. A mitochondrial fraction which contains the vacuoles is sedimented (15 minutes, 20,000 g) and carefully resuspended in medium (0.5 M sorbitol, l0 mM Tris .HC1 buffer pH 7.6). The densely packed white layer at the bottom of the sediment which contains mainly starch is not resuspended. After a second sedimentation under the above conditions the washed mitochondrial fraction is resuspended in ca. 4 ml of medium and loaded in portions of 1 ml onto discontinuous gradients of sucrose [e.g., 2 ml layers of 40% and 15% (w/v) sucrose]. After centrifugation in a swing-out rotor (2 hours, 125,000 g) mitochondria and peroxisomes are present in the sediment; larger vacuoles are trapped at the surface of the 15% sucrose layer, small vacuoles at the surface of the 40% sucrose layer, respectively. The corresponding bands are isolated with a Pasteur pipette, diluted with medium, and the vacuoles collected by sedimentation (15 minutes, 20,000 g). If continuous linear gradients of sucrose (15% to 40%) are used instead of discontinuous gradients, the vacuoles heavier than 15 % sucrose are diffusely distributed in the gradient. This behavior is due to the gradual changes of physical properties of vacuoles in the course of vacuolation which takes place in the root meristem. Properties of Isolated Meristematic Vacuoles. The preparations are practically free of soluble cytoplasmic, mitochondrial, and peroxisomal enzymes. The lysosomal nature of these vacuoles is demonstrated by the presence of a variety of hydrolases in a concentrated form. 1° Suitable Objects. Similar techniques have been used for isolating lysosomes from root meristem, 1~ tomato, 12 and other tissues. ~,14 Of interest for the isolation of vacuoles are the laticifers of certain species whose latex contains numerous small vacuoles. In this case, tissue extraction can be achieved simply by tapping the extended system of communicating laticifers.~,~6 Isolation of Aleurone Vacuoles Aleurone grains (protein bodies) represent specialized vacuoles in reserve tissues of seeds. They are packed with reserve proteins and phytate 11p. Coulomb, C. R. .4cad. Sci. Ser. D 267, 2133 (1968). 12E. Heftmann, Cytobios 3, 129 (1971). ~P. Coulomb, C. R. .4cad. Sci. Ser. D 269, 2543 (1969); 1. Microsc. (Paris) 11, 299 (1971). ~4W. Iten and P. Matile, 1. Gen. Microbiol. 61, 301 (1970). 1~S. Pujarniscle, Physiol. Veg. 6, 27 (1968). ~*P. Matile, B. Jans, and R. Rickenbacher, Biochem. Physiol. Pflanzen 161, 447 (1970).
576
SUBCELLULARFRACTIONS DERIVED FROM PLANT TISSUE
[59]
which, in resting seeds, provide the organelle with considerable stability. Although intact aleurone grains are usually obtained upon grinding of reserve tissues, the vacuolar membrane may be injured and, therefore, soluble constituents be lost. Mobilization of reserves taking place in the course of germination results in a considerable swelling of aleurone vacuoles. Extraction of intact organelles from germinating seeds is therefore difficult to achieve. Moreover, these vacuoles are labile after extraction. The physical properties of aleurone grains from ungerminated seeds are similar to those of starch grains and fragments of cell walls. Hence, isolation and purification require centrifugation techniques that take advantage of small differences in density or sedimentation velocity. In the isolation technique ~7 described below, some possibilities for overcoming the difficulties mentioned above are evident. A l e u r o n e Grains f r o m Cotton Seeds. Cotton seeds contain an oleaginous reserve tissue (contaminating starch excluded). Loss of water-soluble constituents from extracted aleurone grains is avoided by employing glycerol as a nonaqueous medium. The extraction of 10 g of dehulled dry seeds is carried out in a Waring Blendor in the presence of 30 ml of glycerol. The extract is strained through cheesecloth. Large debris is subsequently removed by centrifuging the extract at 1100 g for 5 minutes. Thereafter aleurone grains are sedimented at 41,000 g (20 minutes). The high viscosity of the glycerol allows a satisfactory separation of the large and heavy aleurone grains from other cytoplasmic particles. The sediment of aleurone grains is resuspended in glycerol and sedimented again under the above conditions. Result: 75% of the total proteins present in the precentrifuged extract is obtained in the fraction of isolated aleurone grains. Acid protease ( 1 0 0 % ) and acid phosphatase ( 7 7 % ) are concentrated in this fraction whereas a soluble cytoplasmic enzyme, ethanol dehydrogenase, is completely absent. 17 Suitable Objects. The following seeds have been used for the isolation of intact aleurone grains, or subfractions thereof, using a variety of centrifugation techniques: wheat, TM barley, 19 rice, 2° pea, 21,22 soybean, 23,2~ broad bean, 25 cotton, z6 hemp, ~7,2s and squash. 29 I~L. Y. Yatsu and T. J. Jacks, Arch. Biochem. Biophys. 124, 466 (1968). ~sj. S. D. Graham, R. K. Morton, and J. K. Raison, Aust. J. Biol. Sci. 16, 375 (1963). ~gR. L. Ory and K. W. Henningscn, Plant Physiol. 44, 1488 (1969). ~oH. Mitsuda, K. Yasumoto, K. Murakami, T. K. Kusano, and H. Kishida, Agr. Biol. Chem. 31, 293 (1967). 21j. E. Varner and G. Schidlovsky, Plant Physiol. 38, 139 (1963). 22p. Matile, Z. Pflanzenphysiol. 58, 365 (1968).
