Planit Phlysiol. (1906) 41, 181-189

Trhe Regulation of Sugar Uptake and Accumulationin Bean Pod Tissue

J. A. SacherDepartment of Botany, California State College at Los Angeles

Received May 24, 1965.

Suomlmaitry. The identity, localization and physiological significance of enzymes in-\-olved in sugar uptake anid accumulationi were determined for endocarp tissue of pods ofKenitucky XVonder pole beans (Phaseolhs vulgaris). An intracellular, alkaline invertase(H optimum, 8) was assayed in extracted protein, as well as enzymes involved in-lcrose synthesis, namely, uridinediphosphate (UDP-glucose pyrophosphorylase andU DP-glucose-fructose transglucosylase). Indirect evidence indicated the presence also ofhlexokinase, phosphohexoseisomerase and phosphoglucomutase. The data suggested tnatsucrose synthesis occurred in the cytoplasm, and that both sugar storage and an alkalineinlvertase occurred in the vacuole. The latter functions to hydrolyze accumulated sucroseAn outer space invertase (pH optimum, 4.0) was detected, but was variable in occilrr-ence. Although its activity at the cell surface enhanced sucrose uptake. sucrose mav betaken up unaltered.

Over a wide range of concentrations of exogenous glucose the sucrose/reducing sugarratio of accumulated suigars remained unichanged a-t about 20. Synithesis of sucrose ap-pears to be requisite to initial accumulation fromi glucose or fructose, as free hexoses (lonot increase at the apparent saturating concentration for uptake. Sucrose accumulationifrom exogenious hexose represents a steady-state value, in which sucrose is transportedacross the tonoplast into the vacuole at a rate equivalent to its rate of synthesis. Evi-dence indicates that this component of the accumulation process involves active transportof sucrose against a conicentration gradient. The ratio of sucrose/reducing sugars in theaccumulated sugars immilediately after a period of uptake was inversely related to the levelof inner space inivertase. WVithin 16 hours after a period of accumulation. practicallyN allof the sugar occurs as glucose and fructose.

The absence of comnpetition among hexoses and sucrose indicated that a conlmllollcarrier was not involved in their uptake. From a series of studies on the kinetics ofuptake of glucose and fructose, including competition studies, the effects of inhibitors,radioactive assay of accumulated sugars and the distribution of label in accumulatedsucrose it appeared that rate limitationi for glucose or fructose uptake resides in the se-luence of reactionis leading to sucrose synthesis, rather than in a process miediated by acarrier proteini.

The tissues of several higher planits and somiie ani-mals are similar iin that uptake of glucose is abouit 3times faster tlhan fructose (1. 8. 9. 18. 21). For planttissues glucose inlhibits fructose uptake considerably.while fructose does not inhibit glucose uptake (8:,Sacher. Hatch and Glasziou. unpublished data onsugo,arcane). There is considerable evidence from theuse of metabolic inhibitors an(I anaerobiosis thatenergy coupling is essential to sugar tuptake (2. 3. 8).Sugar accumulation froml solutionls of glucose orfructose occurs principally as sucrose ( 1. 2. 6. 8. 15.17).

For yeast and sugarcane it has been shown thathvdrolysis of sucrose at the cell surface is prerequisite

Supported by Research Grant GB-287 from the Na-ti,nal Science Foundation.

181

to uptake (5, 19. 21) while in tobacco leaf discssucrose is taken up unaltered (16). There are -re-ports of considerable invertase in the outer space ofother higher plants (4. 9. 13. 17). In the latter, how-ever, it has not beeni establishe(d that hydrolysis ofsucrose is essential to tuptake.

Bean endocarp tissue differs from the tissnes re-ferred to above in that sugar is stored almzost entirelyin the form of glucose and fructose, 'rather thansucrose. Yet in this tissue considerable sucrose issynthesized during the onset of senescence, attendingchanges in membrane permeability which affect cell-ular compartmentalizationi (7). IThis p)al)er reportson the enzymes involved in sugar uptake and accum-ulation and their distribution in the tissue. Evidenceis presented that the rate-limiting step and energycoupliplg requirements for hexose ul)take miay be atthe sites of hexose I)hosphorxlation and(1 transformiia-tions leadIing- to sucrose synthesis.

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Materials and Methods

T'hee(olterexocarp was remove(l from thepo)0ds ofmature greeni Kentucky wonider pole beans (Phascohli

(suflgariis). 'T'he renmaining endocarp), a homogenoustissue, wastisel for assay of enzymes and pl)take and

acccumulation of sugars.

Reagqcuits. Sucrose-V( C'4 (6.8nic/n i), glucose-U_C'4 (14.4 mic/nvi)anid fructose-U-Cl'I (10.0Imic/m'm)) were obtained froml Volk Radiochmical,lBur-bank. California; uridinediphosphateglucose (IJDP-glucose), UTP. .kTP and glucose-1-P fron Calbio-chem Corporation,L os Angeles, California: andanalytical invertase fromii Nutritioinal BiochemicalCorporation, Cleveland. ('hio. Fructosvl-U-C14sucrose wasprepared as described previously (21).using a UI)P-glucose-fructose transglticosylase prep-aration froini bean en(locarl).

Preparation of Enz;yines. Eindocarp tissue was

groundl wvith glass beads iin a chilled imiortar, thehomogemiate expressed through fiine imiusliii, and theresultant jutice centrifuged at 30,000 x g at 20 for30minutes. FE,nzyme prepared froni the superniatantfraction byprecipitatingprotein with ammiloniumiii sul-fate (described in text) wasused for deterniiinationsof Kiiis aiid levels of enzymes.

A4SSav of E5mz-ynics. All enzymies \\ ere assayedusinig radioactive stubstrates. Aliquots (> l) of re-actioni Imix'iri-es were applied at zero time and atvarious intervals onlto \VhatmaI -No. 1 paper anid co-chromatographed with nonradioactive sucrose, gIn-cose and fructose imarkers, using descending chroma-tography with ethyl acetate: pyridine: xvater (8: 2: 1.

v/v) as the elutinlg solvent. Chromatogramls were

(levelope(I withl p-anisidine phosphate and radioactivestigars assayed directly onl the papers with a thin end-vindow Geiger-Muller tube. Enzyme activity was

miieasured (luring the zero order phase of the reac-tions.

