Plant Physiol. (1986) 82, 916-9240032-0889/86/82/09 16/06/$0 1.00/0

Electrophoretic Analysis of Protoplast, Vacuole, and TonoplastVesicle Proteins in Crassulacean Acid Metabolism Plants'

Received for publication June 27, 1986 and in revised form August 5, 1986

WILLIAM H. KENYON AND CLANTON C. BLACK, JR.*Shell Agricultural Chemical Co., P.O. Box 4248, Modesto, California 95352 (W.H.K.), and BiochemistryDepartment, University ofGeorgia, Athens, Georgia 30602 (C.C.B.)

ABSTRACr

Protoplasts and vacuoles were isolated and purified in large numbersfrom the CAM plants Ananas comosus (pineapple) and Sedum telephiumfor protein characterization. Vacuoles were further fractionated to yielda tonoplast vesicle preparation. Polypeptides of protoplasts, vacuoles,and tonoplast vesicles were compared to whole leaf polypeptides fromboth plants by one-dimensional sodium dodecylsulfate-polyacrylamidegel electrophoresis. Approximately 100 vacuole polypeptides could beresolved of which 25 to 30% were enriched in the tonoplast vesicles. Theproteins of protoplasts, vacuoles, and tonoplast vesicles from A. comosuswere analyzed further by two-dimensional gel electrophoresis. When one-dimensional electrophoretograms of A. comosus polypeptides werestained with a glycoprotein-specific periodic acid Schiff stain, very fewpolypeptides appeared to be glycosylated, whereas a large number ofglycosylated polypeptides were detected with a silver-based glycoproteinstain particularly in tonoplast vesicles. Analysis of the enzymic contentof vacuoles from both plants indicated the presence of a variety ofhydrolases, including bromelain as a major constituent of A. comosus.No substrate-specific ATPase, however, could be detected in vacuoles ortonoplast vesicles from either plant.

lutoids and sugar uptake into vacuoles from beet root and peamesophyll also have been shown (8, 15). The association ofATPase and permeases with the tonoplast suggests that thismembrane may have a more complex composition than previ-ously thought. The polypeptide composition ofthe tonoplast hasbeen examined in other plant species, e.g. beet root (16) andHippeastrum petals (27), but not in CAM plants.The vacuoles of CAM plants are unique because they are

directly involved in photosynthetic carbon assimilation (9-1 1).CAM leaf vacuoles participate in the nocturnal accumulationand diurnal release of large amounts of malic acid (10) and inpineapple hexoses and malate fluctuate reciprocally each day (9).During the night malic acid accumulates against a steep concen-tration gradient implying that malic acid is actively transportedinto vacuoles. Evidence is available for a malate permease onthe tonoplast of K. daigremontiana (4). The relationship betweenthis permease, ATPase activity known to be present on tonoplastvesicles (2, 23), and malate transport, however, remains to bedetermined.

Characterization of the protein composition of the vacuoleand tonoplast is an initial step in understanding the processes bywhich the vacuole functions as an active metabolic compartmentwithin the plant cell. This study was undertaken to characterizethe proteins and polypeptides found in tonoplast vesicles andvacuoles from two CAM plants.

The vacuole in higher plant cells is a multifunctional organellewhich often may occupy more than 90% of the mature cellvolume. It is involved in osmotic regulation of cell volume,storage of ions and metabolites, compartmentation of specificenzymes including hydrolases, sequestration ofsecondary metab-olites, and senescence (17). The tonoplast mediates the myriadfunctions of the vacuole either directly by transporting selectedcompounds or indirectly by maintaining a barrier restricting theexchange of vacuole contents with the rest of the cell.

Evidence is accumulating which suggests that tonoplast pro-teins are actively involved in the transport of various cellularconstituents between the cytoplasm and vacuole. ATPase2 activ-ity has been associated with the tonoplast from beet roots (6, 13),Hippeastrum and tulip flower petals and leaves (14, 29), tobaccoleaves (22), Hevea lutoids (5), sugar-cane suspension cells (25),and Kalanchoe& daigremontiana (2, 23). ATP-dependent protontransport into vacuoles has been reported for Hevea lutoids (15),and tulip leaves (28). ATP-stimulated citrate uptake into Hevea

'Supported by National Sciences Foundation grant PCM8023949 toC.C.B.

2Abbreviations: ATPase, adenosine 5'triphosphatase; EGTA, ethyl-enebis (oxyethylene nitrilo)-tetraacetic acid; pHMB, p-hydroxymercuri-benzoate; PEPC, phosphoenolpyruvate carboxylase; MDH, NAD-malatedehydrogenase.

MATERIALS AND METHODSPlant Material. Ananas comosus (pineapple) and Sedum tele-

phium plants were grown as isogenic clones of a single parent inpots with an artificial potting soil in a greenhouse. The pottingsoil consisted of pine bark, vermiculite, and granular fertilizercontaining both micro- and macronutrients. All plants were wellwatered and fertilized alternately, biweekly with one-halfstrengthHoagland solution and a commercial (15-15-15) (NPK) fertilizer.One month preceding the experiments, plants were transferredto growth chambers maintained at a 30°C/15°C and 15 h/9 hday/night cycle with 70 to 80% RH and 200 to 350 ,uE m 2 s-'PAR.

