Plant Physiol. (1988) 88, 259-2650032-0889/88/88/0259/07/$0 1.00/0

Analysis of the Reaction Products from Incubation of SugarcaneVacuoles with Uridine-Diphosphate-Glucose: No Evidence forthe Group Translocator

Received for publication December 23, 1987 and in revised form March 2, 1988

JOACHIM PREISSER AND EWALD KOMOR*Pflanzenphysiologie, Universitat Bayreuth, D-8580 Bayreuth, West Germany

ABSTRACT

Isolated sugarcane (Saccharum spp. hybrid H50-7209) vacuoles in-corporate radioactivity during incubation with labeled UDP-glucose by amechanism which was postulated to be responsible for sucrose storagein the vacuoles (UDP-glucose group translocator). Analysis of the reac-tion products in the medium revealed that several enzymic processes aregoing on during incubation with UDP-glucose such as production ofhexose phosphates, UMP, and sugars, all of which seem unrelated to theincorporation of radioactivity into vacuoles. The incorporated radioactiv-ity was identified mainly as (1-.3)-,-glucan (callose) of polymerizationgrades up to more than 20. Callose occurs as a contaminant at the surfaceof isolated vacuoles coming from the plasmalemma. The properties ofUDP-glucose incorporation into the vacuolar preparation compared fa-vorably with known properties of callose synthase. The low mol wtglucans that are found are probably degradation products of labeledcallose due to hydrolases, which are liberated by centrifugation of vacu-oles. The labeled disaccharide, which chromatographically had beenformerly identified as sucrose, is laminaribiose. No sucrose (or sucrosephosphate) could be identified in the vacuole preparation after incubationwith UDP-glucose. Thus, the mechanism of sucrose storage in sugarcanevacuoles is still open.

The vacuoles are a preferred compartment for storage of sugarsin higher plant cells. Most prominent examples are the storageof sucrose in sugar beet and sugarcane (reviewed in Ref. 28) andthe intermediate storage of sucrose in oat mesophyll cells (1 1).Different mechanisms for sucrose transport into the vacuoleshave been reported. Whereas catalyzed diffusion operates inmesophyll vacuoles (12), sucrose accumulation occurs in sugarbeet and sugarcane vacuoles. Briskin et al. (3) found a sucrose-proton antiport system in sugar beet vacuoles and Thom andMaretzki (25) detected sucrose formation in sugarcane vacuolesby incubation of isolated vacuoles with UDP-glucose. Theypostulated a "UDP-glucose group translocator," a tonoplast-bound enzyme complex that could achieve formation offructose-6-phosphate from UDP-glucose and subsequently a vectorialsynthesis of sucrose-phosphate from the generated fructose-6-phosphate and UDP-glucose. This postulated mechanism ofsucrose storage in sugarcane vacuoles attracted much attentionbecause it constituted an entirely new type of transport throughmembranes. Furthermore, the operation of this mechanism insugar beet vacuoles was also shown (8, 23).According to the postulated scheme of Thom and Maretzki

(25) no reaction that required transmembrane energization ofthe tonoplast was involved, neither a pH-gradient nor a mem-

brane potential. On one side, addition of sucrose in presence ofMgATP to vacuoles from sugarcane stalk gave barely perceptibleuptake of sucrose (14), while on the other hand MgATP eveninhibited the UDP-glucose group translocator (26). The delicatecoexistence of tonoplast energization by the ATPase (22) andsucrose storage by the UDP-glucose group translocator was puz-zling, especially in the case of sugar beet vacuoles, where thecoexistence of the proton-driven sucrose-proton antiport systemand the UDP-glucose group translocator was visualized (23).Therefore, as a first step to obtain ideas about the mutualinteraction of energization and sucrose storage, a careful analysisof the reaction products, which are produced by incubation ofsugarcane vacuoles with UDP-glucose, was performed.