Stucrose synthesis wxas assayed in 0.2 ml reactioi-imllixtures comitaiiiing enzymile (0.05-0.2 mg protein).(.05 nil 0.2 a\ Tris-HCl, pH 8. additives as indicatecdbelowv anid icicubated at 300 in a shaker. Tris (0.05\i) was tse(l as it iniliibited (94 %) the alkaline in-vertase in this tissue, as was shown for sugarcaiie('11). For assay of UDP-glucose-fructose transglu--cosylase, reactioni niiixtures contained 1.0 ymoleUDPG amid 1.3 /.nmole fructose-U-C14 (1.2 puc). For(letection of UDP-glucose pyrophosphorylase reactioiimiiixtures contained 2.5 /Amoles UTTI- 5 ymoles glu-cose-l-P, 2 ,umoles MgCl., and 1.3 umiole fructose-U-C'1 (1.2 nc). Sucrose phosphorylase was assayed

in reaction mixtures comitaining 5 /xnioles glucose-i-Pand 1 .3 ,unmole fructose-U-C'4 (1.2 c). Enzyme ac-

tivity was meastire(l by countilig the baseline spotsbefore chromatography, the suicrose spots after chiro-muatograpliv. arnd ealcuilatimig sucrose synthesis as thepercentage of haseline radioa;ctivity appearing inisuicrose.

Invertase was assayed in 0.2 ml reaction mixtures

containing em,zvrne (0.1-0.7 mg protein). 5.9 jUmolessI('rfls( IT ( 1 (1.2 n) (n 0 nil pi,lcsphate citrate

l)uffer,plH 8.0. Proce(duries foi assay of inivertase in

Cell-residue fractionis are(descril)e(l in the text.For assay ofinvertase in the ouiter sl)plce,( annula

sectionis (1.2-ii-mm (liameter) fromiieindocarp tissuewere wvashed for 1 hour in running tal) water.Batches of 200 iig freslh xeiglht\ ere inculbated for3 hours in 0.3ml reactioni mixtuires in1H X 70-mlmitest tubes at 300in ashaker. RZeactionl miiixtures.buffered to various)l1's, cointailne(l 9 /inmoles sucrose

U-C14 (1.5,uc). Atzero time and various intervaloaliquots (5 ul) were applied to\\ latman No. 1 papel-for radioactive assay of sucrose hydrolysis.

Sutgar Uptake anid ,4ccoioolotioi. LUptake de-scribes the total amiiount of'lgciar removed from thlmedium and retainedl by the tissnie afterNashing foi-1 hour, irrespective of its form (21). This ;was as-sayed in a 70 % ethaniol extract. That part of thetotal uptake which appeared as sugar in the ethanolextract is defined as accumulationi. Active transportdescribes transfer of sugars againist a gradienit. Tis-sue sections were prepared andl washed as describedabove. Batches of 200 mg each wvere incubated in ashaker for 3 hours at 30° in 0.3 ml of radioactivetsugar solutionis. Ulnless otherwvise specified, ul)takewas assayed in reaction imiixtures buffered atpH 6.1After incubation the tissuie sections were \washed fotl hour in riniiig tal) water, blotted gentlv, aind ex-tracted witlh 3 volunmes of 95 % ethalnol overnight ill

shaker. For determination of tlptake. aliquots ofthe ethaniol extract were applied onto l)Ianclhets andcounted with a Baird-Atomic thini end-windowx gasflow counter. For (letermininig the formii in whiclsugar was stored, aliquots of the ethainol extract were

applied to WVhatmanl.\No. I paper and chromiiatographed as descril)edl above, followed bv radioactiveassay of glucose, fructose and sucrose.

The distribution of radioactivity in the lhexosemoieties of stored sucrosc was assayed as dlescribedllpreviously (' 20).

Results

Il)z'ert(1a(c. An invertase was (leternimiedI in thitsul)ernatanit fluid fromii a homogenate of endocaritisstue after- ceintrifugation at 30.000 X . 'lhe eni-zvme \x as characterize(d uISillg protein precipitate(dfromii the siperlnatant fractioni with 35 % (/v) am-imiomium sulfate. The enzymle is optimally activeover a broad pH range froml 7.5 to 10 (fig 1). andshows 66 % of maxinmal activity at pH 7. Th'le Kmqfor this alkaline invertase was (letermined to be 2.6X 10-2 M\. The amotunit of enzymiie activity varie(lseasonally, generally froml 1.2 to 5.1 jumoles sucrosehydrolyzed/g fresh weight per hour, although in 2of 1 3 assay's condlucted over a1 1-yea- p)erio(l no en'vine was dletected. The enzyvmie is simiiilar to an inlvertase occurring in sugarcaile (11 ) in its Km fo-sucrose and in beitl- inihibited (()4 % by (0.0)5\i Trin5pH 8.

\VVlen endocarp I ections were inicubated in buftere(l soliutioi ' conittiiing (.03 \1 sucrose U C'l con1si(lerahlc fre oli1(coe a11(1 licwtose ;Ometiuiiee ap-

182 P',.SLAN I'HY'S10 LOGY

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S.\HHER-REGULATION OF SUGAR UPTAKE AND ACCUMULATION

2.0 peared in the amiibient solution. The increase in-sucrose hydrolysis with decrease in pH was paralleled|by increased uptake (fig 2). Similar results were

N / > obtained in 2 other experimiienits. A 1)1H lower thanI.5 * / \ 4 was not used for fear of injtury to the tissue. That

> / \ invert sugars formed a considerable l)art of the utp-take miiay be indicated also by the simiiilarity shown

0 0 \hbetween the plot for free space invertase activity and1.0 the plot for free space invertase activity adjusted for

uptake (fig 2). For the latter, radioactivity takenup was added to outer space invertase activity meas-ured by assay of redtucing sugars in the amiibient solu-

0.5 - tion.E

O,Oo \ |/To ascertaini the site of the invertase a sanmple ofthe tissue wtas homlogenized and the juice expressedthrough fine muslin. The dialyzed juice contained

