[21] Isolation of amyloplasts from suspension cultures of soybean

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lmmunodots We have successfully used the Bio-Dot microfiltration apparatus (BioRad) for quantitative analyses of envelope membrane polypeptides. The procedure described in the Bio-Rad instruction manual works very well with envelope membranes, in the range of 5-10/zg to a few nanograms envelope polypeptides with antibodies diluted to 1/500 to 1/I000.

Comments The procedures described above--polyacrylamide gel electrophoresis, immunoblotting, immunoelectrophoresis, or immunodots--are widely used for protein characterization. The specific problems related to the application of these methods to envelope membranes are due to the very high glycerolipid content of the outer envelope membrane. 3 Most of these glycerolipids should be removed prior to solubilization of the envelope polypeptides. The chloroform/methanol-diethyl ether procedure described above is a convenient but critical step. Treatment should be done very carefully and should be controlled to prevent any specific removal of hydrophobic polypeptides by this treatment. Acknowledgments ProfessorNam-Hal Chua is gratefullyacknowledgedfor his collaborationin the preparation of antibodies to El0, E24, E30, and E37. Our thanks are also owed to Dr. Agnrs BiUecocq for her collaboration in the optimization of the procedure for immunochemical studies on isolated, intact chloroplasts.

[21] I s o l a t i o n o f A m y l o p l a s t s f r o m S u s p e n s i o n C u l t u r e s of Soybean

By ToM AP REES and FRASER D. MACDONALD Insufficient is known of the structure and properties of nonphotosynthetic plastids for us to be able to define precisely the stage of differentiation at which they may be called amyloplasts. In this chapter we define amyloplasts as nonphotosynthetic plastids in which starch accumulation is the dominant activity. As starch is widespread in nonphotosynthetic cells and is made only in plastids, it follows that amyloplasts are widely METHODS IN ENZYMOLOGY, VOL. 148

Copyright © 1987 by Academic Press, Inc. All riots of reproduction in any form reserved.

[21]

AMYLOPLASTS FROM SOYBEAN CELLS

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distributed. However, the most obviously developed amyloplasts are found in the major starch storing tissues such as the endosperm of cereals and the tubers of potatoes. Amyloplasts vary in size according to their stage of development and to the plant species. In maize roots the differentiation of amyloplasts involves a 25- to 50-fold increase in plastid volume and results in a mature amyloplast that has a diameter of 2.17/zm. 1 In barley endosperm amyloplasts of up to 35/~m in diameter are found. 2 Amyloplasts are bounded by an envelope that consists of two membranes, 1,3 but, compared to chloroplasts, the internal membrane systems are poorly developed. Recent studies of amyloplast structure suggest the existence of two types of membrane. 1 First is the amyloplast envelope and a series of vesicles and tubules apparently in continuity with the envelope. Second is a separate series of "thylakoid-like" vesicles within the stroma of the amyloplast. In mature amyloplasts by far the greater part of the plastid is occupied by starch granules. The number and size of the latter vary. Amyloplasts from barley endosperm contain a single starch granule,3 those from maize roots may contain up to 8 granules, ~whereas those from the endosperm of oat have been reported to contain up to 100. 3 Despite the fact that the bulk of mankind's food is formed within amyloplasts, there have been very few reports of attempts to develop methods for the isolation of amyloplasts. The ultimate aim of any such study must be the preparation, in reasonable yield, of intact, functional, pure amyloplasts. At the time of writing this has not been reported. If we consider the enormous amount of work that was required to achieve this aim for chloroplasts, it is perhaps not surprising that the more difficult problem of isolating functional amyloplasts has not been solved. The prime difficulty is the extreme fragility of amyloplasts, which almost certainly results from their very high ratio of starch to stroma. The method that we describe below will give appreciable yields of pure amyloplasts, but the method is limited in that, as yet, we have no proof that these purified amyloplasts have retained all the physiological and biochemical properties that they possessed in the cell. Notwithstanding this limitation, the procedure can be used to characterize the basic composition of the amyloplasts, an essential step in understanding their function. The method that we describe for amyloplast isolation involves sucrose density gradient centrifugation of lysates of protoplasts made from suspension cultures of soybean cells. i p. W. Barlow, C. R. Hawes, and J. C. Home, Planta 160, 363 (1984). 2 j. M. Williams and C. M. Duffus, Plant Physiol. 59, 189 (1977). 3 M. S. Buttrose, J. Ultrastruct. Res. 4, 231 (1960).