MATERIALS AND METHODS

Isolation of Protoplasts and Vacuoles. Protoplasts were iso-lated from 8- to 1 -d-old sugarcane suspension cells (a subcloneof Saccharum spp. hybrid H50-7209 grown in supplementedWhite's basal salt medium) as described by Thom et al. (24) andsuspended in White's basal salt medium containing 0.5 M man-nitol at pH 5.6. For isolation of vacuoles, the protoplast suspen-sion was layered over a cushion of 12% Ficoll made up inprotoplast suspension medium and centrifuged for 1 h in aKontron TST 54 Rotor at 45,000 rpm. Vacuoles were recoveredat the 0/12% Ficoll interface and washed three times in White'sbasal salt medium containing 0.5 M mannitol at pH 6.5.Uptake Measurements. For uptake measurements, vacuoles

were suspended in White's basal salt medium plus 0.5 M mannitoland the suspension was adjusted to a protein concentration ofabout 0.5 mg ml-', as determined by the method of Bradford(2). Uptake measurements were done in the same medium,usually containing 2 mm CaCl2 and 5 mM MgCl2 on a rotaryshaker at 25°C. Uptake was initiated by addition of radioactiveUDP-glucose. Effectors were added immediately prior to addi-tion of the labeled substrate. Vacuoles were separated from themedium by centrifugation through silicone oil. Aliquots of themedium were boiled for 2 min immediately after centrifugation.Vacuoles were suspended in 70% ethanol or water and thenheated for 2 min. Aliquots of the resuspended vacuoles and ofthe medium were used for scintillation counting (5 g 2,5-diphen-yloxazole, 100 g naphthalene in 1 L dioxane).

Preparation of '4C-Labeled UDP-Glucose. Depending on thepurpose of the experiment, the vacuoles were incubated eitherin [glucose-'4C(U)] uridine diphosphate glucose (UDP-['4C]glu-cose) obtained from NEN-Dupont, Dreieich (FRG), or in [uri-dine-4-'4C]uridine diphosphate glucose (['4C]UDP-glucose),which was prepared enzymically as described by Wright andRobbins (29) using commercial UDP-glucose-pyrophosphorylasefrom Boehringer. Two ,umol ['4C]UTP (2.5 ,uCi) from Amer-

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PREISSER AND KOMOR

sham, Braunschweig (FRG), were incubated with 3 ,umol ofglucose- 1-phosphate, 2 Amol of MgCl2, and inorganic pyrophos-phatase (Sigma). After boiling for 2 min and centrifuging, thereaction products were separated on a Dowex 1 anion exchangecolumn (Serva) by stepwise elution with 0.025 M NH4Cl, 0.01 NHCI, and 0.1 M NaCl in 0.01 N HCI (4, 5). Fractions weremonitored for UV-absorption and radioactivity. The UDP-glu-cose fraction was concentrated by absorption on charcoal, fol-lowed by elution with 50% ethanol containing 1% ammonia.After neutralization with HCI the UDP-glucose was further con-centrated by evaporation. The purity of UDP ['4C]glucose wastested by PEP-paper chromatography or TLC on PEI-cellulosefoils (Sigma).Chromatography of Products. Nucleotides in the medium were

identified by paper chromatography on PEI-impregnated What-man No. 1 cellulose according to the method of Verachtert et al.(27). Chromatograms were first developed with water, dried, andthen developed with 0.4 M LiCl. Unlabeled reference nucleotideswere applied together with the labeled samples and localized witha UV-lamp. The spots were cut and radioactivity was determinedby scintillation counting.

Products in the medium after incubation with UDP-glucose14C labeled in glucose were chromatographed on PEI-celluloseready foils from Sigma. In the first development with water,mannitol and free sugars migrate with the front, while in thesecond elution with 0.4 M LiCl, sugar phosphates are separatedfrom UDP-glucose.