All samples were taken at the end of the light period whendiurnal acid levels were lowest and protoplast and vacuole yieldswere highest (9-1 1).

Protoplast and Vacuole Isolation. Protoplasts and vacuolesfrom S. telephium leaves were isolated and purified as previouslydescribed (9-1 1). Briefly, protoplasts were isolated from S. tele-phium and lysed by exposure to hypotonic media in the presenceofEGTA. The released vacuoles were purified on a discontinuousFicoll gradient. Extra washing steps were included to improvethe cleanliness of protoplast and vacuole preparations. Proto-plasts and vacuoles from pineapple leaves were isolated by amodification of this procedure. Leaf tissue was prepared bywashing the surface of the leaf in water with a soft-toothbrush to

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VACUOLE AND TONOPLAST PROTEINS IN CAM PLANTS

remove superficial raphide-containing cells. Leaves were slicedinto 0.5 x 5 mm pieces and digested in a medium containing0.5 M mannitol, 50 mM Mes-NaOH, 5 mM MgCl2, 2% w/vCellulysin (Calbiochem), 8% v/v Pectinase (Sigma), and 0.1%w/v defatted BSA. Tissue to digestion medium ratio was 5 to 6g per 25 ml of medium. Digestion was carried out at 30°C in areciprocating shaker with gentle shaking under a floodlight (200,E m-2 s-') for 3.5 to 4 h.

Digested tissue was swirled gently to complete the release ofprotoplasts and then filtered through four layers of cheeseclothand a 130 gm nylon net. Additional protoplasts could be re-covered by gently squeezing the cheesecloth. Following this step,all preparations were carried out at 4°C. The protoplasts werewashed by centrifugation (90 g for 5 min), resuspension in washmedium (0.6 M mannitol and 25 mm Tris-HCl [pH 8.0] at 22°C)and recentrifugation at 90g for 5 min.The resulting protoplast pellet was resuspended in 12 ml of

20% w/v Ficoll-400-DL in wash medium and overlayered firstwith 16 ml of 10% w/v Ficoll-400-DL in wash medium and then3 ml of wash medium. The discontinuous gradients were spunat 164g for 10 min and protoplasts were recovered at the 0/10%Ficoll interface.

Pineapple vacuoles were isolated from the protoplasts by firstlysing the protoplasts in 0.5 M mannitol, 25 mM Tris-HCl, and 5mM EGTA (pH 8.0) (2 ml protoplasts suspension: 15 ml lysisbuffer) for 10 min at 30°C. The lysed protoplasts were cooled to4°C and mixed (5 ml lysed protoplast preparation:30 ml Ficoll)with 3.5% w/v Ficoll-200-DL in 0.5 M mannitol and 25 mMTris-HCl (pH 8.0) at 22°C. They then were overlayered with 3ml of 0.5 M mannitol and 25 mm Tris-HCl (pH 8.0) at 22°C.The gradients were spun at 25,000 rpm (90,000g average) in aBeckman SW 27 rotor for 30 min. Vacuoles were recovered atthe 0/3.5% Ficoll interface, washed by dilution in 0.6 M mannitoland 25 mM Tris-HCl (pH 8.0) at 22°C, and concentrated bycentrifugation at 1 64g for 5 min.Tonoplast Isolation. Washed vacuoles were lysed by dilution

into ice-cold 15% w/w sucrose in 25 mM Tris-HCl (pH 8.0) at22°C, 1 mm pHMB, and sonicated for 5 s at 4C. The membranewas concentrated by centrifuging the sonicated preparation overa pad of 35% w/w sucrose in 25 mm Tris-HCl (pH 8.0) at 22C,for 2 h at 1 50,000g average in a Beckman SW4 1 Ti rotor at 5°C.The tonoplast was recovered from the 15%/35% interface andfurther washed by diluting into a 12-fold excess of 25 mm Tris-HCI and 1 mm pHMB (pH 8.0) at 22°C, and centrifuging at1 50,000g average for 90 min.Enzyme Assays. The following marker enzymes, associated

with various organelles and the cytoplasm, were assayed accord-ing to published procedures: cytoplasm, PEPC (30); cytoplasmand mitochondria, MDH; chloroplasts, Gal-3P DH; mitochon-dria, Cyt c oxidase; peroxisomes, OH-pyruvate reductase andcatalase (26). Hydrolytic enzymes were assayed essentially asdescribed by Boller and Kende (3). Peroxidase was assayed bythe method of Stafford and Bravinder (24). All enzyme assayswere linear with time and protein concentration.

Electrophoresis. One-dimensional SDS-PAGE was performedusing the buffer system of Laemmli (12) in a Hoefer modelSE600 slab gel apparatus (Hoefer Scientific Instruments). Slabgels 0.75 mm thick were constructed as a 10 to 17% w/v linearpolyacrylamide gradient and a 4% w/v acrylamide stacking gel.Electrophoresis was performed at 8°C and 5 mamp per gel.Two-dimensional electrophoresis was performed according to

O'Farrell (19) with samples loaded on the basic end of thefocusing gel. The second dimension was prepared and run asdescribed above for SDS-PAGE.Sample Preparation. Samples for one-dimensional SDS-PAGE

were prepared by 1:1 dilution in 2x solubilization buffer con-

6.8) at 22°C. Samples were immediately heated for 3 to 4 min at100°C. Pineapple samples were solubilized in the absence ofreductant and in the presence of the sulfhydryl proteinase inhib-itorpHMB (2 mM) to inhibit proteolytic digestion during heating.Preliminary experiments indicated that reduction after boilingdid not significantly alter the migration of either BSA or oval-bumin (a glycoprotein) on gels. The solubilized samples wererapidly cooled and aliquots were taken for protein assays.