[59]
VACUOLES AND SPHEROSOMES
577
Spherosomes Spherosomes represent vacuole-like organelles that are characterized by a high content in tdglycerides (lipid droplets). They are ubiquitous in the plant kingdom. The oleaginous reserve cells of certain seeds contain numerous large spherosomes that are extremely rich in lipids. Since the density of these organelles is less than unit, they can easily be flotated in appropriate media. The following procedure has been worked out for tobacco endosperm? °,3' It closely resembles techniques that have been used for isolating spherosomes from other seeds. Isolation of Spherosomes from Tobacco Seeds A layer of resting, soaked, or germinated seeds is placed on a cooled glass plate. The seeds are covered with medium containing 0.4 M mannitol and I 0 mM Tris-HCI pH 7.4. They are now gently squeezed under a second glass plate, which is lightly pressed and moved. The soft endosperm can thereby be extracted without damaging the embryos or seedlings. The resulting milky suspension is filtered through cotton wool (removal of seedlings and seed coats). The extract is subsequently placed in centrifuge tubes and carefully overlayered with an equal volume of 0.2 M mannitol. After centrifugation (30 minutes, 700g) the flotated spherosomes are aspirated with a Pasteur pipette. Result. At low temperature isolated tobacco endosperm spherosomes are stable for many hours if kept in the mannitol medium or even in distilled water. At room temperature they rapidly burst and coalesce to smaller lipid droplets. Over 90% of the total endosperm lipids are contained in the isolate. The existence of a limiting membrane is indicated by the presence of phospholipids. Spherosomes isolated from resting and germinated seeds contain lipase and other hydrolase activities in a concentration form. Suitable Objects. Both oleaginous reserve tissues and nonoily tissues have been used for isolating spherosomes: Douglas fir seeds, 32 peanut
~'M. P. Tombs, Plant Physiol. 42, 797 (1967). aK. Saio and T. Watanabe, Agr. Biol. Chem. 30, 1133 (1966). ~G. F. I. Morris, D. A. Thurman, and D. Boulter, Phytochemistry 9, 1707 (1970). ~N. S. T. Lui and A. M. Altschul, Arch. Biochem. Biophys. 121, 678 (1967). 2TA. J. St. Angelo, L. Y. Yatsu, and A. M. Altschul, Arch. Bioehem. Biophys. 124, 199 (1968). 28A. J. St. Angelo, R. L. Ory, and H. J. Hansen, Phytochemistry 8, 1135 (1969). 2~j. N. A. Lott, P. L. Larsen, and J. J. Darley, Can. J. Bot. 49, 1777 (1971). ~op. Matile and J. Spichiger, Z. Pflanzenphysiol. 58, 277 (1968). 31j. U. Spichiger, Planta 89, 56 (1969). '2T. M. Ching, Lipids 3, 482 (1968).