Radioactive spots were detected by scanning the plate with aBerthold TLC scanner and were counted with a Geiger tube(Berthold). Extraction of vacuoles was made with hot water orethanol. Vacuoles were incubated for 1 h at 60°C either in H20or 70% ethanol and centrifuged for 5 min in a microfuge. Fordetermination ofincorporated products, the soluble extracts wereconcentrated and spotted on Avicel ready foils (Schleicher &Schull). Thin layers were developed three times either in ethyl-acetate-pyridine-water (100/35/25) for ethanol extracts or n-butanol-ethanol-water (3/2/2) for water extracts (9). Both waterextracts and ethanol extracts were also chromatographed onAvicel in n-butanol-acetic acid-water (4/1/5 upper phase). Ra-dioactive bands were localized with the TLC scanner andcounted. For detection of unlabeled sugars the TLC-foils weresprayed with aniline-diphenylamine-phosphoric acid spray re-agent (9).Enzymic Treatment of Extracts. Labeled compounds in the

vacuolar extracts were treated with alkaline phosphatase (Sigma,bovine intestinal mucosa), invertase (Serva, yeast), Onozuka-cellulase (Serva), cellulase from Trichoderma viride (Worthing-ton), a-amylase (Boehringer),,3-glucosidase (Sigma), or laminar-inase (Sigma). Incubation with enzymes was usually for 60 minat 38C. Ethanolic extracts were evaporated to dryness anddissolved in water before enzymic treatment. Incubation withalkaline phosphatase was made in 20 mm ethanolamine buffer,pH 9.6, containing 1 mm MgCl2. When phosphatase treatmentwas followed by incubation with invertase the pH was adjustedto 5.0 with HC1 and incubation was continued for another 60min. The incubations with other enzymes were in sodium ace-tate, pH 5.0. Incubation with enzymes was terminated by boilingthe samples for 2 min. Samples were centrifuged in a microfugeand the supernatants were concentrated and spotted on TLC-foils. Specificity of the polysaccharide-hydrolyzing enzymes wastested under the same conditions using commercial substrates.Cellulose MN 300 (Macharey and Nagel), lichenan (Sigma),laminarin (Sigma), and soluble starch (Fluka) were used at con-centrations of 2.5 mg ml-'. Liberation of reducing sugars fromthese substrates was measured by the method of Nelson (16),

modified by Somogyi (I19), or by the dinitro-salicylic acid-method(1).Both cellulases from Serva and Worthington hydrolyzed all

four substrates, a-amylase hydrolyzed only starch, and laminar-inase exclusively hydrolyzed laminarin. If cellulase from Wor-thington (5 mg ml-') was boiled for 60 min, it still hydrolyzedlichenan to a great extent but none of the other substrates. Thisheat-stable activity was called "lichenase."Enzymic Determination of Sugars and Sugar Phosphates. Sug-

ars and sugar phosphates in the medium after incubation ofvacuoles with UDP-glucose were determined enzymically bytransformation to glucose-6-phosphate and oxidation with glu-cose-6-P-dehydrogenase according to Stitt et al. (20).Assay of UDPase. For measurement of UDPase activity,

vacuoles were incubated in White's basal salts in 0.5 M mannitolwith 5 mM MgC92, 0.1 mm ammonium molybdate, and 0.05 to1 mm UDP. After 10 min, the reaction was terminated by boilingand centrifugation. UDP in the supernatant was determined withpyruvate kinase as described by Salerno and Pontis (18).

Staining with Aniline Blue. Vacuoles were stained on a micro-scopic slide with a few microliters ofa 0.01% aniline blue solution(Sigma) in 60 mm phosphate buffer, pH 8.5. Stained vacuoleswere examined for fluorescence with a Zeiss fluorescence micro-scope using the filter combination G 436/FT5 I0/LP 520 or BP450-490/FT 510/LP 520.

