- Email: [email protected]
[66] Preparation and use of protoplasts for studies of lipid metabolism
774
CELL AND TISSUE TECHNIQUES
[66]
[66] P r e p a r a t i o n a n d U s e o f P r o t o p l a s t s for S t u d i e s o f Lipid Metabolism B y D. N. KUHN and P. K. STUMPF
Protoplasts are cells derived from either plants or fungi that have had their cell wall removed. They offer two major advantages for plant research. First, they can act as a homogeneous, single-celled, higher plant for in vivo metabolism studies and ensure the even distribution of labeled precursors into the cells by avoiding the problem of transport through epidermis, xylem, or phloem cells. Although suspension cell cultures offer a similar advantage, protoplasts can be released from fresh tissue and retain the differentiated function of that tissue. Moreover, production of suspension cell cultures from tissue takes months, and the cells become undifferentiated. Second, protoplasts are an excellent starting material for isolation of intact organelles, since they can be easily lysed. Separation of organelles from protoplast lysates on density gradients allows complete accounting for enzyme activity and is superior to differential centrifugation for subcellular localization studies. Takebe et al. 1,2 have reported the protoplast isolation conditions for a wide variety of plants, using a two-step isolation procedure that produces first isolated cells and then protoplasts. Power and Cocking3 have simplified this method to a single incubation of tissue with a mixture of pectinase and cellulase to produce protoplasts directly. Gamborg and Wetter's "Plant Tissue Culture Methods ''4 booklet gives basic information on protoplast isolation and culture media. The use of protoplasts for isolation of intact organelles is covered in articles by Nishimura et al. 5 and Rathnam and Edwards. 6 Haas et al. 7 have presented an excellent description of the use of protoplasts in lipid and flavonoid metabolic studies. A comprehensive review of plant protoplast methods is planned for an upcoming volume of M e t h o d s in E n z y m o l o g y . 1 I. Takebe, Y. Otsuki, and S. Aoki, Plant Cell Physiol. 9, 115 (1%8). 2 y. Otsuki and I. Takebe, Plant Cell Physiol. 10, 917 (1969). 3 j. B. Power and E. C. Cocking, J. Exp. Bot. 66, 64 (1970). 4 O. L. G a m b o r g and L. R. Wetter, eds., " P l a n t Tissue Culture M e t h o d s . " National Research Council o f Canada, Prairie Regional Laboratory, Saskatoon, Sakatchewan, 1975. '~ M. N i s h i m u r a , D. G r a h a m , and T. A k a z a w a , Plant Physiol. 58, 309 (1976). 6 C. K. M. R a t h n a m and G. E. E d w a r d s , Plant Cell Physiol. 17, 177 (1976). r R. Haas, E. Heinz, G. Popovici, and G. Weissenb6ck, Z. Naturforsch. 34, 854 (1979).
METHODS IN ENZYMOLOGY, VOL. 72
Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181972-8
[66]
PROTOPLASrS IN LIPID METABOLISM STUDIES
775
Preparation of Protoplasts from Spinacia oleracea Leaf Tissue Protoplasts have been isolated from a wide variety of plant tissues (including the bark of white pine) and probably any tissue will yield some protoplasts. Leaves and tissue culture cells are the two most commonly used sources owing to their greater yield of protoplasts. Of particular interest for lipid synthesis studies, protoplasts can be made from germinating castor bean endosperm s and the mesocarp of avocado. 9 The following procedures for protoplast isolation are for spinach leaf tissue. This material was chosen because of its year-round commercial availability in California and because much previous research on lipid metabolism in plants has been done using spinach chloroplasts. Thin, flat, young leaves of spinach (4-8 cm in length) are used since they provide the greatest yield of intact protoplasts with the shortest incubation in the cell wall degrading enzymes. If commercial spinach is used, the leaves should be picked from the bunches, washed, and kept in the dark overnight to remove starch from the chloroplasts before isolating protoplasts. Starch-filled chloroplasts have a high sedimentation coefficient (s) value and can cause lysis of protoplasts during centrifugation by bursting through the plasmalemma. If either greenhouse or growth chamber or hydroponically grown spinach is used, place the whole plant in the dark before isolation. Unfortunately, protoplast yield and quality depend strongly on the quality of the flesh material and the season. With spinach of poor quality, (old, near flowering, or during the winter months), an increase in the length of incubation and amount of starting material are needed to obtain the same yield of protoplasts. The primary concern in preparing material for protoplast isolation is increasing the surface area of the tissue that can be acted on by the cell wall degrading enzymes. For leaf tissue, removal of the upper and lower epidermis by peeling or abrasion followed by slicing into 1-mm strips gives a greater surface for enzyme activity and increases the rate of release of protoplasts. Peeling of the lower epidermis of young spinach leaves, though tedious, can be accomplished, but the yield of protoplasts or rate of release is not much different than with abraded leaves. The epidermis of leaves can be removed by dusting the leaves with Carborundum (320-mesh powder), followed by brushing with a soft artist's brush.l° Similar results can be obtained by using a soft nylon toothbrush, and this avoids the problem of removal of Carborundum from the protoplasts. The
s M. Nishimura and H. Beevers, Plant Physiol. 62, 40 (1978). 9 j. Harwood, personal communication. ~o H. Beier and G. Bruening, Virology 64, 272 (1975).