578
SUBCELLULARFRACTIONS DERIVED FROM PLANT TISSUE
[60]
cotyledons, 33 castor bean endosperm, 34 cotyledons of legume seeds, as cotton seeds, onion bulbs, and cabbages. 36 nT. J. Jacks, L. Y. Yatsu, and A. M. Altschul, Plant Physiol. 42, 585 (1967). ~4R. L. Ory, L. Y. Yatsu, and H. W. Kircher, Arch. Biochem. Biophys. 123, 255 (1968). ~H. H. Mollenhauer and C. Totten, J. Cell Biol. 48, 533 (1971). ~L. Y. Yatsu, T. J. Jacks, and T. P. Hensarling, Plant Physiol. 48, 675 (1971).
[60] The Isolation of Plant Protoplasts B y EDWARD C. COCKING
It has recently been emphasized that isolated higher plant protoplasts are naked cells and that being cells they can under suitable conditions be induced to grow and divide. 1 These naked cells are in many respects ideal single-cell cultures. They have considerable potential for the cloning of plant cells generally. They are also well suited for studies on the fusion of plant cells, and the resultant heterokaryons serve as starting cultures for experimental investigation of the possibility of somatic hybridization of plants. 2 One remarkable attribute of these naked cells is that they can rebuild a cell wall. This resynthesis has been fully described and discussed previously2a; physiologically it results in the development of cells with the growth potential of cells as ordinarily cultured. Increasingly, plant biochemists and plant physiologists have become interested in such isolated protoplasts; moreover the removal of the cell wall exposes the plasmalemma directly to the influence of the culture medium. The presence of a cell wall is a complicating factor in studies on uptake generally, particularly so if the investigator is interested in the possible uptake of macromolecules or particles by the isolated protoplasts since the cell wall acts as a very efficient ultrafilter. Studies on the fusion and endocytotic activity of these naked cells have focused attention on the properties of the plasmalemma. Willison et al. 3 have postulated a mechanism for the pinocytosis (endocytosis) of latex spheres by tomato fruit protoplasts. Protoplasts were isolated from tomato fruit locule tissue obtained from tomato plants grown under controlled conditions. 4 Watering of plants should not be carried out for several hours 1E. C. Cocking, Annu. Rev. Plant Physiol. 23, 29 (1972). 2E. C. Cocking, Scienza Tecnica 74 (in press). 21E. C. Cocking, in "Dynamic Aspects of Plant Ultrastructure" (A. W. Robards, ed.), 1973 (in press). 3j. H. M. Willison, B. W. W. Grout, and E. C. Cocking, J. Bioenerg. 2, 371 (1971). ~J. W. Davies and E. C. Cocking, Planta 67, 242 (1965).
SUBCELLULAR FRACTIONS DERIVED FROM PLANT TISSUE
[59]
[59] V a c u o l e s a n d S p h e r o s o m e s
By PHILIPPE MATILE and ANDRES WIEMKEN Vacuoles Plant vacuoles cannot be isolated according to a universal scheme of cell fractionation. This circumstance is chiefly due to the wide variation of physical properties of these organelles (size, shape, density, etc.). In addition, the mechanically resistant cell walls require drastic procedures of tissue homogenization which inevitably result in the destruction of large vacuoles. The disintegration of parenchymatous tissues using conventional techniques will always result in bursting and fragmentation of these organelles. The soluble substances contained in these vacuoles are, therefore, mainly present in the soluble fraction of extracts. On the other hand, meristematic cells may contain small vacuoles that are not destroyed upon grinding or blending of the tissue. It appears that the preparation of vacuoles depends largely on the selection of suitable objects. One of these is represented by spheroplasts I whose fragility permits a gentle lysis and the release of even large vacuoles. Isolation of Vacuoles from Spheroplasts The procedure involves the following steps: (i) preparation of osmotically stabilized spheroplasts by enzymatic digestion of the cell walls, (ii) osmotic lysis of spheroplasts and release of intact vacuoles, and (iii) isolation and purification of vacuoles, la The following method has been successfully used in several laboratories. Spheroplasts. A growing population of Saccharomyces cerevisiae is collected by centrifugation. After washing with distilled HzO, the cells are suspended in a medium containing buffered osmoticum (0.6 M sorbitol, 10 mM citrate buffer, pH 6.8) supplemented with 0.14 M cysteamineHCI (approx. 1 × 109 cells/ml). The suspension is indubated for 20 minutes at 27 °. The cells are now sedimented and washed once with the above osmoticum (no supplement). The subsequent incubation in the presence of snail gut enzyme (Helicase, lyophilizdd product of l'Industrie Biologique Fran~aise S.A. Gennevilliers, Seine, France; Glusulase, or corresponding preparations) is carried out under the following conditions: 1 ml of Helicase solution (30 mg of enzyme p~r milliliter of 1The term spheroplast has now been generally adopted in cases wh~re the question whether the wall has been completely eliminated or not is irrelevant. lap. Matile and A. Wiemken, Arch. Mikrobiol. 56, 148 (1967).