RESULTSReaction Products in the Medium. The incubation of isolated

sugarcane vacuoles with labeled UDP-glucose led to the produc-tion of several compounds in a biphasic time course. Initially,within the first 15 s, a fraction of UDP-glucose was transformedto sugar phosphates and UMP (Figs. 1 and 2). In a second phase,the slow decrease of UDP-glucose was accompanied by a similarincrease of UMP, free sugar, and the incorporation of radioactiv-ity into the vacuoles. UDP and UTP appeared only in tracequantities. More than half of the sugar phosphate was glucose-1-phosphate, the remainder, glucose-6-phosphate and fructose-6-phosphate; sucrose phosphate was not detected. The labeled freesugar was mostly sucrose (Table I); hardly any labeled glucoseand fructose were found. There was, however, nonlabeled glucoseand fructose in the medium in the range of 0.2 to 0.4 mm,because mannitol, which was used as osmoticum, is contami-nated with hexoses.The first reaction was a phospho-diesterase reaction, which

split UDP-glucose to UMP and glucose-l-phosphate. This reac-

100

C 00

C 50-C UDP-gic

0

sugar phosphatesLd incorporated into vacuoles

sugar

00 2 4 . 6 8 10

minFIG. 1. Time course of labeled compounds in the medium from UDP-

['4C]glucose. Isolated vacuoles (0.45 mg protein ml-') were incubated in100AM UDP-[4Clglucose in presence of 2 mm CaCl2 and 5mM MgC12.

260 Plant Physiol. Vol. 88, 1988

I Abbreviation: PEI, polyethylenimine. www.plantphysiol.orgon June 17, 2018 - Published by Downloaded from

Copyright © 1988 American Society of Plant Biologists. All rights reserved.

REACTION PRODUCTS FROM UDP-GLUCOSE WITH VACUOLES

100 9

0

0C-

Li4-

0J

L.

v_F

2 4 6 8 10min

FIG. 2. Time course oflabeled compounds in the medium from UDP-['4C]glucose. Isolated vacuoles (0.52 mg protein ml-') were incubated in100 gM UDP-['4C]glucose in presence of 2 mm CaCl2, 5 mM MgCl2 and0.1 mM molybdate.

Table I. Composition ofLabeled Sugar Phosphates and Labeled Sugarin Medium from Labeled UDP-['4C]Glucose

Isolated vacuoles were incubated with 100 AM UDP-['4C]glucose for 8min. The medium was analyzed by enzymic tests for sugar phosphatesand by TLC and Geiger counting for free sugars.

ConcentrationSugar Percentage of Labeled

CompoundsM

Glucose- I -phosphate 47 16.8Glucose-6-phosphate 38 13.7Fructose-6-phosphate 15 5.4Total hexose phosphates 100 35.9

Sucrose 80 0-10Glucose 10 1Fructose 10 ITotal sugar 100 2-12

tion had no relationship to the UDP-glucose-dependent grouptranslocator, because it occurred in the first seconds ofincubationwhen hardly any label is incorporated into the vacuoles. Thesecond phase of UDP-glucose consumption might be related tothe incorporation of labeled glucose into vacuoles. UMP seemsto be the reaction product of the uridine part of UDP-glucose,together with trace amounts of UTP and UDP. Some doubtshave to be raised, however, concerning the primary productionofUMP, because sugarcane vacuoles possess a very active UTP-and UDP-splitting enzyme (21). This uridine-nucleotide phos-phatase was inhibited by pyrophosphate (Fig. 3). If pyrophos-phate was used to inhibit UDP-hydrolysis in the UDP-glucoseincorporation experiments, there was a very rapid decrease inUDP-glucose and an equally rapid increase in UTP, UDP, andUMP (Fig. 4). The greatly increased rate of UDP-glucose con-sumption, resulting in UTP and sugar phosphate, seems due toa UDP-glucose pyrophosphorylase. UDP was the major uridinenucleotide produced, but still there was much UMP. Additionof UDP or UTP was found to inhibit the conversion of UDP-glucose to sugar phosphates (data not shown).

Because a large proportion of UDP-glucose was already con-verted by enzymic reactions unrelated to the group translocator,it was not possible to elaborate the fate of the uridine part ofthose UDP-glucose molecules of which the glucose part wasincorporated into the vacuoles.

