Plant Physiol. (1976) 58, 656-662

Isolation of Vacuoles from Root Storage Tissue ofBeta vulgaris L.1

Received for publication June 29, 1976 and in revised form August 9, 1976

ROGER A. LEIGH2' 3 AND DANIEL BRANTONThe Biological Laboratories, Harvard University,

ABSTRACT

Morphologically intact and osmotically active vacuoles were isolatedfrom root storage tissue of the red beet Beta vulgaris L., and the factorsinfluencing both yield and stability of the vacuoles were determined.Successful isolation depended upon slicing the tissue in an apparatusspecifically designed to cut open plant ceUls without the use of high shearforces and to liberate cellular organelles into an undisturbed reservoir ofosmoticum. The resulting brei was centrifuged at 2,000g for 10 min toyield a pelet which contained many vacuoles but which also containedtissue fragments, nudei, mitochondria, and plastids. The vacuoles werefurther purified by accelerated flotation through a Metizaumide stepgradient. Biochemical assays, light microscopy, and electron microscopyconfirmed that there was only trace contamination of the final vacuolepreparation by other organelles. Isolated vacuoles were intact and re-tained their in vivo coloration.

Although vacuoles are the largest membrane-bound organ-elles in the plant cell, they remain poorly characterized becausethere are few methods for their large scale isolation and purifica-tion in an intact and physiologically active state. Because expo-sure to the high shear forces used to disrupt cell walls duringtissue homogenization also disrupts the fragile vacuoles, at-tempts to isolate vacuoles using conventional cell fractionationschemes have not been successful (5) or have yielded a "prova-cuole" fraction which may or may not have the same propertiesas mature vacuoles (17, 21).

Preparation of vacuoles by osmotic lysis of protoplasts hasbeen more fruitful, and this method has been used to preparevacuoles from yeasts and other unicellular microorganisms (11,18, 20, 25). Cocking (4) and Strobel and Hess (28) reported theisolation of vacuoles by osmotic lysis of enzymically derivedplant protoplasts, but they did not attempt to assess contamina-tion by other organelles. More recently, Wagner and Siegelman(29) described the large scale isolation of mature plant vacuolesfrom protoplasts. Although protoplast techniques can producelarge quantities of apparently pure vacuoles, several difficultiesremain. These methods can be applied only to relatively softtissue (e.g. petals, leaves, fruits) from which protoplasts can beenzymically prepared, and it is not known whether the distribu-tion of vacuolar contents, particularly small ions, is altered bythe prolonged exposure of tissues to cell-wall-degrading en-zymes.

I This work was supported by funds from the Maria Moors CabotFoundation for Botanical Research.

2 Present address: Botany School, Cambridge University, DowningStreet, Cambridge CB2 3EA United Kingdom.

3 Recipient of a Maria Moors Cabot Fellowship in Botanical Re-search.

16 Divinity Avenue, Cambridge, Massachusetts 02138

To overcome these problems, we have developed a largescale, mechanical apparatus to liberate intact, mature vacuolesfrom untreated plant tissue. Our approach is based on theobservation that small numbers of vacuoles can be prepared byslicing tissue with a razor blade into a suitable osmoticum on amicroscope slide (9, 27). Slicing action by a fine, sharp edgeminimizes shear, but is not employed by any commercial deviceknown to us. Our apparatus provides such a cutting action andliberates organelles directly into an undisturbed reservoir ofosmoticum exposing each cell only once to the cutting edge. Themethod is useful for those firmer tissues not suitable for enzymicprotoplast production, and the rapidity of the techniques relievesconcerns about the possible redistribution of vacuolar contents.

MATERIALS AND METHODS

Plant Material. Root storage material of the red beet Betavulgaris L., purchased locally, was used because its cells havelarge microscopically visible vacuoles containing the water-solu-ble pigments, betacyanin and betaxanthin (15).