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Growth of Suspension Cultures The cultures of soybean cells, derived originally from cotyledons of germinating soybeans (Glycine max [L.] Merr. cv. Acme), were obtained from Dr. Miller in 1970 and have been maintained in Cambridge ever since. 4 The culture is best maintained as a callus grown at 25° in the dark on the medium shown in Table 15 but solidified with 1% agar; subculturing should be done at monthly intervals. To obtain a suspension culture add 10 g fresh weight of callus to a 500-ml Erlenmeyer flask that contains 100 ml of the medium shown in Table I, then incubate the flask for 7-14 days on an orbital shaker at 100 rpm at 25° in the dark. The resulting cell suspension (30 ml) is then used to inoculate fresh medium (100 ml) in a similar flask that is then incubated as described above to give a suspension culture. The latter should be subcultured every 7 days. The suspension cultures are intolerant of hypoxia and require constant agitation to ensure adequate aeration. It is prudent to maintain callus cultures on solid medium so as to be able to replace suspension cultures if the latter are lost.

Preparation of Protoplasts

Reagents Sorbitol medium: 0.35 M sorbitol, 4 mM CaH4(PO4)2, l0 mM MES (pH 5.7) Digestion medium: 0.35 M sorbitol, 4 mM CaH4(PO4)2, 10 mM MES (pH 5.7), 270 U pectinase (polygalacturonase, EC 3.2.1.15, from Sigma Chemical Co.), and 5% (w/v) Driselase (Uniscience, Cambridge)

Method Use cultures that are 4 to 5 days old. Working at room temperature (20°), take 100 ml of cell suspension (4 to 5 g fresh weight of cells) and collect the cells by gentle suction onto glass fiber filter paper (Whatman GF/C) in a Biichner funnel. Rinse the cells in the funnel with 10 ml sorbitol medium and then use a further 10 ml of the same medium to wash the cells off the paper into a 50-ml beaker. Incubate the resulting cell

4 C. O. Miller, in "Modem Methods of Plant Analysis" (H. F. Linskens and M. V. Tracey, eds.), Vol. 6, p. 194. Springer-Verlag, Berlin and New York, 1963. 5 C. O. Miller, Ann. N . Y . Acad. Sci. 144, 251 (1967).

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TABLE I MEDIUMa FOR GROWTH OF SOYBEAN CULTURES

Component Sucrose

myo-Inositol KNO3 NH4NO3 Ca(NO3)2 • 4H20 KH2PO4 MgSO4 • 7H20 KC1 MnSO4 - 4H20 Ethylenediaminetetraacetic acid, ferric monosodium salt

(mg/liter)

Component

(rag/liter)

30,000 100 1,000 1,000 500 300 71.5 65 14 13.2

ZnSO4 • 7H20 H3BO3 Cu(NO3)2" 3H20 (NH4)6MoTO24 - 4H20 KI Nicotinic acid Pyridoxine-HCl Thiamin-HC1 Naphthalene-l-acetic acid Kinetin

3.8 1.6 0.35 0.1 0.8 0.5 0. l 0.1 2.0 0.5

Macronutrients are added as solids, micronutrients are added from stock solutions. The latter are kept at - 2 0 ° except that those for KI, naphthalene-l-acetic acid, and kinetin are always freshly prepared. Naphthalene-l-acetic acid and kinetin are dissolved by boiling for 1 hr in distilled water. The pH of the medium is not adjusted; sterilization is by autoclaving for 20 min at 121 ° at 104 kN m -2. Adapted from Miller)