[59]
VACUOLES AND SPHEROSOMES
573
osmoticum) is used for suspending 1 g of packed cells; incubation at 30 ° in an oscillating water bath. Within 45-90 minutes the cells are converted into protoplasts (controls in phase contrast microscope; note the spherical shape of spheroplasts) which are now cooled down to 0 °, the subsequent working temperature. Separation of spheroplasts from the Helicase solution is achieved by centrifugation in a swing-out rotor (20 minutes, 1000 g) through a gradient ranging from 0.6 M sucrose to 0.6 M sorbitol. The sedimented protoplasts are washed twice with a solution of 0.7 M sorbitol. Note that the successful conversion of yeast cells into protoplasts is possible only in growing cells. Resting cells (e.g., commercially available cakes of bakers' yeast) must be incubated in an appropriate culture medium for a few hours prior to the preparation of protoplasts. Liberation of Vacuoles. Osmotic lysis is induced by placing the packed protoplasts in a medium containing 0.1 M mannitol or sorbitol, and 8% (w/v) Ficoll (Pharmacia, Uppsala, Sweden). Resuspension is carried out by gently stirring and swirling with a glass rod. Microscopical controls are necessary in order to check the completeness of lysis and release of vacuoles upon gentle agitation of the suspension. It is important that vacuoles are freed as completely as possible from adhering cytoplasm. This can be achieved by moving the suspension repeatedly through a Pasteur pipette. Isolation and Purification of Vacuoles. The product of lysis is placed in centrifuge tubes and carefully overlayered with about equal volumes of 7.50% and 7.0% (w/v) Ficoll dissolved in 0.1 M mannitol or sorbitol in 10 mM citrate buffer, pH 6.8. After centrifugation in a swing-out rotor (15 minutes, 8500 g) the vacuoles have formed a white layer on top of this system from where they are carefully removed. Turbidity in the Ficoll layers indicates the presence of small vacuoles and lipid granules which have not flotated to the top under the conditions used. The sediment contains, among other products of lysis, vacuoles that have not been completely liberated from the protoplasts. If the preparation of flotated vacuoles is contaminated with lipid granules, a further purification may be achieved by sedimenting the vacuoles (30 minutes, 2000 g) after having slowly diluted the Ficoll with 1.5 volumes of 0.6 M sorbitol in 10 mM citrate buffer, pH 6.8. Critique. Osmotic lysis seems to change the permeability properties of the tonoplast. The above procedure, therefore, is not suitable for investigating the micromolecular contents of vacuoles. To study the low molecular weight contents osmotic lysis can be substituted by metabolic lysis of yeast protoplasts in the presence of glucose and chelating agents. ~,3 In 2K. J. Indge, J. Gen. Microbiol. 51, 433 (1968). ~K. J. Indge, J. Gen. Microbiol. 51, 441 (1968).