Small Scale Preparation of Vacuoles. Five-mm long, 5-mmdiameter cores (average weight: 50 mg) were cut from the beetroot storage tissue using a cork borer and were plasmolyzed in 2M sucrose for 20 min, unless otherwise stated. The tissue wasremoved from the sucrose, gently blotted, and placed on amicroscope slide in 0.1 ml of collection medium whose composi-tion varied and is specified for each experiment. The tissue wasthen sliced into 20 pieces with a new razor blade. Vacuoles wererinsed from the tissue onto the slide with a further 0.1 ml ofmedium. The tissue was discarded, and a No. 1, 22-mm squarecover glass was placed over the vacuole suspension. The numberof vacuoles observed at a magnification of 100 in the lightmicroscope on each of three different scans across the slide wasrecorded and the average number of vacuoles per scan calcu-lated. In experiments in which vacuole stability was investigated,a scan across a single section of the slide was repeated at varioustimes after cutting. All experiments were repeated at least threetimes. Although the methodology of these small scale experi-ments was crude, the results were consistent from experiment toexperiment and were confirmed using the large scale isolationtechnique.

Tissue Slicing Apparatus. The tissue slicing apparatus is illus-trated in Figure 1. It was constructed from stainless steel. Theside arm of the blade housing was rigidly fixed to the uprightcolumn of a drill press and the paddle wheel was engaged in thedrill press chuck via a universal joint. The cutting blades wereimmersed in collection medium. Tissue was loaded into thesector-shaped compartments of the paddle wheel so that whenthis wheel rotated, the tissue passed over a stationary array ofsingle-edged razor blades on the bottom plate. The orientationand protrusion of the blades could be varied by using differentblade holders. The piston, which fitted into, and turned with, thepaddle wheel, was hand-held by its bearing-mounted handle.

656

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ISOLATION OF BEET VACUOLES

-Shaft

!,>I, --Handle

X-Pis-tPistonX I

:

PaddleWheel

uardBlade

1 1J Housing

ades

CollectionMedium

FIG. 1. Tissue-slicing apparatus.

The applied hand pressure gradually fed the tissue into theblades. The cut tissue, vacuoles, and other cell organelles floweddirectly into the undisturbed reservoir of collection medium. Thecircumferential guard around the blade housing prevented therotation of the paddle whec. from translating its turbulence tothe collection medium.

Large Scale Isolation of Vacuoles. The procedures describedbelow are those found to be optimum for the isolation of beetvacuoles.Four hundred fifty g of fresh, peeled beets were cut into

approximately 5-mm thick slices and loaded into the slicingapparatus (Fig. 1). They were sliced at 150 rpm into 1 liter ofcollection medium (1 M sorbitol, 5 mm EDTA, 0.1 mg/ml Na 2-mercaptobenzothiazole [13], 50 mm tris-HCI, pH 7.6) at roomtemperature. The tissue pieces were separated out of the result-ing brei on Miracloth supported on a stainless steel kitchen sieve,and these pieces were then sliced a second time into 1 liter offresh collection medium. Tissue was again removed by filtrationand the two filtrates were combined to yield about 1800 ml offiltered brei. All subsequent operations were performed at 4 C,and the suspensions were handled very gently at all stages inorder to minimize vacuolar breakage. Eight 220-ml aliquotswere centrifuged at 2,000g for 10 min (GSA rotor; Sorvall RC2-B centrifuge). The supematants were discarded, and the pelletswere very gently resuspended and combined in 16 ml of 15% (w/v) Metrizamide (Nyegaard Co., A/S Oslo, Norway, brand of (2-[3-acetamido-5-N-methylacetamido-2,4,6-triodobenzamido]2-deoxy-D-glucose) in isolation medium (1.5 molar sorbitol, 1 mmEDTA, 10 mm tris-HCl, pH 7.6). Three-ml aliquots of thisresuspended 2,000g pellet were placed in 1 5-ml glass centrifugetubes and overlaid with S ml of 10% (w/v) Metrizamide, 2.5ml of 2.5% (w/v) Metrizamide, and 2.5 ml of 0% Metrizamideeach in isolation medium. The resulting step gradients werecentrifuged at 650g for 10 min (HB-4 swinging bucket rotor,Sorvall RC2-B centrifuge) and fractions (Fig. 2) were trans-ferred into individual tubes for further analysis. The majorityof the vacuoles were recovered from the interface of the 10%Metrizamide and 2.5% Metrizamide (Fig. 2). If further concen-tration was required, the vacuole-containing bands were dilutedwith 2 volumes of isolation medium and centrifuged at 650g for