suspension for 20 min without shaking. Next, collect the cells onto a glass fiber filter paper as described above and wash them off the paper with 15 ml digestion medium. Divide the resulting suspension into thirds and incubate each lot of 5 ml at 25 ° in a 25-ml beaker for 3 hr on a reciprocal shaker at 60 strokes/min for the first hour and 10 strokes/min for the last 2 hr. Protoplasts appear after about 1 hr; after 3 hr, 40-60% of the cells are converted to protoplasts. The purification of the protoplasts is done at room temperature (ca. 20°). First, filter by gravity the suspension of cells in digestion medium through stainless steel gauze (mesh size 43/zm). In this and all subsequent transfers of protoplasts use a Pasteur pipet with a wide mouth (2-3 mm). To recover protoplasts from the above filtrate centrifuge at 100 g (ray 11 cm) for 1 min. Gently resuspend the protoplasts in 10 ml sorbitol medium with a fine paint brush. Centrifuge as above and gently resuspend the protoplasts in I0 ml 42% (w/w) Percoll (Pharmacia, Ltd.) in sorbitol medium. N o w carefully overlay the protoplast suspension with 5 ml sorbitol medium and centrifuge at 200 g (ray 11 cm) for 5 min. Intact protoplasts float up to the interface and should be removed carefully with a Pasteur pipet and added to 15 ml sorbitol medium.

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Fractionation of Protoplasts

Lysis Sediment the purified protoplasts by centrifugation for 1 min at 100 g (ray 11 cm) and then resuspend the 0.5 to 1.0 ml packed volume of protoplasts in 1.0 ml homogenization medium [50 mM Tricine, pH 7.4, 10 mM KC1, 1 mM MgCl2, 1 mM EDTA, 0.1% bovine serum albumin, 21% (w/w) sucrose] at 4 °. This suspension is loaded via the open end into the barrel of a 2-ml disposable syringe in which a piece of Miracloth (Calibiochem, San Diego, CA) has been put between the barrel and the needle (0.7 × 40 ram). The protoplast suspension is now forced out through the cloth and needle. Repetition of this process twice, to give three passages through the needle in all, generally gives almost complete lysis of the protoplasts. At this stage it is important to check with a microscope that lysis is almost complete; repeat the process until it is. The lysate is finally filtered by gravity through two layers of Miracloth and made up to 15 ml with icecold homogenization medium to give the unfractionated lysate. Lysis is carried out at room temperature.

Fractionation A portion (8 ml) of the unfractionated lysate is placed carefully onto a 27-ml linear gradient of 16-60% (w/w) sucrose supported by 2 ml 60% (w/w) sucrose. It is important to ensure that the density of the unfractionated lysate is the same as that of the solution at the top of the gradient. 6 The sucrose in the gradient is dissolved in homogenization medium. The lysate is at 4 ° and the gradient is precooled to 4°. Gradients should be prepared and cooled within 8 hr of and 2 hr prior to use. The gradient plus lysate are centrifuged at 4 ° for I hr in an SW-27 rotor, Beckman L5-50 centrifuge, at 21,000 rpm (90,000 g at rmax 16.1 cm). Fractionate the gradient from the bottom. It is convenient to take 1.0-ml fractions except for the gradient pellet, which should be resuspended in 2.0 ml homogenization medium containing 21% (w/w) sucrose, and for the top 8 ml of the gradient, equivalent to the volume of lysate added, which is best treated as a single fraction.

Distribution of Marker Enzymes To assess the effectiveness of the fractionation the following markers may be used to locate the amyloplasts and possible contaminants in the gradient fractions. The best indication of the presence of amyloplasts is 6 M. Emes and M. W. Fowler, Planta 144, 249 (1979).

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T A B L E II ACTIVITIES OF MARKER ENZYMES IN AMYLOPLAST PREPARATIONS°

Enzyme Starch synthase ADPglucose pyrophos-

phorylase Nitrite reductase Alcohol dehydrogenase Cytochrome-c oxidase Cytochrome-c reductase Catalase

Percentage of activity in unfractionated lysate that was recovered in:

Activity in unfractionated lysate (nmol/min/ ml lysate)