776
CELL AND TISSUE TECHNIQUES
[66]
leaves should be brushed until a color change is seen from light, waxy green to a dark, liquid green. Although it may seem that many cells are damaged by this brushing, microscopic observation will show the vast majority of cells to be still intact and undamaged even after vigorous brushing. The abraded leaf material is then deribbed, weighed, and sliced into 1-mm strips with a new razor blade. Even slightly dull razor blades will crush the thin leaves rather than cut them and cause much more cell damage than the brushing. L e a f slices are then floated on 20 ml of 0.7 M mannitol in a 15 × 100 mm petri dish for at least 0.5 hr. This removes cell contents from broken cells and plasmolyzes the protoplasts, pulling them away from the cell wall. The 0.7 M mannitol is removed and replaced with the enzyme solution; the enzyme solution is vacuum infiltrated into the plasmolyzed leaf tissue. Petri dishes are placed in a small desiccator and house vacuum is applied twice for a period of 3 min each. Usually, bubbles can be seen forming on the surface indicating that the intracellular air spaces of the spongy mesophyll are being filled with the enzyme solution. Plasmolyzing the leaves before vacuum infiltration allows the enzyme to be more efficiently moved to the cell wall, the site of its action. Incubation is at 30° in a shaking water bath at one excursion per second or less. Incubation can be in the light or dark, the dark being more convenient, as the temperature in the petri dishes is more easily controlled in a covered water bath.
Enzyme Solutions A number of cell wall degrading enzymes are available commercially. Cellulases isolated from the fungus Trichoderma viride work quite well in isolating protoplasts from fresh leaf tissue, when used in conjunction with a mixture of pectinases and hemicellulases. For tissue culture cells, cellulase alone is sufficient for protoplast isolation. The commercially available cellulases (cellulase Onozuka R-10, Kinki Yakult Manufacturing Co., Nishinomiya, Japan; and cellulysin, Calbiochem-Behring Corp., La Jolla, California) and pectinase-hemicellulases (macerozyme R-10, Kinki Yakult; and macerase, Calbiochem) are impure and vary in activity from lot to lot. If a number of protoplast experiments are planned, it is best to sample the available lots of macerase and cellulase and buy enough of both so that all experiments will be done with protoplasts produced by the same enzymes. Because of contamination by proteases, the hydrolytic enzymes should be made the day of the isolation. If a stock is made, it should be stored frozen. The enzyme solution used for isolation of spinach protoplasts consists of 2% cellulysin, 1% macerase, 0.7 M mannitol, and 50 mM
[66]
PROTOPLASTS IN LIPID METABOLISM STUDIES
777
MES, pH 5.5. The pH optima for the cellulases is around 4, but the cells seem to survive the incubation best at pH 5.5. Dextran sulfate has been added to enzyme solutions to increase protoplast release, 2 but is not essential to isolation of protoplasts. For sterile isolation of protoplasts/ the enzyme solution can be filter sterilized with a 0.45-/~m mesh. The ratio of enzyme solution to fresh weight of material is important in the rate of release of protoplasts. Rate of release is critical, since contaminants in the enzyme solution may damage the protoplasts. Therefore, the shortest incubation giving a reasonable yield is desired. Twenty milliliters of enzyme solution per gram fresh weight should give a good yield of protoplasts without being too expensive in terms of ceUulase and macerase.
Protoplast Purification After several hours of incubation at 30° in the cell wall degrative enzymes, protoplasts will begin to appear in solution. Microscopic observation of the tissue slices will show them as aggregates of protoplasts. At 4 hr, protoplasts are harvested by filtering the enzyme solution and softened tissue slices through a 53-/xm mesh. (Any mesh from 50 to 80 /zm is suitable.) The remaining tissue can be stirred in a 0.7 M mannitol solution to release more protoplasts, but violent shaking usually causes greater lysis of protoplasts, and the extra yield from this step is minimal. Protoplasts are pelleted by centrifugation at 133 g for 4 min at room temperature. If Carborundum was used to abrade the tissue, the protoplast-enzyme solution should be overlaid on a pad of 30% sucrose to allow the Carborundum to pellet and to concentrate the protoplasts at the mannitolsucrose interface. Resuspend the pelleted protoplasts in 1 ml of 0.7 M mannitol by gently shaking the tube. Vortexing or pipetting of the pellet causes a great deal of lysis and reduces the yield. Add 40 ml of 0.7 M mannitol, and respin at 133 g for 4 min at room temperature. Wash protoplasts twice in this manner and resuspend in a small volume of 0.7 M mannitol. Plastic centrifuge tubes should be used, as the protoplasts are sticky and will coat glass surfaces. Yield can be determined in milligrams of chlorophyll and compared to chlorophyll per gram fresh weight of spinach. Chlorophyll is estimated by extraction of the protoplasts in 80% acetone as described by MacKinney. 11 After a 4-hour incubation at 30°, approximately 20% of the chlorophyll in the leaf slices is found in the intact protoplasts. Protoplasts tend to clump and to aggregate with membrane fragments, free chloroplasts, and other cell debris. Aggregates can be removed by ~ G. MacKinney, J. Biol. Chem. 140, 315 (1941).