10 min. The supernatant was discarded, and the pellet contain-ing the vacuoles was gently resuspended in the desired volumeof isolation buffer.A total particulate fraction was prepared by centrifuging a

sample of the filtered brei at 100,000g for 1 hr.Enzyme and Chemical Assays. Vacuoles were counted with a

Levy ultraplane improved Neubauer hemocytometer. Betacy-anin was estimated spectrophotometrically as follows. One ml ofdistilled H20 and 0.2 ml of 10% (w/v) SDS were added to 0.3 mlof sample. Absorbance was measured at 550 nm against waterand 1 unit of betacyanin/ml of the original sample was defined asthat concentration which gave an A of 0.01. Cytochrome oxidaseand NADH-Cyt c oxidoreductase were assayed according toHodges and Leonard (10). Glucose-6-P dehydrogenase was as-sayed according to Kornberg and Horecker (12). Glutamateoxaloacetate transaminase was determined with a commercialassay system (Sigma Chemical Co.). DNA was assayed by thefollowing modification of the method of Burton (3). To 0.3 ml or0.5 ml of sample was added 0.2 ml ice-cold 42% (w/v) trichloro-acetic acid, and the volume was made up to 1 ml with distilledH20. DNA was precipitated overnight at 4 C. Samples werecentrifuged for 10 min at full speed in a clinical centrifuge andwashed twice with ice-cold 7% (w/v) trichloroacetic acid. Thewashed pellet was dissolved in 0.2 ml 1 M perchloric acid byheating at 70 C for 15 min. The sample was cooled and 1 ml ofdiphenylamine reagent (1.5g diphenylamine, 100 ml glacialacetic acid, 0.5 ml of 16 mg/ml acetaldehyde) was added. Colorwas allowed to develop for 16 hr at 20 C. Absorbance at 595 nmwas measured, and DNA content was determined from a stan-dard curve prepared using highly polymerized calf thymus DNA(Sigma Chemical Co.). Protein was determined by the method ofLowry et al. (14), after the samples had been precipitated withtrichloroacetic acid. BSA was used as a standard.

Neutral red absorbtion was observed in the light microscopewhile gently flowing a 1% solution of neutral red in isolationmedium under the coverslip.

Electron Microscopy. Vacuole pellets were frozen in theirisolation medium and freeze-fractured using standard techniques(8).

RESULTS

Our early experiments were directed toward defining the basiccomposition of a suitable medium for the collection and eventualisolation of beet vacuoles. We felt that the medium should beisotonic or slightly hypertonic to the vacuoles, be at the correctpH and temperature to stabilize the vacuoles, contain any factorswhich enhanced vacuole yield and stability, and be of lowenough density and viscosity to permit initial concentration ofthe vacuoles by sedimentation. Unless otherwise stated, experi-ments to define the most suitable medium were conducted usingthe small scale vacuole preparation technique.

Figure 3 shows the yield of vacuoles as a function of sucrose

0% Metrizamide

2.5% Metrizamide

10% Metrizamide

650g10 min

Fraction IFraction 2:Fraction 3Fraction 4

Froction 5

:Pink

Red

2,000g Pellet in Fraction 6 X Red15% Metrizamide Pellet

FIG. 2. Metrizamide step gradient before and after centrifugation at650g for 10 min.