9 only the alkaline invertase. The remiiaininig cell-resi-4 6 8 lo 12 due fraction xwas washed 3 timiies wvith deionized water

pH and then assayed for sucrose hydrolysis in standard3.0 reaction mixttures for invertase assay, buffered at

various pH's (fig 3). The pH-activity curve indi-o FS. invertase corr. cated that the hy-drolvsis was enzvmliic. The similar-

2 \ foruptake ity in specific activity (u1ioles sucrose hydrolvzed/g2.5 x.F.S. invertas 05 fr wvt per hr) of sucrose hyclrolysis at pH 4.0 by tis-

\ uptake suc sections anid by washed cell-residue preparationsX (compare fig 2 anid 3) indicated that unider both con-

ditioins the inversion of sucrose was miediated by the\ same e2.z0me and that the enlzynme is tlherefore in the

o \ \ * a outer space.Assays for outer space invertase in bean enidocarp

>1.5 x1\ 0.3 * over a 1-year period showed that amiiong 12 assays theo enizyme level was high (0.6-2.5 umoles sucrose hy-

o \ \^ o drolyzed/g fr wt per hr) in 3 samples, low in 4 and.\\; u) niot detectable in 5 batches of tissue. The basis for

" I.0 \ 5< \ 0.2 * this variability is not knowni. The pH optimum of\ \ h E the enzy-,e detected in the ouiter space and cell resi-

Eo dticdue fractionl wvas markedly different from that of theE inltracellular inivertase, which inidicated that its origin

0.5 0.1 could niot b)e attributed to loss of initracellular enzyme.-t Afi s Although the occurrenice of ouiter space hydrolysis of

4 5 6 7 8 sucrose miiay enlhanice uptake fronm a solution of su-pH crose (fig 2). the tissue is lnot comllpletely depenident oii

J 2-5 X A FIG. 1. pH-Activity curve for inner space invertase0 / \ of bean endocarp. Tris-HCI, 0.05 ri, pH 8.0, inhibited

> 2.0 F / \ . hydrolysis of sucrose 94 %.X FIG. 2. pH-Activity curve for outer space sucrose

w / \ hydrolysis and sucrose uptake. WVashed tissue sectionsUn / \ (0.2 g) were incubated in 0.3 ml solutions of 0.03 M su-o / \ crose-U-C14 at various pH's. A sucrose uptake as-

1.5 | CELL RESIDUE sayed as radioactivity in ethanol extract; X = outeren / INVERTASE space invertase activity measured by assay of invert

sugars in ambient solution; 0 - relationship shown be-o /0 tween hydrolysis of sucrose in the outer space and uptakeE 1.0 o- by plotling the sum of uptake plus outer space invertase

activity assayed as describeed abooye.0.752 3 4 5 6 7 8 FIG. 4. ipH-Activity curve for sucrose hydrolysis by

xvashed cell residue fraction of tissue used for assay ofpH outer space invertase activity in figure 2.

183)

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PLAN'l PHYSIlULOL

rumoles/g fr wt per hr

2.190.990.220.130.0

0.371.890.901.310.92

hydirolysis prior to uptake as in yeast (5. li)) or sugar-cane (21). This is illustrated by the results of sev-eral experimlenits (table I) wlhich indicate that sucroseinay be taken up readily in the absence of outer spaceinvertase activity.

SiScrose YSynthesizing Enzymies. 'rhe 2 terminal

enzymes in the pathway of sucrose synthesis, namelyUDP-gltucose pyrophosphorylase and UDP-glucose-fructose trainsglucosylase, wvere demonstrated in beanendocarp, assayed in ainmiiionium sulfate-precipitatedprotein fractions in the presence of 0.05 m Tris, pH8.0. TI'he latter enzymiie wvas (listributed about equallybetween 35 % and 35 to 70 % amimonium sulfate frac-tions. Fq'or both enzymes sucrose syvithesis was deter-iiine(l by assay of the incorporationi of radioactivefructose-U-C14 into sticrose. For synithesis of su-

crose involving UDP-glucose p)yrophosphorylase,ATP could not substitute for UTP. Hydrolysis ofthe sucirose synthesize(d and( subsequent assay of theradioactive invert sugars showed all of the radio-activity in the fructosyl moiety. The activity ofUD P-glucose-fructose tranisglucosylase varied from0.62 to 4.7 (av = 1.1) tmnioles sucrose synthesized/gfresh weight per hour. Activity of UDP-glucosepyrophosphorylase (assaye(d in a 70 % ammoniumsulfate fraction) was measured by the rate of sucrose

formiiation from glucose-l-'. UTP. fructose-U-C'4adlcl MIgCl,. The formationi of 0.34 ,umole sticrose/gfreslh wveight per hour inidicated that DI)P-glucosefrtictose transglticosylase was lnonlimiting, as the ac-

tivitv of the latter enzyme is tsubstantially higher thanthis. Synthesis of sucrose fromii fruictose-U-C4, UTPalnd AgCI, in a 70 % aninmonium sulfate fractioln)cctIrre(l at a low rate (0.14 ,umole/g fr wt per 1r).wvhich couldlc have been dIue inl part to high phosphataseactivity, inasmiiuch as conisiderable free gltucose wva.s

formedl. The synthesis of sucrose under these con-(litions indicates the presence in the tissue of hexo-kinase. phosphohexoseisomerase and phosphogluco-muttase. The rate at which sucrose is accumulated b!tissue froni a solution of fructose or glucose, as willbe shown in A -following section, indicates a much

greater capacity for suicrose synithesis thaii has beendemonistrated in these cell-free preparations.

No sucrose phosphorylase could be detected inbeau endocarp. This enzyme s,eemsl to be lacking inhlizher plantts (of. 11)

Uptake and Accumnulation promlJI (ilutcose anid Frtic-lose. Sugar taken up by bean endocarp from soluitions of glucose-U-C14 or fructose-U-C14 was accumulated as uniformly labeled sucrose (cf. 21).Since bean endocarp contains considerable glucoseand fructose it is apparent that sncrose synthesis oecurs at a site where the labeled sugar is not significaintly diluted by the large pool of endogenous hexoses.Cellular compartmentation of sugars has beeni re-ported previously (2, 6, 8, 14, 21). For uptake ofeither hexose, over a wide range of concentrationis(0.005-0.18 m) the sucrose/redntcing sugar ratio ofaccumulated (labeled ) sugar scarcely varied from]about 20 (table II). Thus, it appears that sucrosesynthesis is a requisite for accumulationi froimi solutions of glucose or fructose, and that the radioactivreducing sugars arise from hydrolysis of sucrose.