After solubilizations all sample. were desalted over SephadexG-25 in 25 mM Tris-HCl pH 8.0 at 22°C by using a rapid,centrifuge-column technique (20) and brought to a final concen-tration of 10% v/v glycerol and 30 mm DTT before loading ontogels. Care was taken to minimize sample dilution at each step.

Protein samples for two-dimensional electrophoresis were sol-ubilized in a urea solubilization buffer containing 2% v/v am-pholines (pH 5-10), 5% v/v ,B-mercaptoethanol, 2% v/v NP-40,0.5 M urea, 2 mm pHMB, and 10 mM methylamine. The meth-ylamine inhibited carbamylation ofproteins by breakdown prod-ucts of urea. When necessary, electrofocusing gels were stored at-70°C until run in the second dimension.Gel Staining. Gels for staining of total protein were fixed in

50% v/v methanol and 10% v/v acetic acid and stained witheither 0.025% w/v Coomassie brilliant blue R250 in 25% v/vmethanol, 10% v/v acetic acid, or a modification of the silver-based stain of Oakley et al. (18) Stain gels were photographedimmediately.

Gels for staining glycoprotein were fixed as above and stainedwith either PAS or a modification of the Ag-based glycoproteinstain from Dubray and Bezard (7).

Protein Determination. Protein was assayed according to Pe-terson (21) with BSA as the standard protein.

Bromelain. Two sources of bromelain were used. Pineapplestem bromelain from Sigma and affinity-purified pineapple leafbromelain. Briefly, whole pineapple leaves were homogenized ina buffer containing 50 mm citrate, 50 mM Na2HPO4, and 1 mMMgCl2 (pH 6.0) and centrifuged at 10,000g for 15 min to removeparticulate material. The supernatant was brought to 80% v/vacetone and kept at -20°C for 30 min. Precipitated protein wascollected by centrifugation, resuspended in the homogenizationbuffer and applied to a PCMB-Agarose (Sigma) column equili-brated with the same buffer, 1 mM DTT, and 1 M KCI. Bromelainwas eluted with 5 mM HgCl2.

RESULTS

Protoplast and Vacuole Purity. The sensitive protein stainingtechniques employed for SDS-PAGE required that exceptionalcare be taken to minimize cross-contamination of separate frac-tions prepared for electrophoresis. Protoplasts were washed re-peatedly and purified on a Ficoll gradient before using directlyfor electrophoresis or for isolating vacuoles. The flotation tech-nique used to purify pineapple vacuoles decreased the yield(approximately 2-4% of whole leaf malate was recovered in thevacuole fraction) but assured that contamination by protoplastswas minimized.

Routinely, vacuole preparations with greater than 0.05% pro-toplasts by number were discarded. A comparison of markerenzymes associated with various subcellular compartments pro-vides an estimate of the amount of cross-contamination (TableI). The vacuole preparation was virtually free of cytoplasmic andorganellar contamination. Similar results were obtained whenvacuoles and protoplasts were ruptured in the presence of either0.1% w/v BSA or 1 mm pHMB to inhibit proteinase activity(data not shown). Mixing vacuoles with protoplasts did notdecrease enzyme activities recovered in protoplast preparations.Comparison of Coomassie Blue and Silver Stains for Proto-

plast, Vacuole, and Tonoplast Vesicle Proteins. Initial attemptstaining 125 mM Tris-HCl, 4% w/v SDS, and 1 mM EDTA (pH

917

to use Coomassie brilhant blue to stain acrylamide gels loaded

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918KENYON AND BLACK

Table I. Marker Enzvme Activity in Purified Protoplasts and Vacuolesfrom Pineapple LeavesEnzymes Protoplasts Vacuoles Recovery

Mumol h-' gmol h-' Mmol h -' ,imol h-'mg-' protein (IO' protoplast) mg9' protein (104 vacuole)-' %

NADH-malate de-hydrogenase 886 8.3 45 8.1 x 10-6 0.1

PEP carboxylase 40 0.04 NDa ND 0OH-Pyruvate re-

ductase 2.9 0.03 ND 0Triose-P dehydro-

genase 12.6 0.12 ND ND 0Catalase 1141 21.4 ND ND 0Cyt c oxidase 1.0 9.6 ND ND 0Chl 0.22 ng/protoplast ND nd 0aND = not detectable. Detection limit for NAD(P)-linked enzymes = 0.097 gmol h-' ml-'.