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LEIGH AND BRANTON

0.5 1.0 1.eSUCROSE CONCENTRATION (M)

FIG. 3. Effect of sucrose concentration on the recovery of beet vacu-oles prepared by the small scale isolation technique. The sucrose wasunbuffered.

concentration. At low sucrose concentrations (<0.3M) vacuoleyield was low but increased with increasing sucrose concentra-tion up to 1 M sucrose. Increasing the sucrose concentrationabove 1 M had no effect on vacuole yield, but did decrease thesize of the vacuoles, indicating that they were responding osmot-ically to the medium. We later repeated the experiment byresuspending pellets of vacuoles, produced using the large scaletechnique, in solutions with different sucrose or sorbitol concen-trations. The results were similar to those described above.

Vacuole yield was found to be independent of pH between pH6 and 9 (Fig. 4), but was significantly reduced at lower pHvalues. For instance, at pH 4, about 75% fewer vacuoles wererecovered than at pH 8. At pH 7.6, the buffer used (tris, TES, orphosphate) did not significantly affect the vacuolar yield. Thetemperature of the collection medium affected the number ofvacuoles recovered. When using ice-cold medium, there was a30 to 50% decrease in yield of vacuoles relative to comparableexperiments in which the collection medium was at room tem-perature. Plasmolysis of the tissue prior to slicing did not in-crease the vacuole yield. This was surprising since plasmolysisdecreases the size of the vacuole in situ and thus would beexpected to increase the probability of cutting through a cellwithout destroying the vacuole. The tissue may be so rapidlyplasmolyzed as it comes into contact with the collection mediumupon cutting that plasmolysis prior to slicing is unnecessary.The numbers of vacuoles in a preparation displayed a time-

dependent decrease with apparent first order kinetics. From theslope of the semilogarithmic plots of per cent survival versustime, we were able to calculate the half-life of the vacuoles. Thisprovided a reasonable basis on which to compare the effects ofdifferent media on vacuole stability. Figure 5 shows a plot ofvacuole half-life versus pH. Vacuole stability had a definiteoptimum pH, in the range pH 7.5 to 8, in contrast to the effect ofpH on yield (Fig. 4). At pH 7.6, the stability of the vacuoles wasgreatest in tris-HCl and phosphate buffers (34 and 30 hr, respec-tively) and least in tris-maleate buffer (17 hr). Decreasing thetemperature was found to increase vacuole stability. At 20 C,the vacuole half-life was about 24 hr but it increased to over 80hr at 4 C. Stability was unaffected by any other manipulation ofthe collection medium, e.g. addition of EDTA, Ca2+, Mg2+,dithiothreitol, mercaptoethanol, or BSA.From the above data, we concluded that a reasonable collec-

tion medium would be 1 M sorbitol containing 50 mm tris-HCl,pH 7.6. However, when using this medium for large scale isola-tions, the presence of endogenous or exogenous Ca2+ or Mg2+caused aggregation of vacuoles and other cell organelles result-

ing in unacceptable surface contamination of the vacuoles. Theaddition of 0.1 mg/ml of Na 2-mercaptobenzothiazole (13) and 5mM EDTA to the collection medium and 1 mm EDTA to theisolation medium reduced this aggregation. Sorbitol was usedinstead of sucrose because its lower viscosity and density permit-ted more rapid sedimentation of the vacuoles. Although vacu-oles were significantly less stable at 20 C than at 4 C, we rou-tinely sliced tissue at 20 C because of the compensatory in-creases in yield and convenience.

Figure 6 shows the recovery of vacuoles after centrifuging thefiltered brei at different g-min values. Highest recoveries wereobtained when using 5OOg to 2,000g for 20 min. Greater accel-erations and longer times resulted in a decreased yield of intactvacuoles although spectrophotometry showed that there was noconcomitant reduction in the total amount of pigment in theresuspended vacuole suspensions. We, therefore, assumed thatthe lower recovery of intact vacuoles at higher g-min was due tobreakage of vacuoles during resuspension of the increasinglycompacted pellets. We routinely centrifuged the filtered brei at2,000g for 10 min. Final vacuole yield was also affected by thevolume of medium used to resuspend this 2,000g pellet. Ingeneral, the larger the volume of resuspension medium, thegreater the number of intact vacuoles recovered.