Table II. Relationship between Exogenous Concentra-tion of Glucose, the Rate of Uptake, and the Sutcrose!Reducing Sutgar Ratio of Sugars Acciumulated byt

Bean Endocarp Tissu(e Sectionis

Conc ofg-lucose (m)

0.0050.010.030.060.120.18

Glucose uptake(Lmoles/g fr wt

per hr)

0.430.841.853.385.325.54

Ratio of C14 insucrose/reducing

sugars inaccumulated sugarv

19.019.021.220.224.520.0

Glucose uptake was on the average (11 assays) 3times as fast as that of fructose wvhen comparingulptake from 0.03 M solutions. The Km for fructoseuptake averaged 2.1 X 10-2 mx' for 5 assays (tableIII). For uptake of glucose the average Km for 6assays was 2.1 X 10-' M, and the range of variabilitywas greater than for fructose. KCN or dinitroplhenolat 5 X 10-4 Mi inhibited glucose uptake substantially(52-84 %, table IN'). There was no significant dif-ference betweeni the effect of these inhibitors. Forpotato slices, exposed to similar concentrations of glu-cose and these inlhibitors, total absorption (cpm re-moved from ambient solution) was inhibited 45 % byKCN and 32 % by dinitrophenol (14).

Table III. Kom Values for Uptake of Gluicose andFrutctose by Bean Endocarp Tissue Sections

Glucose

Dateof assay Km (M)*3/2/644/30/64i/l/645/7/645/15/6410/9/64

8 X 10-'

8 X 10-22.9 X 10-13.3 X 10-i3.3 X 10-9.4 y 0-"

Fructose

Dateof assay

3/2/643/25/643/31/644/20/644/21/64

Km (m))*2.6 X 10-21.7 X 10-22.2 X 10-1.7 X 10-22.6 X 10-Y'

* I)tl-ermined I ineaver Burke plots.

Table 1. Rclationship between Outer Space InvertaseA1ctivity antd Sucrose Uptake for

Endocarp Tissuie Sectionis

IncubationExpt medium

Outer spacesucrose

hydrolysisUptake

sucrose-U-C] 4

0.03 Wm sucrose-U-C i t.. .. ..

IIIIII

V

184

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SACHER RE(G.I.ArTION- OF SUGAR UpPTAKE AND ACCU-MULATION-18

Table IV. Effect of KCN anid 2,4-Dinitrophenol onUptake of Glutcose

% Inhibition of uptake*

ExptExogenous 4 X 10-5 M 4 X 10-5 M

glucose conc KCN** Dinitropheniol

I 8.7 X 10-5AtII 1.74 X 10-4 M

1.0 X lr2 MIII 3.0 X 10-2 M

81 %67%52%

84 %63%53%65 %

* Assayed in 70 % ethanol extract of tissue after a1-hour washing.

** Incubated in tightly stoppered 15 ml tubes to preventloss of KCN.

For yeast cells 10-4 M uranyl nitrate greatly in1-hibited uptake and fermentation of sucrose and hex-oses, yet respiration of acetate, pyruvate, lactate andethanol, as well as endogenous respiration was in-sensitive to uranium (19). From this and other evi-dence it was concluded that uraniunm acted at thesurface of yeast cells. Since there was nio effect of10-5 to 10-S M uranyl nitrate on uptake of 0.03 M glu-cose (or sucrose) by bean endocarp, it appears thata different mechanism is involved in sugar perme-ation of the plasma membrane of yeast cells and cellsof higher plants.

Seven experiments were conducted in which tissuesections were incubated in solutions of 0.03 M withrespect to both glucose and fructose, with only one ofthe sugars labeled. Unlabeled fructose had no effectoIn uptake of glucose-U-C14, while unlabeled glucoseinhibited uptake of fructose-U-C14 an average o045 %. Lineweaver-Burke plots showed that the in-hibition by glucose was comipetitive (fig 4). In 4experimeits the hexoses galactose or mannose in-hibited fructose uptake from equimolar (0.01 m) solu-tions from 14 to 33 %.A few experiments, however, were contrary to the

typical nioninhibition of glucose-U-C14 uptake by uIn-labeled fructose and the inhibition of fructose-U-C14uptake by unlabeled glucose. In one, fructose clearlyinhibited glucose competitively (fig 5). The inhibi-tion at 0.03 M concentration of each sugar was 32 %,and the Km for glucose uptake was 3.3 X 10-2 M.which is one order of magnitude below the highestKm reported above (table III) for 6 other deter-miniationis. In another instance miiarked changes wereobserved in the effect of glucose, galactose and milan-nose oll fructose uptake after short-termii storage ofwhole beans at 50. Tissue lharvested fromii a freshbatch of beans showed an uptake of 40 tAg fructose/gfresh weight per hour from 0.01 AI solution of fruc-tose-U-C'4 which was inhibited 34 % by an equimolarconcentration of unlabeled glucose. In tissue re-moved fromi the beans 2 days later the rate of fruc-tose-U-C14 uptake had increased 75 % and glucose,galactose and mannitol each slightly (ca. 15 %)stimulated fructose-U-C'4 uptake. Thus, variationsof this kind may result fromi physiological aging dur-ing storage. Sulch variability and lack of specificity

c

S

a10

E4-

2.0

p

a

0

U.

It

I.5

1.0

0.5

33 50 100 200

I/S (M Fructose)

I/S (M Glucose)

FIG. 4. Lineweaver Burke plot illustrating competi-tive inhibition of uptake of fructose by glucose in beanendocarp sections.