with protein of vacuoles and tonoplast vesicles from eitherSedum or pineapple were unsuccessful. It was necessary to over-load gels severely to reveal even major polypeptides. Conse-quently a more sensitive silver-based stain was employed. Acomparison ofCoomassie blue and the silver-based protein stainsof pineapple protein is shown in Figure 1. Lanes 2, 3, and 4 ofFigure 1, A and B, compare protoplasts, vacuoles, and tonoplastvesicles on an equal number basis. Protein from 50,000 proto-plasts and vacuoles and tonoplast vesicles from 50,000 vacuoleswere loaded on the gel in Figure 1A. Protein from 6,500 proto-plasts and vacuoles and tonoplast vesicles from 6,500 vacuoleswere loaded on the gel in Figure lB. Protein on the silver-stainedgel was less in order to avoid overloading protoplast proteins.Comparison of staining intensity gives an estimate of the relativeamounts of protein in each cell fraction. Lanes 5 to 8 of Figure1, A and B, compare protein from whole leaf, protoplasts,vacuoles, and tonoplast vesicles on the basis of equal proteinloaded per lane. The amount of protein per lane depended onthe maximum allowable volume of the tonoplast preparation

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that could be loaded. The silver-based protein stain (Fig. IB)reveals many more polypeptides than does Coomassie brilliantblue (Fig. IA).Comparison of Protoplast, Vacuoles, and Tonoplast Vesicle

Proteins from Pineapple and S. tekphium. Vacuoles in pineappleand Sedum share one major function-both accumulate largeamounts ofmalate nocturnally. A comparison was made betweenpolypeptide profiles of protoplasts, vacuoles and tonoplast vesi-cles from Sedum and pineapple to examine the polypeptidecomposition of the two plants (Fig. 2). Three general mol wtclasses of polypeptides exhibited similar patterns in tonoplastvesicles from the two plants viz., near 45K, 30K, and 14K molwt (Figs. lB and 2). A further comparison between pineappleand Sedum of the amount of protoplast protein in vacuoles andtonoplast vesicles is shown in Table II. CAM vacuoles are proteinrich, 14 and 23% of the protoplast protein in Table II, and maybe a site of cellular storage or turnover but we did not investigatethese functions. Less than 1% of the total pineapple vacuoleprotein was recovered in tonoplast vesicles.

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FIG. 1. Comparison of pineapple proteins stained with either Coomassie blue (A), or ammoniacal silver (B). A, Coomassie-stained gel: lane 1,Bio-Rad low mol wt standards; lanes 2 to 4, 50,000 protoplasts, 50,000 vacuoles, or tonoplast vesicles from 50,000 vacuoles, respectively; lanes 5 to8: 9 gg protein from whole leaves, protoplasts, vacuoles, and tonoplast vesicles, respectively. B, Silver-stained gel: lane 1, Bio-Rad low mol wtstandards; lanes 2 to 4, 6,500 protoplasts, 6,500 vacuoles, or tonoplast vesicles from 6,500 vacuoles, respectively; lanes 5 to 8, 9 ,ug protein fromwhole leaves, protoplasts, vacuoles, and tonoplast vesicles, respectively.

918 Plant Physiol. Vol. 82, 1986

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VACUOLE AND TONOPLAST PROTEINS IN CAM PLANTS

1 2 3 4 5 6 7

FIG. 2. SDS-PAGE comparison of S. telephium andpineapple leaf proteins. Lane 1, Bio-Rad low mol wtstandards; lanes 2 and 5, Sedum and pineapple proto-plasts, respectively; lanes 3 and 6, Sedum and pineapplevacuoles, respectively; lanes 4 and 7, Sedum and pine-apple tonoplast vesicles, respectively. Approximately 7.5ug protein were loaded per lane. The gel was stained withsilver.

Table II. Protein Content ofPineapple and S. telephium Protoplasts and VacuolesPlant Prctoplast Vacuole Tonoplast Vesicle

ng protein/protoplast ng protein/vacuole pr tonoplastPineapple 1.28 0.18 (14%)' 1.68 (0.9%)bS. ielephiumn 3.33 0.75 (23%)r

a Percent of protoplast protein recovered in vacuoles. b Percent of vacuole protein recovered in tonoplastvesicles. Data are averages of triplicate protein assays and 2 replicate experiments for each plant.

Two-Dimensional Analysis of Pineapple Proteins. One-dimen-sional SDS-PAGE limits the number of polypeptides which canbe resolved confidently from a mixture of proteins to less than100. Two-dimensional gel electrophoresis, in which proteins aredenatured in urea and separated on the basis of net charge in thefirst dimension and separated on the basis of mol wt in thepresence of SDS in the second dimension, greatly increasesprotein resolution. In Figure 3, two-dimensional electrophore-tograms of protoplasts, vacuoles, and tonoplast vesicles are com-pared. Proteins were electrofocused from pH 7 to 5 and separatedin the second dimension on a 10 to 17% w/v linear gradientpolyacrylamide gel. The streak on the left side of each gel wasincluded to illustrate the protein that did not enter the electro-focusing gel. Approximately 100 to 125 individual spots couldbe resolved in the vacuoles and from 25 to 35 in tonoplastvesicles on the original gels (9). Tonoplast vesicle proteins elec-trofocused poorly as indicated by the dark streak (Fig. 3C).Furthermore, fewer spots occurred on the two-dimensional geloftonoplast vesicle proteins than were found on one-dimensionalSDS-PAGE (compare Figs. I B and 3). Solubilization oftonoplastvesicle protein with SDS followed by sequestration of the freeSDS with an excess ofNP-40 by the method ofAmes and Nikaido(1) did not improve electrofocusing of these membrane proteins.