Light microscopy revealed that the 2,000g pellet containedmany intact vacuoles. However, whole tissue fragments, nuclei,and numerous small particles were also present (Fig. 7a). Toassess contamination more accurately, we assayed the 200g

z 500Uen

- 400

cnw-j 30000

5 2000

w 1o00

z4.0 5.0 6.0 %0 8.0 9.0 100

pH

FIG. 4. Effect of pH on the yield of beet vacuoles prepared by thesmall scale isolation technique. A: 50 mm citric acid-KH2PO4; *: 50 mmtris-HCI; *: 50 mm boric acid-KCI-NaOH.

24 _ ,

-18

IL

<12

0

6.0 6.5 7.0 7.5 8.0 8.5 9.0pH

FIG. 5. Effect of pH on the stability of beet vacuoles prepared by thesmall scale isolation technique. Collection medium contained 1 M su-crose buffered to the appropriate pH with 50 mm tris-HCI.

U

I I I I I __

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ISOLATION OF BEET VACUOLES

10 20 30 40 50 60 70 80"7 300CENTRIFUGAL FORCE (g-MINUTES X 103)

FIG. 6. Effect of centrifugation at different speeds and for differenttimes on the recovery of vacuoles. Samples of a filtered brei from a largescale isolation were centrifuged at the accelerations indicated in a SorvallRC2-B centrifuge using an HB-4 rotor. The resulting pellets were resus-

pended in isolation medium and the total number of vacuoles in the breiand in the pellet was determined using a hemocytometer.

pellet for biochemical markers known to be associated withother cell organelles, e.g. DNA (nuclei), Cyt oxidase (mitochon-dria), glutamate oxaloacetate transaminase (plastids) (22; Leighand Branton, unpublished), NADH-Cyt c oxidoreductase (en-doplasmic reticulum, nuclear membrane and outer mitochon-drial membrane; 24, 25, 27) and glucose-6-P dehydrogenase(cytosol). In addition, betacyanin content was measured to pro-vide an independent, chemical measure of vacuole yield.

Table I gives the average recoveries of these markers in the2,000g pellet, measured in a number of experiments. Quantita-tive variation between experiments was the result of seasonalvariations in the beets; qualitative reproducibility of experimentswas excellent. As a percentage of the total particulate activity,the 2,000g pellet contained, on average, 48 + 2% (mean SE)of the betacyanin, 23 + 2% of the protein, 26 + 10% of theDNA, 18 + 2% of the Cyt oxidase, 5 ± 1% of the NADH-Cyt c

oxidoreductase, 36 ± 3% of the glucose-6-P dehydrogenase,and 26 ± 2% of the glutamate oxaloacetate transaminase. Ex-pressing results as percentages of the total particulate activitiesrather than as percentages of the filtered brei activities was a

more useful measure of vacuolar yield and contamination be-cause the filtered brei contained large amounts of soluble vac-

uole contents released from vacuoles disrupted by the tissueslicer.To purify vacuoles further, we tested a number of density

gradient media including sucrose, sorbitol, glycerol, Ficoll, silicasols, Metrizamide, and sodium diatrizoate. Only Metrizamideand sodium diatrizoate produced discrete bands of vacuoles aftercentrifugation to equilibrium (100,OOOg, overnight). We usedMetrizamide step gradients and choose a flotation, rather than a

sedimentation scheme (Fig. 2) so that centrifugation could beterminated as soon as the rapidly moving, large vacuoles reachedequilibrium.