80 I

IC

~60O0..E

o40

0.

c \E

@ 20-

o 0

0~D 0 0.2 0.4 0.6 0.8 1.0

pyrophosphate (mM)FIG. 3. Inhibition of UDPase by pyrophosphate. Isolated vacuoles

(0.2 mg protein ml-') were incubated with 200 Mm UDP for 10 min.Decrease ofUDP was measured as described in "Materials and Methods."

80-

:i60

c L0.;:40.ci4.-

C2aou 20-0Li

v0 10

minFIG. 4. Time course oflabeled compounds in the medium from UDP-

["4C]-glucose in presence of pyrophosphate. Isolated vacuoles (0.6 mgprotein ml-') were incubated in 65 AM UDP-["4C]-glucose in presenceof 50 Mm pyrophosphate.

Products of UDP-I'4C]Glucose Incorporation into the Vacu-oles. Incubation of isolated sugarcane vacuoles with UDP-glu-cose, which was '4C-labeled in glucose, yielded a time-dependentincorporation of label into the vacuoles (Fig. 1). Incubation withUDP-['4C]glucose, which was labeled in the uridine, gave noincorporation of label (Fig. 2) (25). Eighty percent of the radio-activity in the vacuoles could be extracted with hot water, 15 to30% with hot ethanol (this figure varied considerably frompreparation to preparation).The ethanol-insoluble label could be solubilized by incubation

with cellulases in a mixture as used for preparation ofprotoplasts;the label then appeared solely in glucose.TLC chromatography of the water-soluble fraction and the

ethanol-soluble fraction revealed at least eight labeled bands (Fig.5). One band appeared at the solvent front (sometimes veryfaintly), suggesting a lipophilic compound (6). Next seemed tobe glucose, then sucrose, hexotriose, tetraose, and up to heptaose(at least according to the distance from the origin) and a spotremaining at the origin. Sephadex G 50-50 chromatography ofthe water-soluble radioactivity revealed a broad distribution oflabeled compounds with the majority of label from mol wt 300to 1100 (disaccharide to heptasaccharide), but also some largeicompounds (up to mol wt 5000). Incubation of the extracts withalkaline phosphatase had only minor effects, one band in th(

°o - \~~ UDP-gic

UMP

.UDPz-- ,UTP

_O)n UDP-gic0- ,-11UTP -

- UMP-UDP

I I I

I I

(I.

261

5

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PREISSER AND KOMOR Plant Physiol. Vol. 88, 1988

water extract

+ -gtU--cosidcise

- nver-,(se

trorn

frugtc

4m stor,"

rE' t cJecl

D 9 c !-) 0r.; It'.,

ethcinolic extrcct

front

IF-tJ i

.,.iM

+ phos-ohatose

4* inverrose

FIG. 5. Autoradiography from TLC of water-soluble (upper) and ethanol-soluble (lower) compounds from vacuoles, which had been incubatedwith ['4C]-UDP-glucose. s stands for sugar test, i.e. the plate was sprayed to detect the sugars, 1 stands for labeled compounds as detected byautoradiography. The third and the fifth columns of water extract analysis (,B-glucosidase treatment and perchloric acid centrifugation) were fromdifferent TLC plates than the other columns of water extract. The white band next to glucose represents mannitol. The locations of fructose (fru),glucose (glc), and sucrose (suc) on each TLC plate are indicated by lines at the side of the columns. The chromatography was developed three timeson Avicel thin layer plates with ethylacetate-pyridine-water (100/35/25) for ethanol extracts and butanol-ethanol-water (3/2/2) for water extracts.The enzymic treatment of extracts was as described in "Materials and Methods." In one treatment (last column), the vacuoles were sedimentedthrough silicone oil into 1.5 M perchloric acid; before extraction, the vacuolar pellet was neutralized with NaOH.