FIG. 5. Lineweaver Burke plot depicting atypical com-petitive inhibition of uptake of glucose by fructose. TheKm for glucose uptake was 3.3 X 10-2 -f in this experi-ment.

appears to speak againist a reactioni mediated by acarrier protein being rate-determiining for uptake ofglucose and fructose. and may indicate that the up-take of hexose is metabolicall- controlled at the siteof sucrose syinthesis.

In botlh carrot root anid corn root tissue L-arabin-ose and D-ribose are accumiulated as such (8). Inbean endocarp L-arabinose and D-ribose (table V)do not inhibit fructose uptake over a vide range ofconcentratioins. It is interestinig to note that thesepentoses. which do not to a significant extent enterthe site of hexose transformations in other plants,offer no comiipetitionl to uptake of fructose.

]85

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Table V. Effecby Bear

Colnc offructose-U-C 4

(M)

0.0050.010.03

Siucrose Uptcincubation mediainhibits outer sposimilar molarity79 %). The K]10-2 M. AppareM,t sucrose. Inspace invertase atake of labeledwas there inhibiThus, it is evidevolved in uptakecontrast to a repsucrose uptake b)experiments in siexplained on theduring the proce

Although susignificant amoul)resent (fig 2),able invertase in(table I). Thustaken up as suchwith sugarcane (fact that when titosyl-U-CI4 suc:0.001) the sucrctially unaltered (sections were incalong with eithe:the sucrose in th

PLANT PHYSIOLO(G\

-t of D-Rijose on tiptake of Fructose labeled. Substantial randomiiization of label oc-ni Endocarp Tissiui Sections curred in sucrose accumulated under such conditions

by sugarcane tissue sections. in which sucrose is hv-Uptake of fructose drolyzed prior to its u)take (21). These results sug-

,unmnles/g fr Nvt per hr)(_moles/gf_ wt per hr) gest that in bean tissue sucrose mav be taken up andNo 0.01 M 0.03 M 0.05 M accumulated without inversion.

Rihose Ribose Ribose Rihose From the following observations, however, it may

0.26 0.30 0.24 0.30 be demonstrated that some sucrose is hydrolyzed in-0.40 0.48 0.49 0.56 tracellularly during its uptake, the amount depending0.88 0.84 0.92 0.89 upon metabolic demands. The ethanol extracts of

tissue incubated in uniformly labeled sucrose, glucose,ke. For studies of sucrose uptake or fructose, as well as mixtures of sucrose with ancontained 0.05 m Tris, pH 8.0, which unlabeled hexose, were chromatographed and radio-ace invertase activitx about 90 % (a activity in sucrose and at the origin was assayed onof phosphatae citrabuffer inhibited the chromatograms, The radioactivity at the origiinm for sucrose uptake was 3.6 X was largely sugar phosphates, which do not migratent saturation occurred at about 0.12 in the solvent used, and hereafter will be called origintissue which had little or no outer compounds (cf. 9). As shown in table VI the ratio.ctivity there was no inhibition of up- of radioactivity in sucrose compared with origin com-sucrose hy glucose or fructose, nor pounds was 3 to 4 times higher in extracts fromii tis-ition in the reciprocal experiments, sue inctubated in sucrose than inl hexose. rFurther.nt that a comiimon carrier is not in- with a mixture of labeled sucrose and unlabeled hex-of hexose and sucrose. This is in lose in the medium the ratio was inicreased substan-

yort of competitive inhibition of C14- tially. The results suggest that wvhen unlabeled hex-y glucose as xvell as for the recil)rocal ose is available to meet metabolic demiiands less sucroseugarcane (2), which has since beenl is lhydrolyzed intracellularly during its uptake. Al-basis of sucrose being hvdrolvzed though it is nlot known whether cytoplasmic hydroly-

ss of uptake (21). .sis of sucrose is mediated by invertase or reversal ofcrose uptake was enhanced when a UDP-glucose-fructose transglucosylase system, theints of outer space invertase were evidence presented herein oln localization of enlzymesits uptake in the absence of detect- stiggests the latter.the free space was often substantial Concentrations of 5 X 10-- M KCN and dinitro-

s, it appeared that sucrose could be phenol inhibited uptake from 3 X 10 I sucroseI, without need for prior inversion as 81 % and 96 %, and from 3 X 10-2 NM sucrose 72 %(21). This is also in(licated by the and 88 % respectively. Thus, it appearedl that theseissue sections were incubated in fruc- compounds inhibit nmarkedly. irrespective of whether-rose (G/F radioactivity ratio = uiptake is witlh or against a concentrationl gradient.)se was taken up and stored essen- Significaitce of Enzymes anid their Distribution toG/F = 0.03). Further, when tissue Sugar Uptake and Accumulation. Although sucroseubated in a solution of sucrose-U-C'4 uptake is enhlanced when free space invrertase is pres-r nonradioactive glucose or fructose ent, sucrose may be taken up and accumlulated with-e ethanol ex-itract wxas svinimetricallv out inversion. Since the tissue contains anl intracell-

Table VI. Relationship between Externtal Sugar(s) and the Ra(ti(o of Rladioactivity illSucrose/Origin Compounds on Chromatograms of Ethanol Extracts of Tissue Sectioni

Tissue sections were incubated for 3 hours in sugar solutions, washed 1 lhour in runninig tap wvater and extracte(dwith 3 volumes of 95 % ethanol. The ethanol extract was chromatographed wvith markers using ethyl acetate-pyridine-H,O (8: 2: 1. ') as thle elutinlg solvent. Radioactivity in the sucrose and origin spots was assayed.The origin spots include sugar phosphates xwhich do not migrate in this solvent. Exogenous sugar conicentrations,Experiment I all 0.03 lxr; Experiment TI all 0.f)l M; Experiment III sucrose 0.03 M, glucose and fructose 0.06 M.