778
CELL AND TISSUE TECHNIQUES
[66]
filtering through a 37-p~m nylon mesh after each resuspension in the washing procedure. Clumping does not appear to affect uptake or metabolism of lipid precursors by the protoplasts, but causes problems when protoplast lysates are prepared for organelle isolation on density gradients. Addition of salts to the 0.7 M mannitol washing medium and incubation medium will stabilize protoplasts and decrease spontaneous lysis. Stabilizing media range from the simple, 20 mM CaCl~ or l0 mM arginine, ~2 to the complex, B5 medium without sucrose as described in Gamborg and Wetter 4 or a salt solution described by Frearson et al. 13The precise mode of action of the salts in stabilizing protoplasts is unknown, although the favorable effects of polycations on membranes is involved. The wash medium should be adjusted to pH 7.0 after addition of salts to minimize aggregation of the protoplasts seen at low pH. Incubation Procedures
Protoplasts can act like single-celled higher plants without a cell wall. Removal of the cell wall does not markedly change the cell's lipid composition 7 or ability to synthesize and desaturate fatty acids or esterify them into phospho- and galactolipids. 14 Metabolism studies for lipid synthesis are done in a temperature-regulated water bath (Aminco 5-4154 photosynthesis refrigerated-heated Warburg bath) with a light source giving 450 ~einsteins/m2/sec in the photosynthetically active range. Concentrations of protoplasts should be kept around 200/zg of chlorophyll per milliliter to prevent shading of protoplasts by other protoplasts. The incubation should be done with very slow shaking (30 excursions per minute if possible) to keep the protoplasts from settling out of the solution and to keep the medium oxygenated. More vigorous shaking will cause protoplast lysis during prolonged incubations. At 23° and 450 p.einsteins/m"/sec, uptake of [i~C]acetate into protoplasts is linear for 3 hr. For spinach protoplasts, or other photosynthetic protoplasts, metabolic activity can be quickly determined by measuring CO2-dependent 02 evolution with an oxygen electrode. '~ Rates of over 100/zmol per milligram of chlorophyll per hour indicate that the protoplasts have retained "normal" metabolic activity. Protoplasts can be stored in the dark at 4° at pH 7.0 for 24 hr with little loss of metabolic activity as measured by CO2-dependent 02 evolution or COz fixation? 4'~
12 A. ~ E. ~4 D. 1~ M.
Altman, R. K a u r - S a w h n e y , and A. W. Galston, Plant Physiol. 60, 570 (1977). M. Frearson, J. B. Power, and E. C. Cocking, Dev. Biol. 33, 130 (1973). N. K u h n , Doctoral Dissertation, University of California at Davis, 1980. N i s h i m u r a and T. Akazawa, Plant Physiol. 55, 712 (1975).
[66]
PROTOPLASTS IN LIPID METABOLISM STUDIES
ACETATE INCORPORATION ~
779
INTO LIPID VS. pH
oPROTOPLASTS
1000
100
r d "7-
I J T
DJ Fb-Ill (.)
W '~ I--klA
co 500 hi
co I,I
O
O
Z
Z
]00
~
CHLOROPLASTS
I
I
60
[
I
70
I
10
I
80
t
pH FtG. 1. Acetate incorporation into lipid [CHCIa: CHaOH (2:1) soluble material] as a function of pH for spinach protoplasts (left ordinate) and spinach chloroplasts (right ordinate). Uptake by protoplasts © © was under conditions listed in this chapter. Spinach chloroplast data (D []) are from Nakamura and Yamada. t"
Incubation Media and Radioactive Precursors
Protoplasts should be incubated in a pH-buffered medium of the same osmotic strength as that in which they were isolated. For spinach protoplasts, 0.7 M mannitol, B5 salts without sucrose, and MES 50 mM pH 5.5 medium is used. Metabolism of lipid precursors such as acetate depend strongly on pH (see Fig. 1). 1~ Incorporation of longer-chain fatty acids ~ Y. Nakamura and M. Yamada, Plant Cell Physiol. 16, 139 (1975).
780
CELL AND TISSUE TECHNIQUES
[66]
(palmitate, oleate) into protoplasts can be accomplished by dissolving them in small amounts of ethylene glycol monomethyl ether (EGME). Addition of 10 /zl of E G M E and fatty acids to 1 ml of protoplasts in solution appears to have no deleterious effect on the protoplasts, and 90% o f the labeled long-chain fatty acids are found associated with the protoplasts after 1 hr of incubation. Incubations up to 8 hr can be accomplished without significant contamination by bacteria as mannitol is a poor growth medium for bacteria. For longer incubations, protoplasts can be isolated under sterile conditions and incubation up to 48 hr accomplished. For such long incubations, a richer culture medium should be used to prolong protoplast life. 4 Extraction o f Lipids f r o m Protoplasts
After incubation, protoplasts can be centrifuged out of the incubation medium and washed with 0.7 M mannitol; the pellet can be extracted for lipids. N o homogenization is necessary, and lipids are easily extracted by the addition of 2 ml of hexane-isopropanol (3:2) '7 or c h l o r o f o r m methanol (1:2), '8 followed by thorough vortexing and centrifugation. If necessary, the pellet can be reextracted by the same methods. Use of Protoplasts in Subcellular Localization Studies Protoplasts are an excellent starting material for isolation of intact organelles and provide a system for subcellular localization of enzymes that can account for all of a particular e n z y m e activity in the cell. t9 By gently lysing protoplasts and separating the organelles on a density gradient, one can determine not only that an e n z y m e is found in an organelle, but whether it is found in only one organelle or in several or in the cytoplasm. Suitable soluble marker enzymes for the organelles facilitate organelle location in the gradient fractions and allow calculation of organelle breakage. By comparing marker e n z y m e activity with the enzyme activity (or protein) of interest in the cytosol fractions at the top of the gradient, one can determine whether the e n z y m e of interest occurs normally in the cytosol or can be accounted for by leakage from an organelle. The shapes and positions o f peaks in the gradient o f marker e n z y m e activity and the e n z y m e being studied can be c o m p a r e d to reveal that e n z y m e ' s subcellular location (Fig. 2). Total e n z y m e activity from the fractions can be corn,7 A. Hara and N. S. Radin, Anal. Biochem. 90, 420 (1978). ,8 E. G. Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959). ,9 j. B. Ohlrogge, D. N. Kuhn, and P. K. Stumpf, Proc. Natl. Acad. Sci. U.S.A. 76, 1194 (1979).