"oligosaccharide fraction" disappeared, and the glucose spotincreased. Incubation with ,8-glucosidase or cellulases shiftedpractically all radioactivity to the place of glucose; fructose didnot appear (Fig. 5 and Tables II and III). The labeled bands wereglucose and oligomers containing glucose.Of special interest was the disaccharide band, which, according

to the location, had previously been identified as sucrose (25).Treatment of the extracted radioactivity with invertase (with orwithout preceding treatment with phosphatase) did not convertthe disaccharide to hexoses, though the nonlabeled sucrose inthe very same vacuolar extract was totally hydrolyzed by thistreatment (Fig. 5). In contrast, incubation with ,B-glucosidase,which converted nearly all labeled bands to glucose includingthe disaccharide (Fig. 5, Table II), did not hydrolyze the unla-beled sucrose of the extract. The disaccharide, therefore, was a

i3-glucoside, either laminaribiose-( 1-3)-fl, cellobiose-( 1--4)-,, orgentiobiose-(1--*6)-,3. From chromatographic behavior, it wasclearly laminaribiose, because cellobiose and gentiobiose aresignificantly less mobile in this solvent system (data not shown).The oligosaccharides (n = 3-7) also seemed to contain ,3-(1-+3)-glycosidic bonds, because laminarinase, which splits (1-.3)-,B-glucans to mono- and disaccharides, does so similarly with thelabeled compounds extracted from the vacuoles (Table II, Fig.5).

Basically, the water extract showed the same composition asthe ethanolic extract, though a larger proportion of the high molwt compounds seemed to be insoluble in ethanol and, conse-quently, a lower percentage of mono-, di-, and trisaccharides andphosphate esters were found in the water extract (Tables II andIII). From these analyses, it can be concluded that incubation of

262

front

frugicsuc

stort

controt + taminarlncse

fruglc

stcirt

controt

..-. 'n-

S_O

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REACTION PRODUCTS FROM UDP-GLUCOSE WITH VACUOLES

Table II. Distribution of Water-Soluble Labeled Compoundsfrom Vacuoles which Were Incubated withUDP-['4Cglucose

Isolated vacuoles (0.5 mg protein ml-') were incubated with 100 gM UDP-['4C]glucose for 7 min, thenseparated from the medium by silicone oil centrifugation, and extracted in hot water; 80% of incorporatedlabel was thereby extracted. Then the extracts were treated with enzymes as indicated below. The treatedextracts were chromatographed on thin layer, scanned for radioactivity, counted, and sprayed for carbohydrates.

Treatment Polysaccharide Trisaccharide Disaccharide Glucose Fructose Front

% ofextracted radioactivityControl 72 11.7 11.3 2.9 0 2.0+ Alkaline phosphatase 65.7 14.5 12.1 5.9 0.3 1.6+ Cellulase/pectinase 4.6 0.9 0.9 94.7 0.2 1.3+ Alkaline phosphatase 62.4 17.3 14.1 4.8 0 1.3and invertase

+ f,-Glucosidase 13.7 3.0 1.1 82.2 0 0+ Laminarinase 0.7 2.0 20.3 77.0 0 0

Table III. Distribution ofEthanol-Soluble Labeled Compoundsfrom Vacuoles which Were Incubated withUDP-'4C]-Glucose

The procedures were the same as in Table IV with the only difference being that the separated vacuoleswere extracted in hot ethanol, which extracted (in this particular experiment) 15% of the incorporated label.

Treatment Polysaccharide Trisaccharide Disaccharide Glucose Fructose Front

% ofextracted radioactivityControl 54.5 18.4 22.0 3.9 0 1.2+ Alkaline phosphatase 22.2 29.0 21.9 19.4 1.6 5.9and invertase

+ Cellulase/pectinase 16.8 2.6 3.8 72.4 0.8 4.2

isolated sugarcane vacuoles with UDP-['4C]glucose yields a seriesof oligosaccharides, some ofthem more soluble in hot water thanin hot ethanol, which all belong to the (1--3)-f3-glucan series. Inaddition, a small amount (2-5%) of glucose phosphate is foundand a small quantity of lipid-like compounds (ca. 2%). Neitherfructose nor sucrose were labeled to any significant extent inthese experiments.