External medium

UnlabeledsugarSugar

Sucrose-U-C''., 9 , Glucose

FructoseGlucose-U-C"4Fructose-U-C ''

Fthanol extractRatio of

cpm iln sucrose

cnmii in origin compound,5

Expt I Expt II Expt III

6.0 2.4 15.015.0 5.9 32.010.0 5.6 18.02.1 0.57 5.02.0 0.53 4'1

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SACHER-REGULATION OF SUGAR UPTAKE AND) ACCUM ULATION1

Tahble Vll. Relationship Betzueen Level of Irner Spact Inmcrtates (1td(1 tlhe Snic(rse! keduci)n,Sugar Ratio of Aceumulatde( Smim-.5

Expt Incubationmedium

Inner space invertase*(umoles s-ucrose hydrolyzed

/g fr wt per hr)

I 0.03 M sucrose-U-C14 5.14III " " 3.04

IV- 0.03 M fructose-U-C' 0.57* Invertase assayed using amimoniumn sulfate (35 %, w-/v) preparatiols.

ular inivertase the observations suggested that this en-zyme occurs in the vacuole. Two lines of experi-mental evidence are consistent with this interpretation.As shownei previously, accumulation by tissue sectionsfrom solutions -of glucose or fructose occurs as su-crose. \Vhen such tissue sections were subsequentlywashed and incubated in a humidity chamber for 16hoturs the sucrose/reducing sugar ratio dropped from22 to 0.6. This observation is consistent with theanalyses of fresh tissue, which invariably show thatsugar is stored as glucose and fructose, with onlytraces (sucrose/reducing sugar ratio = 0.037) ofsucrose present (7, 23). Since the tissue has a highcapacity for sucrose synthesis, which is likely to oc-cur in the cytoplasm because of the need for energycoupling, the vacuolar compartmentalization of in-vertase and sugar storage is indicated (cf. 6, 21).Also, several experiments in which both the sucrose/reducing sugar ratios and the level of intracellular(alkaline) invertase were assayed showed an inverserelationship between the amount of this enzymile andthe sucrose/reducing sugar ratio of the accumulatedsucrose (table VII). When the level of intracellularinvertase w%as high (3.0-5.1 ,umoles sucrose hydro-lyzed/g fr wt per hr) the ratio was about 2.0, whileNvith a low level of invertase (0.6-1.0 Jumole) theratio was about 26. This refers to the ratios im-mediately after removal of the tissue from the sugarsolution. As noted above, the sucrose/reducing sugarratio decreases greatly when the tissue sections areincubated subsequently in a humidity chamber for 16hours. Similar observations were miiade for sugaraccumulation in sugarcane (21: Sacher. Hatch andGlasziou. unpublished data).

Discussion

Four lines of evidence indicate that sucrose miaybe taken up and accumulated without hydrolysis.First, fructosyl-labeled sucrose was taken up andstored without significant alteration of its labeling(see also 16). Secondly, sucrose accumulated fromsolutions of mixtures of sucrose with either glucoseor fructose was uniformly labeled, irrespective ofwhich of the 2 sugars in the external solution waslabeled. WN'ere sucrose hydrolyzed during accumula-tion some randomization of label could be expectedat the site of phosphorylation and conversioni ofhexoses leading to sucrose svnthesis (cf. 21).

Ratio ofsucrose/reducing sugars

in ethanol extract

1.62.7

30.()22.0

Thirdly, it was deimionstrated (20) that the in1CoL-bation of bean endocarp tissue sections in fructose-U-C14 and UDP-glucose, conditions favorable forsynthesis of fructosyl-labeled sucrose extracellularly,caused a marked decline from unity of the G/F radio-activity ratio of the accumulated sucrose. which wasproportional to the amount of sucrose synithesizedextracellularly. The results were explicable in termiisof uptake and accumulation of fructosvl-labeled su-crose unaltered, and of fructose-U-C'4. of which thelatter vas converted intracellularly to uniformlylabeled sucrose.

Fourthlv. the ratio of label in sucrose coimiparedwith origin compounds was about 3-fold greater inethaniol extracts of tissue incubated in sucrose thanin glucose or fructose (table VI). This ratio wasincrease(l substanltially wheni either unlabeled glucoseor fructose was in the incubation medium with su-crose-U-C14. These observations indicate that intra-cellular hydrolysis of sucrose during uptake is con-trolled by the relative demanid for hexose. It appearsthat the presence of hexose increases the ratio ofsucrose/origin compounds by providing a ready--source of miietabolic intermediates. For tobacco leafdiscs part of the evidlenice used in concluding thatsucrose was taken up without hydrolysis was themuch lesser starch formation from exogenous sucrosethan from glucose (16).

Several points may be enumerated which provideevidence about cellular localization of sugar storage.inner space invertase anid sucrose synthesizing eni-zvmes.

1) In fresh cut tissue the en(logenious sugars coni-sist largely of glucose anid fructose, and only traecanmounits of sucrose (7, 23). Since the sucrose ac-cumulated by bean endocarp from solutionis of unii-formlv labeled glucose or fructose is symmetricallylabeled, it appears that the endogenous hexoses doniot (lilute the muetabolic pool w-here sucrose synthesisoccturs.

2) It could be showni from assays of tissue ex-tracted with lhot ethanol immediately after removalfrom a solution of radioactive sugar and washing.that the sucrose/reducing sugar ratio of the accumu-lated sugars is inversely related to the level of innerspace invertase (table VII). Further, practically allof the sucrose accumulated is hydrolyzed within 16hours, after the period of accumulation, as shown bythe marked decrease in the sucrose/reducinig sugar

187

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P TPLANT PHYSIOLOGY

ratio fronm 22-0.6), xvhen tissue sections are store(lovernight in a humidity chamber after uiptake froiimsolution of labeled hexose or sucrose.

3) In senescing beain entdocarl) tissue consideral)lesucrose is synthesized associated with chaniges inpermeability aiid leakage of sugars (7). This ob-servation indicated that the lhexoses are normallyselparated fronti the s.ite of stucrose synlthesis by apermeability barrier, probably the vacuolar mem-brane. It may be indicated from the evidence thatsuicrose is syinthesized whenl hexoses stored in thev.acuole leak into the cytoplasiut.

It is cooncluded that both sugar storage anid theinIner space invertase occur in the same compartment,

hich is believed to be the vacuole (cf. 6, 16, 21).C InSidering the high capacity of endocarp tissue forsucrose synthesis, it is difficult to envisage the hydro-lysis of accuimiulated sucrose and storage of reducingsugars occu1rring in the compartment where synthesisoi stlcrose takes place. The energy coupliiig andinumber of eIIzymi1es involved in sucrose synthesisbe-Ipeaks its occurrence in the cytoplasm].