0.4
1.2
~
0
1.1 "~ Q
0.2
-
-
'
'
v
Q
= 150 > 1-
0.6
O.
o2
6 75
Q:
0.3
15
1.0
ca to.
4.0 go
-o>
o
== 0.5 i
m
2.0 "~
o
<
tO
20 Fraction
30
FtG. 2. Distribution of acy| carrier protein (ACP) (~g/ml), chlorophyll (/~g/ml), ribulosebisphosphate (RubP2) carboxylase (gmollml/min), ADP-glucose pyrophosphorylase, (/~mol/mFmin), and isocitrate dehydrogenase (/~mol/ml/min) in sucrose gradients of ]ysates of spinach leaf protoplasts. All gradient fractions were 1 ml.19
782
CELL AND TISSUE TECHNIQUES
[66]
pared with enzyme activity in the intact protoplasts to determine what percentage of the total activity has been accounted for in the gradient. Yield of intact organelles is greater than with differential centrifugation, and the separation of organelles with similar s values (chloroplasts and mitochondria) is more distinct.
Lysis of Protoplasts Protoplasts that are to be lysed should be prepared without salts in the enzyme solution or washing medium, to reduce aggregation of organelles. Chloroplasts are especially sensitive to aggregation and will form clumps with other chloroplasts, membrane fragments, etc. To obtain high yields of intact organelles, the lysis of the protoplasts should be done as rapidly as possible, followed by the immediate overlaying of the lysate on the gradient and centrifugation. The protoplast sample to be lysed ( - 1 mg of chlorophyll per 40 ml gradient) is pelleted in 0.7 M mannitol and left as a pellet. Immediately before lysis, remove the 0.7 M mannitol, replace with 3 ml of 0.5 M sucrose, 0.5% BSA, 50 mM Tricine, pH 7.6, for 1 mg of chlorophyll of sample, and rapidly resuspend the protoplasts. This breaking medium is hypotonic and the protoplasts will begin to lyse owing to osmotic shock. The 0.5% BSA coats the organelle membranes and helps reduce aggregation. The resuspended protoplasts are drawn up into a 3-ml syringe with a Luer-lok end that is fitted with either a microemulsifying needle (25 gauge × 2~ inch, Bolab Incorporated, 6 Tinkham Ave., Derry, New Hampshire 03038) or a modified Swinnex filter (Millipore Corp., Bedford, Massachusetts 01730). After 3-6 passes through the microemulsifying needle, all protoplasts should have lysed owing to shearing forces. Replace the microemulsifying needle with a regular syringe needle, layer the lysate onto a 30-55% (w/w) sucrose density gradient and centrifuge for 1 hr at 26,000 rpm and 4° in an SW27 rotor. The sucrose contains 0.5% BSA or 10 mM arginine to reduce aggregation of chloroplasts. Within 1 hr, chloroplasts reach their equilibrium density and mitochondria are close to their equilibrium. Longer centrifugation will result in increased breakage of chloroplasts and formation of large aggregates in the intact chloroplast band. Lysis by using a modified Swinnex is as follows: remove the 0.45-~m grid of a Swinnex filter (1.4 cm) that can be attached to a syringe. Replace the grid with a circle of 20-gin nylon mesh and a circle of Miracloth (Calbiochem). The seal can be tightened by adding an O ring on top of the Miracloth and mesh. The protoplasts are pushed through the Swinnex, and lysis occurs as a result of pressure on the protoplasts by the mesh. The Miracloth removes debris and prevents clogging of the mesh. To lyse
[67]
ISOLATION A N D S U B F R A C F I O N A T I O N OF GI.YOXYSOMES
783
the protoplasts completely, three Swinnex should be used, the first with a 20-t~m mesh, the second and third with a 10-tzm mesh. After passing through the third Swinnex, the lysate is immediately overlaid on a density gradient and centrifuged. Microscopic observation at any step will give a rough estimate of protoplast breakage. Summary Spinach protoplasts are useful in lipid metabolism studies because they provide a homogeneous, differentiated plant tissue that can take up precursors directly from the medium. They are the material of choice for subcellular localization because they can be easily lysed and the intact organelles separated on density gradients. Crude fungal enzyme preparations are used to degrade the cell wall of the spinach leaves, which are stripped of their epidermal cells by brushing and sliced finely to increase the surface for enzyme action. Protoplasts are harvested from the digested leaf slices by filtration and washed and purified by repeated centrifugation. If protoplasts are to be used in metabolism studies, they are stabilized with a salt solution containing divalent cations and incubated in strong light with lipid or fatty acid precursors. For subcellular localization studies, salts are not present in the wash medium to reduce chloroplast aggregation in the gradients and protoplasts are lysed by shearing in a microemulsifying needle or passage through 20- and 10-tzm mesh before sucrose density gradient centrifugation. Comparison with marker enzymes for organelles provides a method for the complete subcellular localization of an enzyme activity.
[67] I s o l a t i o n a n d S u b f r a c t i o n a t i o n o f G l y o x y s o m e s 1
By ANTHONY H. C. HUANG Glyoxysomes were first isolated from the storage fatty endosperm of castor bean seedlings. ''8 They are specialized peroxisomes (microbodies) with the unique function of gluconeogenesis from reserve stores of triacylglycerol. The organelle is surrounded by a unit membrane and contains an amorphous matrix with occasionally a dense core. In addition to the peroxisomal enzymes, catalase, glycolate oxidase, and uricase, S u p p o r t e d b y the N a t i o n a l S c i e n c e F o u n d a t i o n . R. W. B r e i d e n b a c h a n d H . B e e v e r s , Biochem. Biophys. Res. Comrnun. 27, 462 (1967). a H . B e e v e r s , Annu. Rev. Plant Physiol. 30, 159 (1979).