Amyloplasts

Sum of all gradient fractions

0.8 + 0.1 3.4 --- 1.4

23.4 + 1.7 28.9 -+ 6.5

102 -+ l0 120 --- 16

23.1 0.2 0.6 1.8 0.3

t07 97 53 110 69

20.9 260 191 71 14,300

--- 4.4 --- 35 --- 36 -4- 11 +-- 300

-+ 1.7 -+ 0.1 -!--0.2 -+ 0.3 - 0.2

+ 6 --- 12 +- 4 --+- 3 --- 5

Protoplast lysates were fractionated as in Fig. 1. For each enzyme the activities in fractions 1 and 2 are summed to give activity in amyloplasts. Values are means -+ SE from at least five lysates. From Macdonald and ap Rees. 8

coincidence between the distribution of starch and the enzymes starch synthase (EC 2.4.1.21), ADPglucose pyrophosphorylase (EC 2.7.7.27, glucose-l-phosphate adenylyltransferase), and nitrite reductase (EC 1.6.6.4). 7 For contaminants the following markers may be used: cytosol, alcohol dehydrogenase (EC 1.1.1.1), phosphoenolpyruvate carboxylase (EC 4.1.1.31), pyrophosphate-fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90)7; mitochondria, cytochrome-c oxidase (EC 1.9.3.1); endomembrane systems, cytochrome-c reductase (NADH dehydrogenase, EC 1.6.99.3); microbodies, catalase (EC 1.11.1.6), hydroxypyruvate reductase (EC 1.1.1.81). 8 A typical fractionation is shown in Fig. 1 and the results from a number of such fractionations are averaged and shown in Table II. Apart from cytochrome oxidase, the recoveries of the enzymes from the gradient are satisfactory. The modest recovery of cytochrome-c oxidase is not due to the presence of an inhibitor in the amyloplast fraction, s but is probably due to the difficulty in obtaining a linear rate when the standard assay for this enzyme is used with unfractionated lysates. Appreciable and comparable proportions of the amyloplast markers, starch synthase, nitrite reductase, and ADPglucose pyrophosphorylase, are recovered in the gradi7 F. D. Macdonald and J. Priess, Planta 167, 240 (1986). a F. D. Macdonald and T. ap Rees, Biochim. Biophys. Acta 755, 81 (1983).

224

PLASTIDS

[21]

16

65

12

5 5 ~t

E8

...ll

o "o 4

li .....

i . ,1~ i

~0

45

I

!

I

c 0

35 ~ 25

0

it

&

15 3¢,, ® 0 L. 0

if) 0

g16

I

I

I

I

I

I

.08 0.

~

4

°o~

~16 k.

:'_, ,,I-

i 10

t 20

. ........................ 30

Fraction N u m b e r FIG. 1. Sucrose density gradient fractionation of lysates of soybean protoplasts. The gradient pellet was resuspended in 2 ml homogenization medium and is fraction 1: the next 29 ml of the gradient was divided into l-ml fractions (numbers 2-30), and the top 8 ml of the gradient was taken as a single fraction, numbered 38. Total activity is plotted for each fraction except fraction 38, where activity/ml of fraction is given. Each fraction was assayed for: sucrose (a--A), alcohol dehydrogenase ( I - - I ) , nitrite reductase (rq--rq), catalase ( i - - - i ) , starch synthase (lt---ll), cytochrome-c reductase (~7--E7), cytochrome-c oxidase (O--O), tx-glucan phosphorylase (It---V).

ent pellet and the fraction immediately above it. These fractions also contained 78% of the starch added to the gradient. These fractions are essentially free of contamination by any of the other marker enzymes assayed. Thus the gradient pellet and the fraction immediately above it may be regarded, collectively, as forming the amyloplast fraction. The

[21]

AMYLOPLASTS FROM SOYBEAN CELLS

225

fact that this fraction contains more of the starch than the stromal enzymes of the amyloplast is almost certainly due to the presence in the fraction of starch released from broken amyloplasts. Evidence that the presence of the stromal enzymes in the amyloplast fraction is not due to selective adsorption of these enzymes onto starch grains is provided by demonstrations that the activities of such enzymes in the unfractionated lysate are both latent, and protected from the action of trypsin. 8 Comments