574
SUBCELLULAR FRACTIONS DERIVED FROM PLANT TISSUE
[59]
this case sedimentation of vacuoles or flotation in isotonic gradients of Ficoll, sorbitol, and sucrose having higher densities than those described above must be applied. Properties of Isolated Yeast Vacuoles. At 0 ° the isolated yeast vacuoles are stable for many hours. They are osmotically active, but the tonoplasts of vacuoles obtained from osmotically lysed protoplasts seem to be rather permeable with regard to micromolecules. The lysosomal nature of yeast vacuoles is demonstrated by the presence of various digestive enzymes in a concentrated form. The preparations are virtually free of enzymes localized in mitochondria or in the cytoplasmic matrix. Considerable fractions of the total amino acid pools and polyphosphates have been detected in fractions containing vacuoles prepared from metabolically lysed protoplasts. 4 Suitable Objects. Fungi which have been used for protoplast formation and subsequent liberation of vacuoles comprehend Saccharomyces cerevisiae, 1 Saccharomyces carlsbergensis, 3 Candida utilis, ~ Fusarum culm o r u m 2 A variety of fungi have been used for preparing protoplasts. 7 Macroconidia of Neurospora crassa whose cell walls had been weakened by the action of lytic enzymes can be osmotically lysed and intact vacuoles be released2 The observation of free vacuoles in preparations of higher plant protoplasts 9 suggests that the above technique may be successfully modified for the isolation of higher plant vacuoles. Isolation of Meristematic Vacuoles from Plant Tissues The subsequently described procedure for the isolation of small vacuoles (up to 2 ~m in diameter) has been worked out using maize seedlings. 1° Tissue Homogenization. Medium: 0.5 M sorbitol, 50 mM Tris.HC1 buffer pH 7.6, 1 mM EDTA. Root tips, ca. 5 mm in length, are washed in ice-cold distilled water and blotted on filter paper. Five grams of wet tips are placed in a mortar together with 5 ml of medium and 2.5 g of quartz sand (grain size 0.1-0.8 mm). The grinding is done with a minimum of pressure; reduction of the quartz to small pieces is avoided. The resulting brei is diluted with ca. 20 ml of medium and subjected to an initial cen' K . J. Indge, 1. Gen. Microbiol. 51, 447 (1968). A. Wiemken, unpublished results. 6 I. Garcia-Acha, F. Lopez-Belmonte, and J. R. Villanueva, Can. J. Microbiol. 13, 433 (1967). ~J. R. Villanueva, in "The Fungi" (G. C. Ainsworth and A. S. Sussman, eds.), Vol. 2. Academic Press, New York, 1966. 8 p. Matile, Cytobiologie 3, 324 (1971). ~E. C. Cocking, Nature (London) 187, 962 (1960). lop. Matile, Planta 79, 181 (1968).
[59]
VACUOLES AND SPHEROSOMES
575
trifugation (10 minutes, 500 g; elimination of sand, starch, cell walls, etc.). Isolation of Vacuoles. A mitochondrial fraction which contains the vacuoles is sedimented (15 minutes, 20,000 g) and carefully resuspended in medium (0.5 M sorbitol, l0 mM Tris .HC1 buffer pH 7.6). The densely packed white layer at the bottom of the sediment which contains mainly starch is not resuspended. After a second sedimentation under the above conditions the washed mitochondrial fraction is resuspended in ca. 4 ml of medium and loaded in portions of 1 ml onto discontinuous gradients of sucrose [e.g., 2 ml layers of 40% and 15% (w/v) sucrose]. After centrifugation in a swing-out rotor (2 hours, 125,000 g) mitochondria and peroxisomes are present in the sediment; larger vacuoles are trapped at the surface of the 15% sucrose layer, small vacuoles at the surface of the 40% sucrose layer, respectively. The corresponding bands are isolated with a Pasteur pipette, diluted with medium, and the vacuoles collected by sedimentation (15 minutes, 20,000 g). If continuous linear gradients of sucrose (15% to 40%) are used instead of discontinuous gradients, the vacuoles heavier than 15 % sucrose are diffusely distributed in the gradient. This behavior is due to the gradual changes of physical properties of vacuoles in the course of vacuolation which takes place in the root meristem. Properties of Isolated Meristematic Vacuoles. The preparations are practically free of soluble cytoplasmic, mitochondrial, and peroxisomal enzymes. The lysosomal nature of these vacuoles is demonstrated by the presence of a variety of hydrolases in a concentrated form. 1° Suitable Objects. Similar techniques have been used for isolating lysosomes from root meristem, 1~ tomato, 12 and other tissues. ~,14 Of interest for the isolation of vacuoles are the laticifers of certain species whose latex contains numerous small vacuoles. In this case, tissue extraction can be achieved simply by tapping the extended system of communicating laticifers.~,~6 Isolation of Aleurone Vacuoles Aleurone grains (protein bodies) represent specialized vacuoles in reserve tissues of seeds. They are packed with reserve proteins and phytate 11p. Coulomb, C. R. .4cad. Sci. Ser. D 267, 2133 (1968). 12E. Heftmann, Cytobios 3, 129 (1971). ~P. Coulomb, C. R. .4cad. Sci. Ser. D 269, 2543 (1969); 1. Microsc. (Paris) 11, 299 (1971). ~4W. Iten and P. Matile, 1. Gen. Microbiol. 61, 301 (1970). 1~S. Pujarniscle, Physiol. Veg. 6, 27 (1968). ~*P. Matile, B. Jans, and R. Rickenbacher, Biochem. Physiol. Pflanzen 161, 447 (1970).