METHODS IN ENZYMOLOGY, VOL. 72
Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181972-8
CELL AND TISSUE TECHNIQUES
[66]
[66] P r e p a r a t i o n a n d U s e o f P r o t o p l a s t s for S t u d i e s o f Lipid Metabolism B y D. N. KUHN and P. K. STUMPF
Protoplasts are cells derived from either plants or fungi that have had their cell wall removed. They offer two major advantages for plant research. First, they can act as a homogeneous, single-celled, higher plant for in vivo metabolism studies and ensure the even distribution of labeled precursors into the cells by avoiding the problem of transport through epidermis, xylem, or phloem cells. Although suspension cell cultures offer a similar advantage, protoplasts can be released from fresh tissue and retain the differentiated function of that tissue. Moreover, production of suspension cell cultures from tissue takes months, and the cells become undifferentiated. Second, protoplasts are an excellent starting material for isolation of intact organelles, since they can be easily lysed. Separation of organelles from protoplast lysates on density gradients allows complete accounting for enzyme activity and is superior to differential centrifugation for subcellular localization studies. Takebe et al. 1,2 have reported the protoplast isolation conditions for a wide variety of plants, using a two-step isolation procedure that produces first isolated cells and then protoplasts. Power and Cocking3 have simplified this method to a single incubation of tissue with a mixture of pectinase and cellulase to produce protoplasts directly. Gamborg and Wetter's "Plant Tissue Culture Methods ''4 booklet gives basic information on protoplast isolation and culture media. The use of protoplasts for isolation of intact organelles is covered in articles by Nishimura et al. 5 and Rathnam and Edwards. 6 Haas et al. 7 have presented an excellent description of the use of protoplasts in lipid and flavonoid metabolic studies. A comprehensive review of plant protoplast methods is planned for an upcoming volume of M e t h o d s in E n z y m o l o g y . 1 I. Takebe, Y. Otsuki, and S. Aoki, Plant Cell Physiol. 9, 115 (1%8). 2 y. Otsuki and I. Takebe, Plant Cell Physiol. 10, 917 (1969). 3 j. B. Power and E. C. Cocking, J. Exp. Bot. 66, 64 (1970). 4 O. L. G a m b o r g and L. R. Wetter, eds., " P l a n t Tissue Culture M e t h o d s . " National Research Council o f Canada, Prairie Regional Laboratory, Saskatoon, Sakatchewan, 1975. '~ M. N i s h i m u r a , D. G r a h a m , and T. A k a z a w a , Plant Physiol. 58, 309 (1976). 6 C. K. M. R a t h n a m and G. E. E d w a r d s , Plant Cell Physiol. 17, 177 (1976). r R. Haas, E. Heinz, G. Popovici, and G. Weissenb6ck, Z. Naturforsch. 34, 854 (1979).
METHODS IN ENZYMOLOGY, VOL. 72
Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181972-8
[66]
PROTOPLASrS IN LIPID METABOLISM STUDIES
775
Preparation of Protoplasts from Spinacia oleracea Leaf Tissue Protoplasts have been isolated from a wide variety of plant tissues (including the bark of white pine) and probably any tissue will yield some protoplasts. Leaves and tissue culture cells are the two most commonly used sources owing to their greater yield of protoplasts. Of particular interest for lipid synthesis studies, protoplasts can be made from germinating castor bean endosperm s and the mesocarp of avocado. 9 The following procedures for protoplast isolation are for spinach leaf tissue. This material was chosen because of its year-round commercial availability in California and because much previous research on lipid metabolism in plants has been done using spinach chloroplasts. Thin, flat, young leaves of spinach (4-8 cm in length) are used since they provide the greatest yield of intact protoplasts with the shortest incubation in the cell wall degrading enzymes. If commercial spinach is used, the leaves should be picked from the bunches, washed, and kept in the dark overnight to remove starch from the chloroplasts before isolating protoplasts. Starch-filled chloroplasts have a high sedimentation coefficient (s) value and can cause lysis of protoplasts during centrifugation by bursting through the plasmalemma. If either greenhouse or growth chamber or hydroponically grown spinach is used, place the whole plant in the dark before isolation. Unfortunately, protoplast yield and quality depend strongly on the quality of the flesh material and the season. With spinach of poor quality, (old, near flowering, or during the winter months), an increase in the length of incubation and amount of starting material are needed to obtain the same yield of protoplasts. The primary concern in preparing material for protoplast isolation is increasing the surface area of the tissue that can be acted on by the cell wall degrading enzymes. For leaf tissue, removal of the upper and lower epidermis by peeling or abrasion followed by slicing into 1-mm strips gives a greater surface for enzyme activity and increases the rate of release of protoplasts. Peeling of the lower epidermis of young spinach leaves, though tedious, can be accomplished, but the yield of protoplasts or rate of release is not much different than with abraded leaves. The epidermis of leaves can be removed by dusting the leaves with Carborundum (320-mesh powder), followed by brushing with a soft artist's brush.l° Similar results can be obtained by using a soft nylon toothbrush, and this avoids the problem of removal of Carborundum from the protoplasts. The
s M. Nishimura and H. Beevers, Plant Physiol. 62, 40 (1978). 9 j. Harwood, personal communication. ~o H. Beier and G. Bruening, Virology 64, 272 (1975).