Glycoprotein Staining of Protoplast, Vacuole, and Tonoplast

Vesicle Proteins. Two previous electrophoretic studies of vacu-oles presented data on the extent ofglycosylation on the tonoplastproteins. Analysis of Hippeastrum tonoplast indicated that, onthe basis of PAS staining, virtually no glycoproteins were asso-ciated with the tonoplast (27). Conversely, electrophoresis ofbeetroot tonoplast and detection ofglycoproteins using fluorescentlylabeled lectins suggest that nearly all of the tonoplast proteinswere glycosylated (16). An effort was made to resolve this dis-crepancy and extend these observations to include the pineapplemesophyll cell tonoplast. Protoplasts, vacuoles, and tonoplastvesicles including known glycoprotein standards were electro-phoresed and glycopeptides were stained with both PAS and asilver-based glycoprotein stain (7).

Initial attempts to identify glycoprotein with PAS necessitatedseverely overloading polyacrylamide gels with pineapple proteins.Only two faintly stained regions around 30K and 70K mol wtcould be found (data not shown). The mobility of the 30K molwt region approximated that of the PAS-stained area in a lanecontaining commercial pineapple stem bromelain. As a control,ovalbumin, a known glycoprotein, also was electrophoresed.PAS-stained ovalbumin could be detected, but again only whenthe gel was extremely overloaded (data not shown).Dubray and Bezard (7) reported the sensitive, glycoprotein-

specific, silver-based stain. The procedure is based on the ability

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KENYON AND BLACK

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The staining density ofmany polypeptides was different in FigureUr"4, B and C, for both standards and pineapple proteins. OtherX10-3 bands, however, remained unchanged.

Comparison of Figure 4, A and C, indicated a decrease in-93 staining density of standard proteins with the exception of pine--66 apple stem bromelain in lane 7, and the complete disappearance

in Figure 4C of some polypeptides seen in Figure 4A. Most-45 vacuole and tonoplast vesicle proteins seen in Figure 4A (lanes

5 and 6) also can be identified in Figure 4C (lanes 5 and 6),Identification of Pineapple Proteins. The comigration of 4

-31 known polypeptide bands with major protein bands from pine-apple is shown in Figure 5. Lane 2 contains a combination of

-22 corn PEP carboxylase and spinach RuBP carboxylase from com-mercial sources. Major bands in whole leafand protoplasts (lanes

-14 3 and 4) line up with PEPC (approximately lOOK mol wt), thelarge subunit of RuBPC (approximately 55K mol wt) and thesmall subunit of RuBPC (approximately 15K mol wt).

Affinity-purified bromelain from pineapple stem (lane 7) andpineapple leaf(lane 6) comigrate with a dominant staining regionin the whole leaf protoplasts and vacuoles (lanes 3, 4, and 5,

t93 respectively) as well as tonoplast vesicles (see Figs. 1 and 2).Compartmentation of Hydrolases in Pineapple and Sedum.

-68 The compartmentation of various hydrolases in pineapple andSedum leaves was investigated by comparing enzyme activitiesin whole leaves, protoplasts and vacuoles. The vacuoles weremajor repositories of the hydrolases (Tables III and IV). Peroxi-

-31 dase and a-mannosidase in pineapple and virtually all the en-zymes tested in Sedum showed significant activity, associated

-22

with the vacuole. Interestingly fl-fructosidase activity was unde--22 tectable in any subcellular fraction of pineapple leaves (Table-14 III).

Substrate-Specific ATPase Analysis. Attempts to assay a sub-strate-specific ATPase from pineapple and Sedum vacuoles wereunsuccessful. Activity of an ammonium molybdate-insensitiveATPase associated with either vacuoles or tonoplast vesicles wasvery low and inconsistent. A variety of vacuole isolation proce-dures (including vacuole isolation in the absence of osmotic

- 93 shock, pHMB, or EGTA) and enzyme assay conditions were- 66 tried without success. It remains to be determined why ATPase

activity was found in tonoplast vesicles from another CAM plantK. daigremontiana (2, 24) and not in Sedum or pineapple.

31

-22

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FIG. 3. Two-dimensional electrophoretic analysis of pineapple pro-toplasts (A) (approximately 50 ,g protein), vacuoles (B) (approximately14 /Ig protein), and tonoplast vesicles (C) (approximately 6 ,g protein).

of silver nitrate to stain free aldehyde groups on reducing sugars.Exposure of glycoproteins to periodic acid oxidizes the carbo-hydrate moieties to form free aldehydes which then are stainedwith silver nitrate. Pineapple proteins and standard proteins,both glycosylated and nonglycosylated, were run on one-dimen-sional SDS-PAGE. The gel in Figure 4A is stained for totalprotein. As a control for nonspecific staining, using the Ag-glycoprotein stain, gels were incubated in ammoniacal silverbefore periodic acid oxidation (Fig. 4B). Many of the majorpolypeptides were stained including standard proteins (lanes 1-

3, 7 and 8, Fig. 4B) as well as pineapple proteins (lanes 4-6, Fig.4B). Figure 4C represents the gel depicted in Figure 4B but withsubsequent periodic acid oxidation (which causes completebleaching of the gel) and then treatment with ammoniacal silver.