Differences in the rates of uptake and comiipetitionanm:-nlug glutcose. fructose anl sticrose have led to sug-

ge,stions that the rate-liititinig reactioIn for siugar up-

take involves a coillllioll carrier wvhich hlas a greateraffinity, for onie stigar than aiiother (2, 8). Datafrmni the )resent stutylv liow-ever, suggest that therate-limiting reaction ( s) reside at the site of hlexosephosphorylation anid transformiations leading to su-cnine synthesis (cf. 6). Following are summarize(la numiiiber of lines of exvidence wvhich ntay he mise(I foran evaluatiotn of these 9 alternative interpretations.

I ) (l5ucose is takeni approximiiately 3 timiies fatsterthan frtictose. 2) Glucose inhibits fructose-V ('Cuptake. while frtictose ti(Irmtahlly has ito effect on glt-cose tpl)take. .3) r-Arabiniose and(1 i)-ribose (table V),wvhiclh are accumlitilated t( sticlt (8) an1d tlhtis do1 noteniter the p(ool of hexo.se trainsformuatiois. offer no

comiipetitioin to tiptake (If fructose. 4) Cyaniide and

dlinitropheniol greatly itiltilbit ttptake (If hexose. 'l'hleforegoitig obser-vations arc coinsistenit \with eitiercarrier-nl(lidated process or ait emtzvmitclIroccss at thesijtC of stlcrose synthesis being- rate-liittititt- to lhe \osetillt ake. TI'hle following observatiomi is c*onsistent\xxith the initerllretation that rate limitiatioil occuris at

the site of reactionis leadint, to sviythlcsis of smlcrose.) ver a wide ranige of co(ncetttratiolls of exogenlouis

gtlue.~e(0.005-0.18 M) the smicrllse/redttcing sulgarratio of the acctimulated sugars remainis esselnt iall\

unchianged at about 20 (table TT). If the rate- imlitiing reactioii for glicose otr fructose uptake \v;as car-

rier-mediated. it milight be expected that at the appar-ent saturating concetntratio tfor hlexose uptake (tableII) consi(lerable free glucose or fructose vottld ocetir

amoing the acctlmimlatecl stigars. 'I'lie restults indicatethat sticrose sviithfesis is a re(qltisite for-aectitmlat ionfr a solutimti of glucose or fruictose. Sintihar re-stilts were observed foi s;tigarcale (0).

The observations listed niext are iiiconsisteilt withco-nipetitiomi at at carrier-sitc beitig aim expl.amlatiot for

the faster uiptake of glucose thati fructose aind theinhibition of fructose uptake by glucose. 1) Thelower Km for fructose uptake (2.1 X 10-2 Mq) thang-lucose (2.0 X 10-1 Nt) would suggest fructose as agood inhibitor of glucose uptake, when the reverse isustually observed. 2) The variations in the Km forgltlcose uptake of one order of magnitude appeairunustually hig,h for variations in the Km of a reactionmedia,ted by a carrier l)rotein. Suclh variation miightbe expected if the apparent Kmn's are actually meas-uremients of the rate of sucrose syinthesis fron] glucosc.anid the amount of a rate-limitinig enizymiie among theeInzymes involved (e.g., affecting glutcose phos)horyl-ation) is subject to variation. 3) Of 7 determiinationisof the Michaelis coinstaInt for glucose uptake, only withthe lowest observed Km (apparent) wsxa the inhibi-tion of glucose by fructose observed (fig 5). It (loesnot seem that the specificity of a carrier proteini conm-ImloIn to both glucose and fructose would change tothis extenit, as glucose normally inihibits fructose up-take substantially. The observationi, however, wouldseeiri consistenit with what is suggested above.

Most of the observations above are explicab)le iiiterms of glucose niormally beinig a better source ofg^,lucose- p-hosphate and fructose thani frtictose is ofglutcose-I-phosphate. It is suggestedI that the vari-able kinetics observed for uptake of glucose and fruc-tose and mixttures of hexoses niax,be due to variationsin the anounlits (f enzymes ini the s(equenice foIr sticrosesvn-the.sis showxi below. anid thliu> to differential ef-fects of enizyme pro(Iducts oni reactltios iHi the sequieince.This cotu(ld restilt ini Onle or aniothier (If the reactioiti:being rate-limiting. ( )ne possibility is that giucoseor (glucose,I' inlhibits frulctose ph(sP11loryIati"l.

G

F (. F-6-l' ¢ G-6-P ± GC -1-'G-I-I - UTP -- UDPG - PI'UD)G L F -s Sucrose

Iiiniature sugarcane tissue resemIlles b)eani pod)dtissute iii the relative rates of glucose aln frructosuplItake aili(l inihibitionI of fructose uptake bx gluicose.When sugarcalle discs were provided labeledI fructose

l(1 uilnlabeled gltcose the G/F radioacti-ity ratio of-the sucrose accumulated was 0.67. while the ratiorai-ge(d fromii l.58to.3.65 for the reciprocal experimiients.\lso marked asymmli]letry' occurre(l ini .suicrose accumi-milated from mixtures of labeled sucrose anid niilabeledhexoses. In this tissue sticrose is hvdrolyzed in thefree space prior to beiing taken ul). When the hexosewas fructose the G/F ratio was 3.25. while with glu-cose as the unlabeled ssugar the ratio was 0.98 (21;Sacher, Hatch and Glasziou, unpublished data). Forthis tisstue the observed asymmetry of accumnulatedsulcrose also, app)eared tlo )e exl)licab)le ini termis ofgltcose being a better source of glucose-1-P and fiuc-tose thani fructose is (If glucose- -P.

In aninmal tissue. \ here gltcose ul)take is alsc,faster than that of fructose, the phosphorylation-rattratios for glucose and fruictose in vitro are vcrv simiii

I 9

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SACIHER-REGULATION OF SUGAR 'P'FAK I. A NI) A-('C'tClMLLA'I'ION9

ilar to their al)sorptioll-rate ratios in vivo (12), and,,glucose andl glucose-P strongly inihibit fructose phos-phorvlationi. while fructose inhibition of glucose phos-phorylation is hardly detectable (22). The phos-l)horylative capacity of bean endocarp for glucoseaind fruictose in vitro has niot been inXvestigated yet.