776
CELL AND TISSUE TECHNIQUES
[66]
leaves should be brushed until a color change is seen from light, waxy green to a dark, liquid green. Although it may seem that many cells are damaged by this brushing, microscopic observation will show the vast majority of cells to be still intact and undamaged even after vigorous brushing. The abraded leaf material is then deribbed, weighed, and sliced into 1-mm strips with a new razor blade. Even slightly dull razor blades will crush the thin leaves rather than cut them and cause much more cell damage than the brushing. L e a f slices are then floated on 20 ml of 0.7 M mannitol in a 15 × 100 mm petri dish for at least 0.5 hr. This removes cell contents from broken cells and plasmolyzes the protoplasts, pulling them away from the cell wall. The 0.7 M mannitol is removed and replaced with the enzyme solution; the enzyme solution is vacuum infiltrated into the plasmolyzed leaf tissue. Petri dishes are placed in a small desiccator and house vacuum is applied twice for a period of 3 min each. Usually, bubbles can be seen forming on the surface indicating that the intracellular air spaces of the spongy mesophyll are being filled with the enzyme solution. Plasmolyzing the leaves before vacuum infiltration allows the enzyme to be more efficiently moved to the cell wall, the site of its action. Incubation is at 30° in a shaking water bath at one excursion per second or less. Incubation can be in the light or dark, the dark being more convenient, as the temperature in the petri dishes is more easily controlled in a covered water bath.
Enzyme Solutions A number of cell wall degrading enzymes are available commercially. Cellulases isolated from the fungus Trichoderma viride work quite well in isolating protoplasts from fresh leaf tissue, when used in conjunction with a mixture of pectinases and hemicellulases. For tissue culture cells, cellulase alone is sufficient for protoplast isolation. The commercially available cellulases (cellulase Onozuka R-10, Kinki Yakult Manufacturing Co., Nishinomiya, Japan; and cellulysin, Calbiochem-Behring Corp., La Jolla, California) and pectinase-hemicellulases (macerozyme R-10, Kinki Yakult; and macerase, Calbiochem) are impure and vary in activity from lot to lot. If a number of protoplast experiments are planned, it is best to sample the available lots of macerase and cellulase and buy enough of both so that all experiments will be done with protoplasts produced by the same enzymes. Because of contamination by proteases, the hydrolytic enzymes should be made the day of the isolation. If a stock is made, it should be stored frozen. The enzyme solution used for isolation of spinach protoplasts consists of 2% cellulysin, 1% macerase, 0.7 M mannitol, and 50 mM
[66]
PROTOPLASTS IN LIPID METABOLISM STUDIES
777
MES, pH 5.5. The pH optima for the cellulases is around 4, but the cells seem to survive the incubation best at pH 5.5. Dextran sulfate has been added to enzyme solutions to increase protoplast release, 2 but is not essential to isolation of protoplasts. For sterile isolation of protoplasts/ the enzyme solution can be filter sterilized with a 0.45-/~m mesh. The ratio of enzyme solution to fresh weight of material is important in the rate of release of protoplasts. Rate of release is critical, since contaminants in the enzyme solution may damage the protoplasts. Therefore, the shortest incubation giving a reasonable yield is desired. Twenty milliliters of enzyme solution per gram fresh weight should give a good yield of protoplasts without being too expensive in terms of ceUulase and macerase.
Protoplast Purification After several hours of incubation at 30° in the cell wall degrative enzymes, protoplasts will begin to appear in solution. Microscopic observation of the tissue slices will show them as aggregates of protoplasts. At 4 hr, protoplasts are harvested by filtering the enzyme solution and softened tissue slices through a 53-/xm mesh. (Any mesh from 50 to 80 /zm is suitable.) The remaining tissue can be stirred in a 0.7 M mannitol solution to release more protoplasts, but violent shaking usually causes greater lysis of protoplasts, and the extra yield from this step is minimal. Protoplasts are pelleted by centrifugation at 133 g for 4 min at room temperature. If Carborundum was used to abrade the tissue, the protoplast-enzyme solution should be overlaid on a pad of 30% sucrose to allow the Carborundum to pellet and to concentrate the protoplasts at the mannitolsucrose interface. Resuspend the pelleted protoplasts in 1 ml of 0.7 M mannitol by gently shaking the tube. Vortexing or pipetting of the pellet causes a great deal of lysis and reduces the yield. Add 40 ml of 0.7 M mannitol, and respin at 133 g for 4 min at room temperature. Wash protoplasts twice in this manner and resuspend in a small volume of 0.7 M mannitol. Plastic centrifuge tubes should be used, as the protoplasts are sticky and will coat glass surfaces. Yield can be determined in milligrams of chlorophyll and compared to chlorophyll per gram fresh weight of spinach. Chlorophyll is estimated by extraction of the protoplasts in 80% acetone as described by MacKinney. 11 After a 4-hour incubation at 30°, approximately 20% of the chlorophyll in the leaf slices is found in the intact protoplasts. Protoplasts tend to clump and to aggregate with membrane fragments, free chloroplasts, and other cell debris. Aggregates can be removed by ~ G. MacKinney, J. Biol. Chem. 140, 315 (1941).