Influence of callose synthesis effectors on UDP-['4Cjglucoseincorporation by isolated sugarcane vacuoles. Since the analysisof reaction products revealed oligosaccharides of the (l-3)-,B-glucan series as main product, it was suspected that the incor-poration of UDP-['4C]glucose is due to glucan synthase II, thecallose-synthesizing enzyme (13). Therefore, several effectors ofcallose synthesis such as Ca2", UDP, UTP, and cellobiose weretested to see whether they affected UDP-['4C]glucose incorpora-tion into sugarcane vacuoles. They all behaved as described forcallose synthase: calcium (and less so magnesium) and cellobiosestimulated UDP-glucose incorporation, whereas UDP, UTP, andATP inhibited it (Table IV). The effect of Ca2" was smaller thanreported in the literature, but it has to be remarked that nocalcium complexing compound was included in the controlpreparation. Pyrophosphate inhibited glucose incorporation be-cause of the decrease of UDP-glucose concentration by UDP-glucose pyrophosphorylase (Fig. 4). The inhibitory effect of py-rophosphate and UDP was additive. According to these proper-ties, most likely glucan synthase II is responsible for glucoseincorporation from UDP-glucose by sugarcane vacuoles. Glucansynthase II is reported to synthesize higher oligomers and poly-mers, but not di- and trisaccharides, which are found only asdegradation products ofthe polymer (7). The question, therefore,was whether the vacuolar preparation really synthesized lami-naribiose and the small oligomers. It could be imagined that thesedimentation of vacuoles by silicone oil centrifugation liberatedintravacuolar ,3-glucosidase, which partly degraded the freshlylabeled polymer to small oligomers during the small time spanbetween sedimentation and extraction (approximately 2 min).Therefore, vacuole sedimentation into perchloric acid was per-

Table IV. Influence ofDifferent Compounds on UDP-[14C]GlucoseIncorporation by Sugarcane Vacuoles

The control condition contains 2 mM CaCk2 and 5 mM MgCk2; 100%corresponds to 3.0 nmol mg protein-' min-'.

Treatment Incorporation

Control 100- MgC2 89- CaCk2 and MgC2 39+ 5 mM Cellobiose-CaCI2 and MgCl2 74

+ 2 mM MgATP 57+lmMUDP 9.8+ I mM UTP 22+ 100 Mm Molybdate 94+ 200 Mm Pyrophosphate 37+ 100 Mm UDP 63+ 200 Mm Pyrophosphate and 100 MM 21UDP

formed, and the water-extractable compounds were then chro-matographed. In that treatment, hardly any labeled laminaribioseor small oligomers were detected (Fig. 5). Nearly all extractablelabel was in the polymer at the origin of the thin layer (Fig. 5).Glucan synthase II is reported as a typical plasmalemma

enzyme, not a tonoplast enzyme (17). Since the preparation ofsugarcane vacuoles contains significant contamination from plas-malemma (24), it was determined by aniline blue stainingwhether callose-like material is found in the preparation, andwhere it is. Figure 6 demonstrates that the fluorescence was solelyfound in the cap-like membranous lumps on the vacuoles andwas not homogenously distributed over the vacuolar surface, aswould be expected for a tonoplast-bound compound. Thus, mostlikely the UDP-['4C]glucose incorporation occurred in these capswhich contain plasmalemma.

Incubation of the UDP-glucose-labeled vacuoles with a pec-

263

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PREISSER AND KOMOR

FIG. 6. Aniline blue staining of isolated sugarcane

procedure was as described in "Materials and Methods."

significant autofluorescence of unstained vacuoles.

was picked for photography where contaminated

bled. Noncontaminated vacuoles showed practically

do not show up in the fluorograph.

tinase-cellulase mixture removed more than half

tivity from the vacuoles within h. Obviously,

was at the external face of membranes.