'I'he experimenltal work thlius far wN-ith beaul tissue(loes not unambiguously negate 'the possibility of acomi-moni carrier being rate-limiting to uptake of glu-cose and fructose. Nonetheless, several lines of evi-dence presente(l, which appear to be reflections of thekinetics of reactions in the cytoplasm, are consistentwith the hypothesis that the rate-limiting reaction tohexose uptake resides at the site of sucrose synthesis.If this is so, the possibility is presented that the cyto-l)lasm is free space to hexose over the range of con-centration used (0.005-0.18 M). This has been sug-gested previously for sugarcane (6). without imply-ing that the cytoplasm is free space to other solutes.

As noted previously the sucrose/reducing sugarratio in ethanol extracts made immediately after the3-hour period -of sucrose or hexose uptake (tableVII) is inversely related to the level of inner space(vacuolar) invertase. From this and other evidencepresented on cellular compartmenitation it imay be de-(luced that sucrose accumutlation froml exogenous glu-cose represents a .steady-state value. in which sucrosesynthesized from glucose is transported across thetonoplast into the vacuole at a rate equivalent to itsrate of synthesis in the cytoplasm. The initial conl-centration of sucrose in the tissue (7) is about 3.4 X10-3 M (or 1.2 mg/g fr wvt). The rate of accumu-lation of sucrose fronm exogenous glucose ranges from0.07 to 0.9 mg/g fresh weight per hour (table II).of which only a fraction would be present in thecytoplasm at any time. Thus, it appears that thesteady-state accumulation involves active transport ofsucrose across the tonoplast. The mechanismii b)which sucrose is transported across the tonoplastagainst a concentration gradient in bean endocarp isnot knlowni. For sugarcane it appears that the energyfor suich a process is derived fromii hydrolysis of su-crose-P. synthesized from hexose by UDP-glucose-fruictose 6-phosphate glucosvltransferase (10).

Acknowledgments

The author thanks Mr. A. J. Kowalski for valuabletechnical assistance.

Literature Cited

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2. BIFIESKI, R. L. 1962. The physiology of sugarcane. V. Kinetics of sugar accum.ulation. Austral.J. Biol. Sci. 15: 429-44.

3. BROWN, R. 1952. Protoplast surface enzymes andabsorption of sugars. Intern. Rev. Cytol. 1: 107-18.

4. CHANG. C. X\. AND R. S. BANDURSEI. 1964. Exo-cellular enzvimes of corn roots. Plant Physiol.39: 60-64.

5. CIRILLO, V. P. 1961. Sugar transport in micro-organisms. Ann. Rev. Microbiol. 15: 197-218.

6. GLASZIou-, K. T. 1961. AccuImiulation anid trals-formation of sugars in stalks of sugarcane. Originof glucose anid fructose in the inner- s)ace. PlantPhysiol. 36: 175-79.

7. GI.Asziou, K. T., J. A. SACHER. ANI) D. McCAAI.I..1960. OIn the effects of auxil1s oIn membrane per-meability and pectic substances in bean enidocarl).Am. J. Botany 47: 743-52.

8. GRANT, B. R. AND H. BEFVERS. 1964. Absorptionof sugars by plant tissues. Plant Phvsiol. 39: 78-85.

9. HARLEY, J. L. AND D. C. SMITHI. 1956. Sugar ab-sorption and surface carbohydrase activity of Pcl-tigera polvdactyla (Neck.) Hoffm. Ann. BotainyN.S. 20: 513-43.

10. HATCH., M. D. 1964. Sugar accumulation by sugarcane storage tissue: The role of sucrose phos-phate. Biochem. J. 93: 521-26.

1 1. HATCH, M. D., J. A. SACHF.R. AND K. T. GLASZIOu.1963. Sugar accumulationi cycle in sugar cane.I. Studies on enzymes of the cycle. PlantPhvsiol. 38: 338-43.

12. HFT E, M. P. 1953. The phosphorylation and al)-.sorption of sugars in the rat. Il. Sugar absorp-tion in vivo, and the relationshil) to the phosphor l-ation of sugar in vitro. Biochem. J. 55: 8646;7.

13. HELLEBUST, J. A. AND 1). F. FORWARD. 1962. Theinvertase of the corn radicle and its activity in suc-cessive stages of growth. Can. J. Botany 40: 113-26.

14. LATIES, G. G. 1964. The relationi of glucose ab-sorption to respiration in potato slices. PlantPhysiol. 39: 391-97.

1 5. PORTER, H. K. 1962. Synthesis of polysaccharidesin higher plants. Ann. Rev. Plant Phvsiol. 13:303-28.

16. PORTER, H. K. AND L. H. MAY. 1955. Metabolismof radioactive sugars by tobacco leaf disks. J.Exptl. Botany 6: 43-63.

17. PUTNUM, E. Wr. AND W. Z. HASSID. 1954. Sugartranisformation inl leaves of Canna indica. 1. Syn-thesis and iniversion of sucrose. J. Biol. Chem.207: 885-902.

18. RIKLIS, E., B. HABER. AND J. H. QIUASTEL. 1958.Absorption of mixtures of sugars by isolated sur-viving guinea lig intestine. Can. J. Piochem.Physiol. 36: 373-80.

19. ROTHSTEIN, A. 1954. The enzymology of the cellsurface. Protoplasmatologia 2: 1-86.

20. SACHER, J. A. 1964. Extracytoplasmic sucrosesynthesis in higher plants. Life Sciences 3:

% 1053-60.21. SACHER, J. A., M. D. HATCH, AND K. T. GLASZIOU.

1963. Sugar accumulation cycle in sugarcane.III. Physical and metabolic aspects of cycle inimmature storage tissues. Plant Physiol. 38: 348-54.

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2.3. WILLIAMs, N., L. M1. FLYNN, ANI) .\. G. HOGAN.1948. Changes in slugar conitent of raw greenbeans during storage. Food Res. 13: 358-63.

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