778
CELL AND TISSUE TECHNIQUES
[66]
filtering through a 37-p~m nylon mesh after each resuspension in the washing procedure. Clumping does not appear to affect uptake or metabolism of lipid precursors by the protoplasts, but causes problems when protoplast lysates are prepared for organelle isolation on density gradients. Addition of salts to the 0.7 M mannitol washing medium and incubation medium will stabilize protoplasts and decrease spontaneous lysis. Stabilizing media range from the simple, 20 mM CaCl~ or l0 mM arginine, ~2 to the complex, B5 medium without sucrose as described in Gamborg and Wetter 4 or a salt solution described by Frearson et al. 13The precise mode of action of the salts in stabilizing protoplasts is unknown, although the favorable effects of polycations on membranes is involved. The wash medium should be adjusted to pH 7.0 after addition of salts to minimize aggregation of the protoplasts seen at low pH. Incubation Procedures
Protoplasts can act like single-celled higher plants without a cell wall. Removal of the cell wall does not markedly change the cell's lipid composition 7 or ability to synthesize and desaturate fatty acids or esterify them into phospho- and galactolipids. 14 Metabolism studies for lipid synthesis are done in a temperature-regulated water bath (Aminco 5-4154 photosynthesis refrigerated-heated Warburg bath) with a light source giving 450 ~einsteins/m2/sec in the photosynthetically active range. Concentrations of protoplasts should be kept around 200/zg of chlorophyll per milliliter to prevent shading of protoplasts by other protoplasts. The incubation should be done with very slow shaking (30 excursions per minute if possible) to keep the protoplasts from settling out of the solution and to keep the medium oxygenated. More vigorous shaking will cause protoplast lysis during prolonged incubations. At 23° and 450 p.einsteins/m"/sec, uptake of [i~C]acetate into protoplasts is linear for 3 hr. For spinach protoplasts, or other photosynthetic protoplasts, metabolic activity can be quickly determined by measuring CO2-dependent 02 evolution with an oxygen electrode. '~ Rates of over 100/zmol per milligram of chlorophyll per hour indicate that the protoplasts have retained "normal" metabolic activity. Protoplasts can be stored in the dark at 4° at pH 7.0 for 24 hr with little loss of metabolic activity as measured by CO2-dependent 02 evolution or COz fixation? 4'~
12 A. ~ E. ~4 D. 1~ M.
Altman, R. K a u r - S a w h n e y , and A. W. Galston, Plant Physiol. 60, 570 (1977). M. Frearson, J. B. Power, and E. C. Cocking, Dev. Biol. 33, 130 (1973). N. K u h n , Doctoral Dissertation, University of California at Davis, 1980. N i s h i m u r a and T. Akazawa, Plant Physiol. 55, 712 (1975).
[66]
PROTOPLASTS IN LIPID METABOLISM STUDIES
ACETATE INCORPORATION ~
779
INTO LIPID VS. pH
oPROTOPLASTS
1000
100
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I
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[
I
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pH FtG. 1. Acetate incorporation into lipid [CHCIa: CHaOH (2:1) soluble material] as a function of pH for spinach protoplasts (left ordinate) and spinach chloroplasts (right ordinate). Uptake by protoplasts © © was under conditions listed in this chapter. Spinach chloroplast data (D []) are from Nakamura and Yamada. t"
Incubation Media and Radioactive Precursors
Protoplasts should be incubated in a pH-buffered medium of the same osmotic strength as that in which they were isolated. For spinach protoplasts, 0.7 M mannitol, B5 salts without sucrose, and MES 50 mM pH 5.5 medium is used. Metabolism of lipid precursors such as acetate depend strongly on pH (see Fig. 1). 1~ Incorporation of longer-chain fatty acids ~ Y. Nakamura and M. Yamada, Plant Cell Physiol. 16, 139 (1975).
780
CELL AND TISSUE TECHNIQUES
[66]
(palmitate, oleate) into protoplasts can be accomplished by dissolving them in small amounts of ethylene glycol monomethyl ether (EGME). Addition of 10 /zl of E G M E and fatty acids to 1 ml of protoplasts in solution appears to have no deleterious effect on the protoplasts, and 90% o f the labeled long-chain fatty acids are found associated with the protoplasts after 1 hr of incubation. Incubations up to 8 hr can be accomplished without significant contamination by bacteria as mannitol is a poor growth medium for bacteria. For longer incubations, protoplasts can be isolated under sterile conditions and incubation up to 48 hr accomplished. For such long incubations, a richer culture medium should be used to prolong protoplast life. 4 Extraction o f Lipids f r o m Protoplasts
After incubation, protoplasts can be centrifuged out of the incubation medium and washed with 0.7 M mannitol; the pellet can be extracted for lipids. N o homogenization is necessary, and lipids are easily extracted by the addition of 2 ml of hexane-isopropanol (3:2) '7 or c h l o r o f o r m methanol (1:2), '8 followed by thorough vortexing and centrifugation. If necessary, the pellet can be reextracted by the same methods. Use of Protoplasts in Subcellular Localization Studies Protoplasts are an excellent starting material for isolation of intact organelles and provide a system for subcellular localization of enzymes that can account for all of a particular e n z y m e activity in the cell. t9 By gently lysing protoplasts and separating the organelles on a density gradient, one can determine not only that an e n z y m e is found in an organelle, but whether it is found in only one organelle or in several or in the cytoplasm. Suitable soluble marker enzymes for the organelles facilitate organelle location in the gradient fractions and allow calculation of organelle breakage. By comparing marker e n z y m e activity with the enzyme activity (or protein) of interest in the cytosol fractions at the top of the gradient, one can determine whether the e n z y m e of interest occurs normally in the cytosol or can be accounted for by leakage from an organelle. The shapes and positions o f peaks in the gradient o f marker e n z y m e activity and the e n z y m e being studied can be c o m p a r e d to reveal that e n z y m e ' s subcellular location (Fig. 2). Total e n z y m e activity from the fractions can be corn,7 A. Hara and N. S. Radin, Anal. Biochem. 90, 420 (1978). ,8 E. G. Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959). ,9 j. B. Ohlrogge, D. N. Kuhn, and P. K. Stumpf, Proc. Natl. Acad. Sci. U.S.A. 76, 1194 (1979).