576
SUBCELLULARFRACTIONS DERIVED FROM PLANT TISSUE
[59]
which, in resting seeds, provide the organelle with considerable stability. Although intact aleurone grains are usually obtained upon grinding of reserve tissues, the vacuolar membrane may be injured and, therefore, soluble constituents be lost. Mobilization of reserves taking place in the course of germination results in a considerable swelling of aleurone vacuoles. Extraction of intact organelles from germinating seeds is therefore difficult to achieve. Moreover, these vacuoles are labile after extraction. The physical properties of aleurone grains from ungerminated seeds are similar to those of starch grains and fragments of cell walls. Hence, isolation and purification require centrifugation techniques that take advantage of small differences in density or sedimentation velocity. In the isolation technique ~7 described below, some possibilities for overcoming the difficulties mentioned above are evident. A l e u r o n e Grains f r o m Cotton Seeds. Cotton seeds contain an oleaginous reserve tissue (contaminating starch excluded). Loss of water-soluble constituents from extracted aleurone grains is avoided by employing glycerol as a nonaqueous medium. The extraction of 10 g of dehulled dry seeds is carried out in a Waring Blendor in the presence of 30 ml of glycerol. The extract is strained through cheesecloth. Large debris is subsequently removed by centrifuging the extract at 1100 g for 5 minutes. Thereafter aleurone grains are sedimented at 41,000 g (20 minutes). The high viscosity of the glycerol allows a satisfactory separation of the large and heavy aleurone grains from other cytoplasmic particles. The sediment of aleurone grains is resuspended in glycerol and sedimented again under the above conditions. Result: 75% of the total proteins present in the precentrifuged extract is obtained in the fraction of isolated aleurone grains. Acid protease ( 1 0 0 % ) and acid phosphatase ( 7 7 % ) are concentrated in this fraction whereas a soluble cytoplasmic enzyme, ethanol dehydrogenase, is completely absent. 17 Suitable Objects. The following seeds have been used for the isolation of intact aleurone grains, or subfractions thereof, using a variety of centrifugation techniques: wheat, TM barley, 19 rice, 2° pea, 21,22 soybean, 23,2~ broad bean, 25 cotton, z6 hemp, ~7,2s and squash. 29 I~L. Y. Yatsu and T. J. Jacks, Arch. Biochem. Biophys. 124, 466 (1968). ~sj. S. D. Graham, R. K. Morton, and J. K. Raison, Aust. J. Biol. Sci. 16, 375 (1963). ~gR. L. Ory and K. W. Henningscn, Plant Physiol. 44, 1488 (1969). ~oH. Mitsuda, K. Yasumoto, K. Murakami, T. K. Kusano, and H. Kishida, Agr. Biol. Chem. 31, 293 (1967). 21j. E. Varner and G. Schidlovsky, Plant Physiol. 38, 139 (1963). 22p. Matile, Z. Pflanzenphysiol. 58, 365 (1968).