DISCUSSIONThe results from this investigation show that the protein

complement of the tonoplast is complex (Fig. 1). This agreeswith the data of Marty and Branton (16) and Wagner (27). Thebeet root tonoplast has 15 major polypeptides and contains 62%of the vacuolar protein (16). In contrast, pineapple tonoplastcontains only a small proportion (-1%) of the total vacuolarprotein (Table II and Fig. 1). Five major and nine minor poly-peptides were distinguished on SDS-PAGE ofHippeastrum petaltonoplasts (27). Comparisons of the Hippeastrum tonoplast towhole vacuoles were not made, however, and comparison tosoluble vacuolar components was difficult because of the lightprotein staining with Coomassie blue. This is similar to thestaining problem illustrated in Figure lA.

In the present study, from 25 to 30 major bands could bedetected in pineapple tonoplast vesicles (Figs. 1, 2, 4, and 6),constituting 0.9% of the total vacuole protein (Figs. 1 and 2, andTable II). Sedum showed a similar or greater complexity of thetonoplast polypeptide pattern (Fig. 2). Comparison of vacuoleswith tonoplast vesicles on a protein basis indicated that mostmajor protein bands do not comigrate. Counting the bands,however, leads to the conclusion that the vacuoles have no morepolypeptides than does the tonoplast (Fig. lB). Comparing cellfractions on the basis of number puts the relative amounts ofprotein in each fraction into perspective (Fig. B). Although

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920 Plant Physiol. Vol. 82, 1986

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VACUOLE AND TONOPLAST PROTEINS IN CAM PLANTS

1 23 456 7 8B

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FIG. 4. SDS-PAGE of pineapple leaf proteins stained with a silver-based glycoprotein-specific stain, A. Total protein stained with silver (5 jigprotein each lane), B. Proteins stained in absence of periodic acid oxidation (5 ;ig protein each lane), C. Lane 1, Bio-Rad low mol wt standards(ovalbumin, 45K mol wt, known glycoprotein; lane 2, a,-plasma proteinase inhibitor (known glycoprotein); lane 3, spinach RuBP carboxylase; lanes4 to 6, protoplasts, vacuoles, and tonoplast vesicles, respectively; lane 7, pineapple stem bromelain (known glycoprotein); lane 8, BSA, ovalbumin,and Cyt c standards.

1 2 3 4 5 6 7

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FIG. 5. Composite SDS-PAGE comparing pineap-ple proteins with known plant protein standards. Lane1, Bio-Rad low mol wt standards; lane 2, PEP carbox-ylase (approximately lOOK mol wt), large subunitRuBP carboxylase (approximately 55K mol wt), andsmall subunit RuBP carboxylase (approximately 15Kmol wt); lanes 3 to 5, pineapple whole leaf, proto-plasts, and vacuoles, respectively; lane 6, affinity-purified pineapple leaf bromelain; lane 7, affinity-purified pineapple stem bromelain.

there are equal numbers of protoplasts and vacuoles in lanes 2and 3, the vacuole polypeptides appear to be nondetectable on abackground of protoplast protein.The unequivocal localization of proteins on the tonoplast

requires that this membrane be free from contamination bysoluble proteins from either the cytoplasm or vacuole and other

membranes. The stringent isolation procedures for pineapplevacuoles was designed to minimize contamination by extravac-uolar proteins. The use of EGTA and large volume washesresulted in vacuoles contaminated to only a minor extent (TableI). But it is possible that soluble vacuole proteins became trappedinside resealed tonoplast vesicles. Consistent with this idea is the

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Table III. Hydrolases in Pineapple LeavesSpecific Activity8 Recovery of Enzymes in Vacuoles

Enzyme BaedonBae onWhole leaf Protoplasts Vacuoles Based onprotein a-mannosidaseb

Acid proteinase 298 451 107 2.2 1.5Acid phosphatase 80.7 174 250 1.4 1.0P-Diesterase 17.7 36.8 87.0 2.4 1.6a-Mannosidase 12.1 9.7 14.6 1.5 1.0a-Galactosidase 14.0 27.2 35.8 1.3 0.9B-N-Acetylglucosaminidase NAC 25.1 47.5 1.9 1.3Peroxidase 173.8 54.3 87.6 1.6 1.1,B-Glucosidase Negligible 5.2 6.8 1.3 0.9,B-Fructosidase NDd ND ND 0 0(3-Galactosidase 0.7 1.3 1.1 0.8 0.6

'Specific Activity: proteinase, A520nm h-' mg-' protein; peroxidase, A46oj)m min-' mg-' protein; others, nmol p-nitrophenol released min-' mg-'protein. b Ratio ofenzyme activity recovered in vacuoles based on a-mannosidase activity (assuming that 100% of the protoplast a-mannosidaseactivity is in the'vacuole). c NA = not assayed. d ND = not detectable.

