429BEAN AND TODD-PHOTOSYNTHESIS AND RESPIRATION IN FRUITS

7. KROTKOv, G., V. C. RUNECKLES, and K. V. THI-MANN. 1958. Effect of light on the CO., absorp-tion and evolution by Kalanchoe, wheat and pealeaves. Plant Physiol. 33: 289-292.

8. KUNITAKE, G., C. STIrrT, and P. SALTMAN. 1959.Dark fixation of CO, by tobacco leaves. PlantPhysiol. 34: 123-127.

9. RACUSEN, D. W., and S. ARONOFF. 1952. Metab-olism of soy bean leaves. V. The dark reactionsfollowing photosynthesis. Arch. Biochem. Bio-phys. 42: 25-40.

10. SALTMAN, P., G. KUNITAKE, H. SPOLTOR, and C.STITT. 1956. Dark fixation of CO., by succulentleaves. Plant Physiol. 31: 464-468.

11. TCHEN, T. T., and B. VENNESLAND. 1955. En-zymatic carbon dioxide fixation in wheat germ.Jour. Biol. Chem. 213: 533-546.

12. THOMAS, M. 1949. Physiological studies on acidmetabolism in green plants. I. CO.. fixation and

CO, liberation in Crassulacean metabolism. NewPhytol. 48: 390-420.

13. THOMAS, M. 1949. Physiological studies on acidmetabclism in green plants. II. Evidence of CO2fixation in Bryophyllum and the study of diurnalfluctuation of acidity in this genus. New Phytol.48: 421-447.

14. THOMAS, M. and H. BEEVERS. 1954. Physiologicalstudies on acid metabolism in green plants. [II.Further evidence of CO9 fixation during darkacidification of plants showing Crassulacean acidmetabolism. New Phytol. 53: 1-30.

15. WALKER, D. A. 1956. Malate synthesis in a cellfree extract from a Crassulacean plant. Nature178: 593-594.

16. WALKER, D. A. 1957. Physiological studies onacid metabolism. IV. Phosphoenol pyruvate car-boxvlase activity in extracts of Crassulacean plants.Biochem. Jour. 67: 73-79.

CARBOHYDRATE METABOLISM OF CITRUS FRUITS I. MECHANISMS OFSUCROSE SYNTHESIS IN ORANGES AND LEMONS'

DEPARTMENT OF PLANT BIOCHEMISTRY.

The basis for controlling the accumulation ofmetabolites during growth of plants is of special in-terest in the study of fruit growth since the flavorand quality of the fruit may depend on the storedmaterials. Oranges and lemons are two fruits havingwide differences in characteristic sugar storage pat-terns. The mature lemon contains little or no sucrose

in any of its tissues whereas sucrose constitutes themajor storage sugar of the orange. It would be ofinterest to determine if such variations are reflectedin synthetic mechanisms, synthetic capacities. or inutilization of sucrose. It would also be valuable tofind the stage of growth where such differences be-come manifest. The synthesis of sucrose in thesefruits has been compared by chemical and radioisotopetechniques using enzyme and tissue preparations as

well as intact fruits. Results of the study suggestthat differences in sucrose storage may not be dueto any deficiency of sucrose synthesizing mechanismswithin the lemon, since active systems were foundto exist in these fruit at all stages of growth. Pro-nounced differences were found, however, in the syn-

thetic capacities of different tissues of the fruit; itappears that the albedo (white peel layer) has thehighest capacity for synthesis in the older fruit.

1 Manuscript received September 22, 1959.

BEANUNIVERSITY OF CALIFORNIA, RIVERSIDE

MATERIALS AND METHODS

Sucrose synthesis was studied in three ways. A.Reaction of fructose or fructose-6-phosphate withuridine diphosphate glucose (UDPG) was determinedin vitro with extracts or particulate preparations fromthe fruit. B. Formation of sucrose in isolated tissuepreparations was traced by incorporation of activityfrom glucose-U-C' 4 or fructose-U-C' 4. C. The tracertechnique was also extended to examine the formationof sucrose from labeled monosaccharides in small, in-tact fruits. Experimental procedures and materialsare given below for each approach.

I. STUDIES WITH EXTRACTS AND HOMOGENATES.Mature Eureka lemon and Valencia orange fruitswere obtained fresh from local groves. The lemonsand oranges were juiced by hand with a glass reamer.The lemon juice was divided immediately into twoparts. One part was neutralized to pH 7 with 1 NNaOH before carrying out subsequent steps whilethe remainder was treated as initially obtained. Thejuices were filtered through birdseye cloth and cen-trifuged at approximately 25,000 X G for 20 minutes.The sediments were resuspended in two volumes of0.01 M phosphate buffer, pH 7, and dialyzed overnightagainst the same buffer. Supernatants from centri-fuging were saturated with ammonium sulfate at pH5 and the precipitates were collected by centrifuging.This precipitate was redissolved in the pH 7 phos-

PLANT PHYSIOLOGY

phate buffer and dialyzed overnight in Visking sau-sage casing against the same buffer.

Soluble extracts of lemon and orange peels werealso prepared by homogenizing one part peel in tenparts water and treating the homogenates as describedabove for the juices.