After flotation on the Metrizamide step gradient at 650g for10 min, pigment was concentrated in three areas of the gradient:in the 15% Metrizamide band, at the interface of the 10% and2.5% Metrizamide, and at the 2.5% and 0% Metrizamide inter-face. For further analysis, the gradient was divided into sixfractions and a pellet (Fig. 2). Analysis of these fractions (TableI) showed that vacuoles were concentrated at the 10% and 2.5%Metrizamide interface (fraction 4). This fraction contained 35 ±

3% (mean ± SE) of the betacyanin originally present in the2,000g pellet, 4 ± 1% of the protein, 1 ± 1% of the DNA, 2 +

0.5% of the Cyt oxidase, 10 ± 1% of the NADH-Cyt c oxidore-ductase, none of the glucose-6-P dehydrogenase, and 3 ± 0.2%of the glutamate oxaloacetate transaminase. As a percentage oftotal particulate activities, fraction 4 contained almost 20% ofthe betacyanin but less than 1% of all other markers. Thus,intact vacuoles had been separated from other cell components

and concentrated in fraction 4. The majority of other cell organ-elles were concentrated in fraction 6 (15% Metrizamide layer)and the gradient pellet, which together contained 86% of theprotein, 98% of the DNA, 92% of the Cyt oxidase, 72% of theNADH-Cyt c oxidoreductase, 100% of the glucose-6-P dehy-drogenase, and 94% of the glutamate oxaloacetate transaminaserecovered from the gradient. The betacyanin present in the 15%Metrizamide band was nonparticulate and derived mainly fromvacuoles broken during resuspension of the 2,000g pellet.When recoveries were analyzed as specific activities (Table

II), the only components found to be concentrated in fraction 4,relative to the 2,000g pellet, were betacyanin (purified approxi-mately 8-fold) and NADH-Cyt c oxidoreductase (purified ap-proximately 3-fold).Examination of fraction 4 in the light microscope after Metri-

zamide dilution and sedimentation at 650g for 10 min indicatedthe presence of large numbers of highly pigmented vacuoles(Fig. 7b). Some nuclei and smaller particles were also visible,confirming the slight contamination indicated by the biochemicalassays (Tables I and II). Electron micrographs of freeze-frac-tured pellets of fraction 4 (Fig. 7, c and d) corroborated the lightmicroscope observations. Examination of cross-fractured vacu-oles indicated no surface contamination by other membranesalthough very occasionally nuclei were seen. The vacuole mem-brane contained typical intramembrane particles, 70 A to 120 Ain diameter. There were approximately 1450 particles/gm2 onthe E fracture face and 3360 particles/Mum2 on the P fracture face(2). Many of the smaller particles visible in the light microscopeappeared to be broken vacuoles which had reformed as small,sometimes inside-out, vesicles. The osmotic fragility of the vacu-oles prevented study of their true membrane surface by deepetching, which requires that the vacuoles be suspended in water.

Vacuoles prepared by our technique absorbed neutral redfrom solution.

DISCUSSION

Beet vacuoles isolated according to our procedure retainedtheir in vivo properties. They were similar in size and degree ofpigmentation to vacuoles in situ, they responded osmotically totheir surroundings, and were able to absorb neutral red fromsolution (30). Freeze-fracture electron microscopy of purifiedvacuoles (Fig. 7, b and c) showed the presence of intramem-brane particles whose appearance, size (70 A to 120 A indiameter) and partition coefficient between the E and P faces(1460/3360 = 0.425) was similar to that reported by others forvacuoles in situ (1, 7, 19).By using an apparatus specifically designed to slice tissue as

does a hand-held razor blade, we have overcome many of theproblems often encountered when attempting to isolate delicateplant organelles by standard mechanical techniques (e.g. 5).Although only a small percentage of the cells cut by our appara-tus yielded intact vacuoles (less than 2% of the pigment releasedfrom beet tissue was particulate), our apparatus cuts tissue sorapidly that we could conveniently use large quantities of startingmaterial (450 g) and obtain relatively large numbers of vacuoles(107-108) in one preparation. Loss of vacuoles, particularly byphysical disruption, occurred at all stages during isolation, but bycareful handling, these losses could be minimized. Loss by os-motic lysis also occurred unless specific steps were taken toensure that vacuoles did not encounter large negative osmoticgradients.The morphological similarity, at light and electron microscope