Table IV. Hydrolases in S. telephium Leaves

Specific Activity' Recovery of Enzymes inVacuoles

Enzyme BasedWhole leaf Protoplasts Vacuoles on Basedon

protein c-mannosidaseb

Acid proteinase NDC ND NDAcid phosphatase 3918 2567 2025 0.8 1.1P-diesterase 160 113 108 1.0 0.9a-Mannosidase 99.4 58.7 48.7 0.8 1.0a-Galactosidase 13.3 4.7 3.7 0.8 1.1,-N-Acetylglucosaminidase 87.8 46.5 38.2 0.8 1.0Peroxidase 238 48.3 66.2 1.4 0.6(-Glucosidase 4.2 24.8 12.6 0.5 1.6,i-Galactosidase 59.5 5.4 3.7 0.7 0.8

a Specific activity: proteinase, A520nm h-' mg-' protein; peroxidase, A406nm min-' mg-' protein; others, nmol p-nitrophenol released min-' mg-'protein. b Ratio ofenzyme activity recovered in vacuoles based on a-mannosidase activity (assuming that 100% of the protoplast a-mannosidaseactivity is in the vacuole). c ND = not detectable.

finding that a polypeptide corresponding in electrophoretic mo-bility with bromelain, a presumably soluble vacuolar protein wasassociated with the tonoplast vesicle preparation (Figs. 1, 2, and5). Large volume dilutions with EDTA and sonication to disruptthe vacuoles were used to decrease the possibility of adsorptionofproteins onto the tonoplast and trapping ofproteins in vesicles.When sonicated vacuoles were fractionated on a linear sucrosegradient (15-40%, w/v) nearly all of the protein (A280) and all ofthe detectable bromelain activity remained on top ofthe gradientor in the first few fractions. Under these conditions, no bromelainactivity was associated with the tonoplast vesicle peak (data notshown). On the other hand, when sonicated vacuoles were runon linear sucrose gradients, the tonoplast fractions collected,sonicated and rerun on another sucrose gradient, the majorpolypeptide bands illustrated by electrophoresis were similar tothose of tonoplast isolated as described in the "Materials andMethods" (data not shown). While bromelain bands have notbeen identified unequivocally on gels, the data suggest that themajor polypeptides around 30K mol wt associated with both thetonoplast vesicle and soluble fraction of the vacuoles are bro-melain.

Glycosylation is a post-translocational modification ofproteinssynthesized on RER. Glycosylated proteins are destined usuallyfor sites outside the cytoplasm and may be transported viavesicles derived from either RER or the golgi apparatus (17). Thevacuole constitutes an extracytoplasmic compartment and it is

conceivable that many of its proteins would be glycosylated.Pineapple stem bromelain for example, contains 2% carbohy-drate by weight. The extent ofglycosylation ofpineapple vacuoleprotein is difficult to assess unequivocally using the techniquespresented in this study. The PAS staining method was tooinsensitive to detect anything but a major quantity of glycopro-tein. The silver-based glycoprotein stain suggests that manyvacuole proteins, both soluble and membrane-bound, are glyco-sylated (Fig. 4). The selectivity ofthis stain is uncertain, however,because control gels not oxidized with periodic acid also exhibitedsome staining. Of the six proteins used as mol wt markers, onlyovalbumin has been reported to be a glycoprotein and it stainedwell. However, four other standard proteins also were stained,albeit lightly, indicating a degree of nonspecificity. Two knownglycoproteins, aI-proteinase inhibitor and bromelain, alsostained in the control gel (Fig. 4B). Whether or not this was dueto previous oxidation ofthe polysaccharide moieties is unknown.Treatment of the control gel with periodic acid caused completebleaching ofthe silver-stained polypeptides (data not shown) andsubsequent restaining revealed the bands exhibited in Figure 4C.Densitometric scans of gels (Fig. 6) stained for total protein andfor glycoprotein more clearly indicate that only a small percent-age ofprotoplast proteins may have carbohydrate associated withthem; but that vacuole proteins, both soluble and membranebound, are to a large extent glycosylated (Figs. 4, A and C, and6, B and C). The scarcity of PAS-stainable material in pineapple

922 'X%rju.-NYON AND BLACK Plant Physiol. Vol. 82, 1986

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VACUOLE AND TONOPLAST PROTEINS IN CAM PLANTS

A

TOP

TOP

c

BOTTOMTOP

FIG. 6. Densitometric scans of pineapple leaf proteins from proto-plasts (A), vacuoles (B), and tonoplast vesicles (C) stained with the silver-based glycoprotein stain. Equal amounts of protein from each tissuefraction were run on two separate SDS polyacrylamide gels. After fixingof gels they were stained with either the glycoprotein stain (. ) or thesilver-based stain for the total proteins ( ). Gels were sliced intoindividual lanes and the lanes were scanned in a Gilford model 2520 gelscanner operated at 600 nm.

gels agrees with the work of Wagner (27) on Hippeastrum tono-plast and Marty and Branton (16). The results from silver-basedstain which show that a majority of tonoplast proteins areglycoproteins (Fig. 6C), also are consistent with work on beet

root by Marty and Branton (16). They found that nearly all ofthe tonoplast and some of the vacuole sap polypeptides adsorbfluorescent derivatives of lectins. Unequivocal determination ofthe extent of glycosylation of pineapple vacuole soluble andtonoplast proteins, however, requires additional work, such asthe use of iodinated or fluorescently tagged lectins to identify theglycoproteins.

Pineapple tonoplast vesicles exhibit an average equilibriumdensity of 1.13 g/ml on linear sucrose gradients (data not shown).This compares closely with other published values such as 1.12g/cm3 for tonoplast from tobacco callus and 1.01 to 1.1 1 g/cm3for Hippeastrum tonoplast (9). The average density for thismembrane corresponds roughly to that of the smooth ER, pre-sumably its progenitor (17).