[59]
VACUOLES AND SPHEROSOMES
577
Spherosomes Spherosomes represent vacuole-like organelles that are characterized by a high content in tdglycerides (lipid droplets). They are ubiquitous in the plant kingdom. The oleaginous reserve cells of certain seeds contain numerous large spherosomes that are extremely rich in lipids. Since the density of these organelles is less than unit, they can easily be flotated in appropriate media. The following procedure has been worked out for tobacco endosperm? °,3' It closely resembles techniques that have been used for isolating spherosomes from other seeds. Isolation of Spherosomes from Tobacco Seeds A layer of resting, soaked, or germinated seeds is placed on a cooled glass plate. The seeds are covered with medium containing 0.4 M mannitol and I 0 mM Tris-HCI pH 7.4. They are now gently squeezed under a second glass plate, which is lightly pressed and moved. The soft endosperm can thereby be extracted without damaging the embryos or seedlings. The resulting milky suspension is filtered through cotton wool (removal of seedlings and seed coats). The extract is subsequently placed in centrifuge tubes and carefully overlayered with an equal volume of 0.2 M mannitol. After centrifugation (30 minutes, 700g) the flotated spherosomes are aspirated with a Pasteur pipette. Result. At low temperature isolated tobacco endosperm spherosomes are stable for many hours if kept in the mannitol medium or even in distilled water. At room temperature they rapidly burst and coalesce to smaller lipid droplets. Over 90% of the total endosperm lipids are contained in the isolate. The existence of a limiting membrane is indicated by the presence of phospholipids. Spherosomes isolated from resting and germinated seeds contain lipase and other hydrolase activities in a concentration form. Suitable Objects. Both oleaginous reserve tissues and nonoily tissues have been used for isolating spherosomes: Douglas fir seeds, 32 peanut
~'M. P. Tombs, Plant Physiol. 42, 797 (1967). aK. Saio and T. Watanabe, Agr. Biol. Chem. 30, 1133 (1966). ~G. F. I. Morris, D. A. Thurman, and D. Boulter, Phytochemistry 9, 1707 (1970). ~N. S. T. Lui and A. M. Altschul, Arch. Biochem. Biophys. 121, 678 (1967). 2TA. J. St. Angelo, L. Y. Yatsu, and A. M. Altschul, Arch. Bioehem. Biophys. 124, 199 (1968). 28A. J. St. Angelo, R. L. Ory, and H. J. Hansen, Phytochemistry 8, 1135 (1969). 2~j. N. A. Lott, P. L. Larsen, and J. J. Darley, Can. J. Bot. 49, 1777 (1971). ~op. Matile and J. Spichiger, Z. Pflanzenphysiol. 58, 277 (1968). 31j. U. Spichiger, Planta 89, 56 (1969). '2T. M. Ching, Lipids 3, 482 (1968).
578
SUBCELLULARFRACTIONS DERIVED FROM PLANT TISSUE
[60]
cotyledons, 33 castor bean endosperm, 34 cotyledons of legume seeds, as cotton seeds, onion bulbs, and cabbages. 36 nT. J. Jacks, L. Y. Yatsu, and A. M. Altschul, Plant Physiol. 42, 585 (1967). ~4R. L. Ory, L. Y. Yatsu, and H. W. Kircher, Arch. Biochem. Biophys. 123, 255 (1968). ~H. H. Mollenhauer and C. Totten, J. Cell Biol. 48, 533 (1971). ~L. Y. Yatsu, T. J. Jacks, and T. P. Hensarling, Plant Physiol. 48, 675 (1971).
[60] The Isolation of Plant Protoplasts B y EDWARD C. COCKING
It has recently been emphasized that isolated higher plant protoplasts are naked cells and that being cells they can under suitable conditions be induced to grow and divide. 1 These naked cells are in many respects ideal single-cell cultures. They have considerable potential for the cloning of plant cells generally. They are also well suited for studies on the fusion of plant cells, and the resultant heterokaryons serve as starting cultures for experimental investigation of the possibility of somatic hybridization of plants. 2 One remarkable attribute of these naked cells is that they can rebuild a cell wall. This resynthesis has been fully described and discussed previously2a; physiologically it results in the development of cells with the growth potential of cells as ordinarily cultured. Increasingly, plant biochemists and plant physiologists have become interested in such isolated protoplasts; moreover the removal of the cell wall exposes the plasmalemma directly to the influence of the culture medium. The presence of a cell wall is a complicating factor in studies on uptake generally, particularly so if the investigator is interested in the possible uptake of macromolecules or particles by the isolated protoplasts since the cell wall acts as a very efficient ultrafilter. Studies on the fusion and endocytotic activity of these naked cells have focused attention on the properties of the plasmalemma. Willison et al. 3 have postulated a mechanism for the pinocytosis (endocytosis) of latex spheres by tomato fruit protoplasts. Protoplasts were isolated from tomato fruit locule tissue obtained from tomato plants grown under controlled conditions. 4 Watering of plants should not be carried out for several hours 1E. C. Cocking, Annu. Rev. Plant Physiol. 23, 29 (1972). 2E. C. Cocking, Scienza Tecnica 74 (in press). 21E. C. Cocking, in "Dynamic Aspects of Plant Ultrastructure" (A. W. Robards, ed.), 1973 (in press). 3j. H. M. Willison, B. W. W. Grout, and E. C. Cocking, J. Bioenerg. 2, 371 (1971). ~J. W. Davies and E. C. Cocking, Planta 67, 242 (1965).