levels, between the product we obtained and the in vivo struc-ture defined as a vacuole was the basis of our purificationscheme. The purity of our vacuole preparations was confirmedby biochemical assays. With the exception of NADH-Cyt c

oxidoreductase, markers known to be associated with otherorganelles were either not detectable or were present at very low

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LEIGH AND BRANTON Plant Physiol. Vol. 58, 1976

EF.

'~~~~~~~~~~~~~~~~~- F .< ,--7 "+. :. 2:, :. 5~~~~

-t

%.A~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~A

FIG. 7. Micrographs of vacuoles. a: Light micrograph of the 2,000g pellet containing vacuoles (V) and numerous contaminant particles (P),x 1,100; b: light micrograph of purified vacuoles (fraction 4), x 1,100; the variable pigmentation reflects variable pigmentation in vivo in the beetroot tissues; c and d: electron micrographs of small freeze-fractured vacuoles from fraction 4. Concave fractures expose the particle-rich protoplasmicfaces (PF) whereas convex fractures expose the explasmic faces (EF). Ruffling and uneveness of the membrane may be due to slight hypertonicity ofthe isolation medium or to artifacts of the freezing process, x 27,000; insets, x 67,500.

levels. The concentration of NADH-Cyt c oxidoreductase in the work reported in this paper and elsewhere (Leigh and Branton,vacuole fraction (Table II) is of interest since other work has unpublished) confirms its association with tonoplast, although itsindicated that this enzyme is associated with the endoplasmic multisite location and vacuole breakage probably account for itsreticulum (23, 26), nuclear membrane, (26), and outer mito- low recovery in fraction 4. On the other hand, the low centrifu-chondrial membrane (6, 24), as well as the tonoplast (16). The gal forces and flotation scheme used in our procedure make large

660

-,4v -,

t

or t,

dk

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Plant Physiol. Vol. 58, 1976 ISOLATION OF BEET VACUOLES 661

Table I. Recovery of vacuoles and biochemical markers

Vacuoles were purified by the large scale isolation technique. The METRIZAMIDE step gradient was fractionated asindicated in Fig. 2. The total particulate fraction was prepared by centrifuging a portion of the filtered brei atlOO,OOOg for 1 hr. All values have been normalized to 1760 ml of filtered brei. LSD = least significant difference(95% confidence limits) between gradient fractions; N = number of separate experiments in which marker was assayed.The major source of variability was the seasonal variation in beets; n.s. = not significantly detected.

GlutamateFraction Vacuoles Betacyanin Protein DNA Cytochrome-c NADH-cytochrome c Glucose-6-phosphate oxaloacetate

oxidase oxidoreductase dehydrogenase transaminase

xl0 units mg iJg limoles/min AA3 40/min

2000 10,038 1600 13.02 670.1 10.11 1.51 1.00 23.95pellet

1 20 16 0.02 n.s. n.s. n.s. n.s. n.s.2 261 80 0.12 n.s. n.s. n.s. n.s. n.s.3 235 87 0.07 n.s. n.s. n.s. n.s. n.s.4 2,666 604 0.54 6.5 0.23 0.15 n.s. 0.655 366 305 1.08 7.0 0.59 0.53 n.s. 0.946 248 638 6.82 180.6 8.50 1.41 0.84 20.68

gradient 7 77 4.19 529.4 1.25 0.35 0.06 2.46pellettotal par

n.s. 334.0 56.09 2555.3 59.58 30.31 2.77 93.73ticulate

LSD 518 335 0.84 94.7 0.92 0.27 0.31 ...

N 8 10 10 4 7 4 3 2

Table II. Specific activities of various biochemical markers in fractions.