LITERATURE CITED

1. AMES, CFL, K NIKAIDO 1976 Two-dimensional gel electrophoresis of mem-brane proteins. Biochemistry 15: 616-623

2. AOKI K, K NISI-lDA 1984 ATPase activity associated with vacuoles andtonoplast vesicles isolated from the CAM plant, Kalanchoe daigremontiana.Physiol Plant 60: 21-25

3. BOLLER T, H KENDE 1979 Hydrolytic enzymes in the central vacuole of plantcells. Plant Physiol 63: 1123-1132

4. BUSER-SUTER C, A WIEMKEN, P MATILE 1982 A malic acid permease inisolated vacuoles of a Crassulacean acid metabolism plant. Plant Physiol 69:456-459

5. D'AUZAC J 1975 Characterisation d'une ATPase membranaire en presenced'une phosphatase acide dan les lutoides du latex d'Hevea brasiliansis.Phytochemistry 14: 671-675

6. DOLL S, F RODIER, J WILLENBRINK 1979 Accumulation of sucrose in vacuolesisolated from red beet tissue. Planta 144: 407-411

7. DUBRAY G, G BEZARD 1982 A highly sensitive periodic acid-silver stain for1,2-diol groups of glycoproteins and polysaccharides in polyacrylamide gels.Anal Biochem 119: 325-329

8. GuY M, L REINHOLD, D MICHAELI 1979 Direct evidence for a sugar transportmechanism in isolated vacuoles. Plant Physiol 64: 61-64

9. KENYON, WH 1983 Dynamic role of the vacuole in intercellular compartmen-tation in Crassulacean acid metabolism plants. PhD thesis, University ofGeorgia, Athens, GA

10. KENYON WH, R KRINGSTAD, CC BLACK 1978 Diurnal changes in the malicacid content of vacuoles isolated from leaves of the CAM plant Sedumtelephium. FEBS Lett 94: 281-283

11. KRINGSTAD R, WH KENYON, CC BLACK 1980 The rapid isolation of vacuolesfrom leaves of Crassulacean acid metabolism plants. Plant Physiol 66: 379-382

12. LAEMMLI UK 1970 Cleavage of structural proteins during the assembly of thehead of bacteriophage T4. Nature 227: 680-685

13. LEIGH RA, RR WALKER 1980 ATPase and acid phosphatase activities associ-ated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.).Planta 150: 222-229

14. LIN W, CJ WAGNER, HW SIEGELMAN, G HIND 1977 Membrane-bound ATPaseof intact vacuoles and tonoplasts isolated from mature plant tissue. BiochimBiophys Acta 465: 110-117

15. MARIN B, JAC SMITH, U LUTTGE 1981 The electrochemical proton gradientand its influence on citrate uptake in tonoplast vesicles ofHevea brasiliensis.Planta 153: 486-493

16. MARTY F, D BRANTON 1980 Analytical characterization of beet root vacuolemembrane. J Cell Biol 87: 72-83

17. MATILE P 1978 Biochemistry and function ofvacuoles. Annu Rev Plant Physiol29: 193-213

18. OAKLEY BR, DR KIRSCH, NR MORRIS 1980 A simplified ultrasensitive silverstain for detecting proteins in polyacrylamide gels. Anal Biochem 105: 361-363

19. O'FARRELL PH 1975 High-resolution two-dimensional electrophoresis of pro-teins. J Biol Chem 250: 4007-4021

20. PENEFSKY HS 1977 Reversible binding for Pi by beef heart mitochondrialadenosine triphosphatase. J Biol Chem 252: 2891-2899

21. PETERSON GL 1977 A simplification of the protein assay method of Lowry etal. which is more generally applicable. Anal Biochem 83: 346-356

22. SAUNDERS JA 1979 Investigations of vacuoles isolated from tobacco. I. Quan-titation of nicotine. Plant Physiol 64: 74-78

23. SMITH JAC, EG URIBE, E BALL, U LUTTGE 1984 ATPase activity associatedwith isolated vacuoles of the crassulacean acid metabolism plant Kalanchoe~daigremontiana. Planta 162: 299-304

24. STAFFORD HA, S BRAVINDER-BREE 1972 Peroxidase isozymes of first inter-nodes of Sorghtum. Plant Physiol 49: 950-956

25. THOM M, E KOMOR 1984 Role of the ATPase of sugar-case vacuoles inenergization of the tonoplast. Eur J Biochem 138: 93-99

26. TOLBERT NE 1971 Isolation of leaf peroxisomes. Methods Enzymol 23: 665-682

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924 KENYON At

27. WAGNER GJ 1981 Enzymic and protein character of tonoplast from Hippeas-trum vacuoles. Plant Physiol 68: 499-503

28. WAGNER GJ, W LIN 1982 An active proton pump of intact vacuoles isolatedfrom Tulipa leaves. Biochim Biophys Acta 689: 261-266

29. WAGNER GJ, P MULREADY 1983 Characterization and solubilization of nu-

4D BLACK Plant Physiol. Vol. 82, 1986

cleotide-specific, Mg2+-ATPase and Mg2+-PPase of tonoplast. Biochim Bio-phys Acta 20: 267-280

30. WINTER K 1980 Day/night changes in the sensitivity of phosphoenolpyruvatecarboxylase to malate during Crassulacean acid metabolism. Plant Physiol65: 792-796

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