DISCUSSION

The incubation of isolated sugarcane vacuoles

glucose leads to several reactions which are partly

each other and which produce many reaction products,

the enzymic processes are difficult to analyze.

glucose pyrophosphorylase and the uridine nucleotide

tase instantaneously convert a significant fraction

cose to UMP, glucose phosphate, and phosphate.

of a UDP-glucose phosphodiesterase (UDP-glucose

cannot be excluded. The addition of pyrophosphate

the fraction of consumed UDP-glucose; UDP and

it. It is not clear, however, whether there

pyrophosphate present (or formed) at the start of

to allow the rapid pyrophosphorylase reaction.

Other reactions are slower, such as phosphoglucomutase

hexose phosphate isomerase, which equilibrate glucose-

phate with glucose-6-phosphate and fructose-6-phosphate.

small amount of labeled sucrose, which was erratically

the medium, probably originates from soluble sucrose-synthase

by using labeled hexose-phosphates and UDP-glucose.

extravacuolar production of sucrose might in

active sucrose uptake into isolated vacuoles

beet, lead to uptake of labeled sucrose, thereby simulating

glucose group translocation.) In addition there is

ration of glucose into the vacuoles, but the rate

is relatively small compared with the rate ofsome

listed above; therefore, the formation of a certain

uct cannot be assigned to the mechanism of glucose

tion. Glucose from UDP-glucose is incorporated

vacuoles in such a way that it is sedimented down

the vacuoles. When the vacuoles are denatured

water or ethanol, all the incorporated glucose

by f3-glucosidase and/or cellulases. Thus, the reaction

could be identified as (l- 3)-3-g1ucan, i.e. callose.

contained in the isolated vacuole preparation in caps

the surface of the vacuoles. Since most of the labeled

accessible to hydrolases from the medium, it

attached to the membranes, not incorporated

vacuolar space. The series of low mol wt (1--3)-fl-glucosides

most likely the product of,B-glucosidase action, which is liberatedduring sedimentation of the vacuoles. The observed propertiesof UDP-glucose incorporation (stimulation by Ca2+, Mg2+, su-crose, cellobiose, inhibition by UDP, UTP, ATP) are similar tothose reported for callose synthase (10, 15). It is an unfortunatecoincidence that the disaccharide laminaribiose shows up inchromatography in nearly the same location as sucrose, so thatit is easily misidentified. The chromatographic separation ofsugar, especially of the medium, is in addition disturbed by themassive presence of mannitol, which squeezes between fructoseand glucose; only by chemical identification and autoradiographyon the same chromatogram could individual sugars be identified.The variability of the amount of labeled disaccharide woulddepend very much on the time elapsed between breakage ofvacuoles during sedimentation and inactivation of theliberatedhydrolases. Since there was no significant quantity of labeledsucrose detected after incubation with labeled UDP-glucose, thequestion how sucrose is transported and accumulated in sugar-cane vacuoles is still open.

Acknowledgment-The very helpful discussions with and suggestions from Dr.H. Kauss (Kaiserslautern), Dr. Th. Boller (Basel), and Dr. Ph. Matile (Zurich) aregratefully acknowledged, also the experimental help by R. Wendler and H. Schroer,and the corrections on the English style by Dr. A. Maretzki and M. Thom (Aiea).

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3. BRISKIN DP, WR THORNLEY, RE WYSE 1985 Membrane transport in isolatedvesicles from sugar beet taproot.II. Evidence for a sucrose/H' antiport.Plant Physiol 79: 871-875

4. COHN WE 1957 Methods of isolation and characterization of mono- andpolynucleotidesby ion exchange chromatography. Methods Enzymol 3: 724-743

5. COHN WE 1957 Chromatographic separation ofATP, ADPand AMP. MethodsEnzymol 3: 867-869

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