0.4
1.2
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-
-
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20 Fraction
30
FtG. 2. Distribution of acy| carrier protein (ACP) (~g/ml), chlorophyll (/~g/ml), ribulosebisphosphate (RubP2) carboxylase (gmollml/min), ADP-glucose pyrophosphorylase, (/~mol/mFmin), and isocitrate dehydrogenase (/~mol/ml/min) in sucrose gradients of ]ysates of spinach leaf protoplasts. All gradient fractions were 1 ml.19
782
CELL AND TISSUE TECHNIQUES
[66]
pared with enzyme activity in the intact protoplasts to determine what percentage of the total activity has been accounted for in the gradient. Yield of intact organelles is greater than with differential centrifugation, and the separation of organelles with similar s values (chloroplasts and mitochondria) is more distinct.
Lysis of Protoplasts Protoplasts that are to be lysed should be prepared without salts in the enzyme solution or washing medium, to reduce aggregation of organelles. Chloroplasts are especially sensitive to aggregation and will form clumps with other chloroplasts, membrane fragments, etc. To obtain high yields of intact organelles, the lysis of the protoplasts should be done as rapidly as possible, followed by the immediate overlaying of the lysate on the gradient and centrifugation. The protoplast sample to be lysed ( - 1 mg of chlorophyll per 40 ml gradient) is pelleted in 0.7 M mannitol and left as a pellet. Immediately before lysis, remove the 0.7 M mannitol, replace with 3 ml of 0.5 M sucrose, 0.5% BSA, 50 mM Tricine, pH 7.6, for 1 mg of chlorophyll of sample, and rapidly resuspend the protoplasts. This breaking medium is hypotonic and the protoplasts will begin to lyse owing to osmotic shock. The 0.5% BSA coats the organelle membranes and helps reduce aggregation. The resuspended protoplasts are drawn up into a 3-ml syringe with a Luer-lok end that is fitted with either a microemulsifying needle (25 gauge × 2~ inch, Bolab Incorporated, 6 Tinkham Ave., Derry, New Hampshire 03038) or a modified Swinnex filter (Millipore Corp., Bedford, Massachusetts 01730). After 3-6 passes through the microemulsifying needle, all protoplasts should have lysed owing to shearing forces. Replace the microemulsifying needle with a regular syringe needle, layer the lysate onto a 30-55% (w/w) sucrose density gradient and centrifuge for 1 hr at 26,000 rpm and 4° in an SW27 rotor. The sucrose contains 0.5% BSA or 10 mM arginine to reduce aggregation of chloroplasts. Within 1 hr, chloroplasts reach their equilibrium density and mitochondria are close to their equilibrium. Longer centrifugation will result in increased breakage of chloroplasts and formation of large aggregates in the intact chloroplast band. Lysis by using a modified Swinnex is as follows: remove the 0.45-~m grid of a Swinnex filter (1.4 cm) that can be attached to a syringe. Replace the grid with a circle of 20-gin nylon mesh and a circle of Miracloth (Calbiochem). The seal can be tightened by adding an O ring on top of the Miracloth and mesh. The protoplasts are pushed through the Swinnex, and lysis occurs as a result of pressure on the protoplasts by the mesh. The Miracloth removes debris and prevents clogging of the mesh. To lyse
[67]
ISOLATION A N D S U B F R A C F I O N A T I O N OF GI.YOXYSOMES
783
the protoplasts completely, three Swinnex should be used, the first with a 20-t~m mesh, the second and third with a 10-tzm mesh. After passing through the third Swinnex, the lysate is immediately overlaid on a density gradient and centrifuged. Microscopic observation at any step will give a rough estimate of protoplast breakage. Summary Spinach protoplasts are useful in lipid metabolism studies because they provide a homogeneous, differentiated plant tissue that can take up precursors directly from the medium. They are the material of choice for subcellular localization because they can be easily lysed and the intact organelles separated on density gradients. Crude fungal enzyme preparations are used to degrade the cell wall of the spinach leaves, which are stripped of their epidermal cells by brushing and sliced finely to increase the surface for enzyme action. Protoplasts are harvested from the digested leaf slices by filtration and washed and purified by repeated centrifugation. If protoplasts are to be used in metabolism studies, they are stabilized with a salt solution containing divalent cations and incubated in strong light with lipid or fatty acid precursors. For subcellular localization studies, salts are not present in the wash medium to reduce chloroplast aggregation in the gradients and protoplasts are lysed by shearing in a microemulsifying needle or passage through 20- and 10-tzm mesh before sucrose density gradient centrifugation. Comparison with marker enzymes for organelles provides a method for the complete subcellular localization of an enzyme activity.
[67] I s o l a t i o n a n d S u b f r a c t i o n a t i o n o f G l y o x y s o m e s 1
By ANTHONY H. C. HUANG Glyoxysomes were first isolated from the storage fatty endosperm of castor bean seedlings. ''8 They are specialized peroxisomes (microbodies) with the unique function of gluconeogenesis from reserve stores of triacylglycerol. The organelle is surrounded by a unit membrane and contains an amorphous matrix with occasionally a dense core. In addition to the peroxisomal enzymes, catalase, glycolate oxidase, and uricase, S u p p o r t e d b y the N a t i o n a l S c i e n c e F o u n d a t i o n . R. W. B r e i d e n b a c h a n d H . B e e v e r s , Biochem. Biophys. Res. Comrnun. 27, 462 (1967). a H . B e e v e r s , Annu. Rev. Plant Physiol. 30, 159 (1979).
METHODS IN ENZYMOLOGY, VOL. 72
Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181972-8