Vacuoles were purified using the large scale isolation technique. The METRIZAMIDE step gradient was fractionatedas indicated in Fig. 2. The total particulate fraction was prepared by centrifuging an aliquot of filtered breiat 100,000j for 1 hr. All values have been normalized to 1760 ml of filtered brei. Values are given as mean ±SEM where 3 or more determinations were made. N = number of separate experiments in which marker was assayedjn.s. = not significantly detected.

Specific Activity

Glucose-6- GlutamateFraction Betacyanin DNA Cytochrome oxidase NADH-cytochrome c phosphate de- oxaloacetate

oxidoreductase hydrogenase transaminase

units/mg protein jg/mg protein hmoles/min mg protein AA340/min mg

2000g pellet 139±95 50.26±13.39 0.86*0.08 0.11*0.02 0.08±0.01 2.06

fraction 1 ... n.s. n.s. n.s. n.s. n.s.fraction 2 494+82 n.s. n.s. n.s. n.s. n.s.fraction 3 1110±133 n.s. n.s. n.s. n.s. n.s.fraction 4 1091+175 17.30+17.30 0.47+0.04 0.30+0.11 n.s. 0.94fraction 5 334+83 9.6 + 9.6 0.57+0.09 0.19±0.07 n.s. 0.94fraction 6 96±15 25.02± 5.20 1.24+0.10 0.20±0.04 0.11+0.02 3.32gradient pellet 25±7 108.27+25.09 0.26+0.03 0.07+0.01 0.01+0.01 0.74

total particulate 60±11 42.95+ 1.63 1.05±0.13 0.51+0.00 0.05±0.00 1.70

N 10 4 7 4 3 2

scale contamination of the vacuoles by slow moving small vesi-cles of endoplasmic reticulum or outer mitochondrial membraneunlikely.Our method yields vacuolar preparations which appear to be

of similar purity to those of Wagner and Siegelman (29) al-though direct comparison of the two techniques is difficult be-cause these authors presented no biochemical analyses of theirpreparations. Our method is rapid, requiring no overnight incu-bation in lytic enzymes prior to vacuole isolation, but is relativelyinefficient and requires large initial amounts of plant tissue. Themethod of Wagner and Siegelman can be used for small amountsof tissue but will probably prove difficult to apply to tissues

which do not readily yield large numbers of protoplasts, tissuesfor which our method would seem particularly suitable. Severaldifferences in the details of the two methods are apparent.Wagner and Siegelman used about a 500 milliosmolar solutionto lyse protoplasts and release intact vacuoles. This is well belowthe minimum osmotic concentration (approximately 1,000 mil-liosmolar) required to stabilize beet vacuoles (Fig. 3). Whetherthis reflects tissue or species variation in vacuolar contents orsome artifact of one or the other of the isolation schemes re-mains to be determined. Also, Wagner and Siegelman usedmuch lower accelerations to sediment vacuoles than were effec-tive for beet vacuoles (lOOg for 3 min compared with 1,000-

Recovery in various fractions

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662 LEIGH AND BRANTON

2,000g for 10 min in our system). These differences may beexplicable, in part, by differences in centrifuge tube lengths, bythe higher viscosity of our medium, and by the smaller size ofbeet vacuoles.

Isolated vacuoles should provide a convenient system in whichto study the precise role of the vacuole in the metabolism of theplant cell. They should also provide a source from which thetonoplast can be isolated and identified unambiguously as aprelude to its biochemical and structural characterization.

Acknowledgments - We wish to thank S. Gooch for her excellent technical assistance and E.Crump for her expert help in preparing the manuscript.

LITERATURE CITED

1. BRANTON, D. 1969. Membrane structure. Annu. Rev. Plant Physiol., 20: 209-238.2. BRANTON, D., S. BULLIVANT, N. B. GILULA, M. J. KARNOVSKY, H. MOOR, K. MUHLE-

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