Particular attention should be given to the following three points. First, the age of the suspension cultures used to make amyloplasts has a critical effect on the yield of amyloplasts. If the cultures are too young and contain too little starch the yield is reduced and much of the plastid markers are spread as a diffuse band between fractions 6 and 17. 9 The latter probably represents plastids that have not fully differentiated into amyloplasts. Second, rupture of the protoplasts must be done carefully so as to ensure that all the protoplasts are broken with the minimum damage to amyloplasts. Unbroken protoplasts are generally recovered in the diffuse band (fractions 6-17) and can be detected by the presence of all the marker enzymes. 9 Third, the integrity of protoplast and amyloplast membranes is severely reduced by detergents, thus all glassware and solutions should be scrupulously prepared to be free of detergent. The above procedure gives a respectable yield of uncontaminated amyloplasts, 23-29%, which compares favorably with yields of chloroplasts from leaves. Protection experiments carded out on the isolated amyloplasts from the gradient indicated that 30% of the purified amyloplasts were intact.l° The extent to which such amyloplasts are functional has not yet been established. Finally we stress that the above method is not the only one available for the isolation of amyloplasts. Echeverria et al. have described a promising method for the isolation of amyloplasts from maize endosperm. ~1In the latter, two of the potential disadvantages of our method, relatively high centrifugal forces and high concentrations of sucrose as osmoticum, are avoided by allowing the amyloplasts to settle under gravity through a gradient of Ficoll. Yields were 5-10% and estimates of intactness of the isolated amyloplasts ranged from 27 to 93% depending upon the methods used to assess it. Journet and Douce have recently reported the successful 9 p. Gross and T. ap Rees, Planta 167, 140 (1986). 10 F. D. Macdonald, Ph.D. Thesis, University of Cambridge (1981). H E. Echeverria, C. Boyer, K.-C. Liu, and J. Shannon, Plant Physiol. 77, 513 (1985).

226

PLASTIDS

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purification of small plastids from cauliflower buds. 12 The yield was low but intactness was very high, 90%. Although these cauliflower plastids contained starch, they were also active in fatty acid synthesis. So the extent to which they represent amyloplasts is not yet known. In terms of yield of intact amyloplasts the three methods do not differ greatly, 30% of 23-29%, 27-90% of 5-10%, 90% of 1%. The crucial comparison will be the extent to which the isolated plastids are functional. No data on this question have been published yet. 12 E . P. J o u r n e t a n d R . D o u c e ,

Plant Physiol.

79, 4 5 8 (1985).

[22] Isolation o f A m y l o p l a s t s f r o m D e v e l o p i n g E n d o s p e r m of M a i z e (Zea mays L.)

By JACK C. SHANNON, EDGARDOECHEVERRIA, and CHARLESBOYER Introduction Amyloplasts are colorless organelles specialized for the synthesis and accumulation of reserve starch. They develop from proplastids, as do chloroplasts, and are bounded by a double membrane. Under certain conditions amyloplasts develop into chloroplasts and vice versa.l Amyloplasts may contain single or multiple starch granules, depending on the species and the tissue within which the amyloplasts occur. 2 Normal maize endosperm amyloplasts generally contain a single starch granule. 2 However, the number and shape of starch granules in amyloplasts vary in different maize endosperm mutants. The reader is referred to the review by Shannon and Garwood2 for a more complete discussion of the genetics and physiology of starch development. Procedures for the successful isolation of relatively pure, intact plastids from castor bean endosperm were reported several years ag o 3'4 and these have been very useful in demonstrating that plastids contain the J. T. O. Kirk and R. A. E. Tilney-Basset, "The Plastids: Their Chemistry, Structure, Growth and Inheritance." Elsevier/North-Holland Biomedical Press, New York, 1978. 2 j. C. Shannon and D. L. Garwood, in "Starch: Chemistry and Technology" (R. L. Whistler, J. N. BeMiller, and E. F. Paschall, eds.), p. 26. Academic Press, New York, 1984. 3 p. D. Simcox, E. E. Reid, D. T. Canvin, and D. T. Dennis, Plant Physiol. 59, 1128 (1977). 4 M. Nishimura and H. Beevers, Plant Physiol. 62, 40 (1978).

METHODS IN ENZYMOLOOY, VOL. 148

Copyright © 1987 by Academic Press, Inc. All fights of reproduction in any form reserved.