Sucrose synthesis was measured in the solublepreparations by modifications of previously publishedprocedures (2, 10, 11, 12). A 50 Al aliquot of theenzyme solutions was incubated at room temperature(21-23° C) for half an hour with 0.37 /,moles ofUDPG and 2.5 Amoles fructose or 2 Amoles of D-fructose-6-phosphate in a total volume of 0.1 ml.Following inactivation by heating in a boiling waterbath for one minute, the residual free fructose or fruc-tose-6-phosphate was destroyed by heating in 1 ml0.1 N NaOH for 15 minutes at 1000 C. The alkali-stable ketose (keto group protected by glycosidiclinkage) was determined by the method of Dische andBorenfreund (8) using a 2-hour incubation ratherthan the specific 24-hour period. Adequate controlswere prepared, omitting UDPG, enzyme or ketose.

For particulate preparations, the procedure wasmodified by doubling the volumes and quantities used.Following incubation, the suspension was centrifugedto remove the particulate material and an aliquot of0.1 ml of the supernatant solution taken for analysisas above.

The UDPG was prepared from yeast extracts bya gradient elution modification of the procedure ofCabib, Leloir and Cardini (7). This material con-tained approximately 25 per cent of uridine diphos-phate A-acetyl-D-glucosamine and uridine diphos-phate. Potassium D-fructose-6-phosphate was ob-tained from the commercial barium salt by treatmentwith Dowex-50 (hydrogen form) followed by neu-tralization with potassium hydroxide.

Il. TRACER STUDIES IN ISOLATED TISSUES.Lemons and oranges of varying ages were obtainedfrom the groves. It was possible to obtain almost afull range of growth of the lemon fruit at a singleperiod due to the continuous setting habit of the lemon.The range examined in oranges was limited to rela-tively young fruit. Each piece of fruit was cut inhalf and the vesicles were carefully excisedl. Sectionsof the flavedo (colored, outer layer of peel) andlalbedo, approximately 1.5 mm thick, were cut perpen-dicular to the core of the fruit. Samples in the rangeof 100 mg of each of these three tissues were weighedinto containers and incubated at room temperature,in the dark, with 0.1 ml of a solution of labeled fruc-tose or glucose. The solutions contained 20 Ag ofthe active sugar with a total activity of 800.000 to900,000 cpm per 0.1 ml aliquot. The sugars wereprepared photosynthetically by the method of Putman,et al (14, 15).

Following incubation for 3 to 4 hours, the isolatedtissues were inactivated and extracted with boilingethanol. The ethanol extracts were concentrated andchromatographed by standard procedures. Water

saturated phenol, butanol-acetic acid-water (52:13:35) and butanol-ethanol-water (52.5: 32 :15.5) sol-vents were used in chromatography. Active sugarswere located by radioautography and the activity ineach sugar determined directly on paper with a Nu-clear D-47, Micro-mil end window, gas-flow counter.Sucrose was eluted from the paper and rechromato-graphed following hydrolysis with invertase. Activi-ty in each of the resulting monosaccharides was de-termined as above.

III. TRACER STUDIES IN INTACT FRUIT. Fruitin the range of 5 g total fresh weight was obtained.The stems and buttons were cut off undler water andeach piece of fruit was weighed quickly. A smallring of polyethylene was attached around the stemscar with the aid of a little silicone grease. The solu-tion of carbon 14 labeled glucose or fructose wasplaced in the cup thus formed. An aliquot of 0.1 mlcontaining 1.1 mg of the sugar was used. Totalactivity with glucose was 9.3 X 106 cpm. For thefructose experiments 8.0 x 106 cpm was used withoranges and 16 X 1061 cpm for lemons. Ac-tivity was measuredl with the equipment mentionedabove. The fruit was allowed to take up the majorportion of this solution in the dark. This requireda period of about 90 minutes for the lemons and 150minutes for the oranges. The residual solution waswashed into boiling ethanol. The flavedo was rapid-ly cut off into hot ethanol. The fruit was then cutin half and the juice sacs excised into hot ethanol.The remaining albedo material was also inactivatedin boiling ethanol after slicing it into thinner sections.After extraction with ethanol, the extracts weretreated as outlined above for analysis of the isolatedtissue extracts. The approximate amount of eachsugar on the paper chromatograms was determinedby a densitometric technique, using the color developedby reaction of the sugars with benzidline dihydro-chloride (4). Density was measuredl with a variableintensity light source and an International RectifierCompany photocell. The signal from the photocellwas amplified through a transistorized amiiplifier andlrecorded with a recording milliammlleter. Theamount of sugar present was determinedl by compar-ing peak heights with those peak heights found forknown quantities of sugars. Using this technique,it was possible to get approximate specific activitiesof the sugars directly on paper.

RESULTS

Results for sucrose synthesis by enzyme prepara-tions from oranges and lemons are given in table I.Data on unprocessed juices and extracts could not beobtained directly since chromogenic materials in theoriginal preparations interfered with determinationof the small amounts of sucrose formed. In the solu-ble extracts, this difficulty could be overcome by am-monium sulfate precipitation of the enzyme except inorange peel preparations. No difficulty was metwith the resuspended particulate fractions.

430

BEAN-CARBOHYDRATE METABOLISM OF CITRIUS FRUITS

TABLE I

FORMATION OF SUCROSE BY PREPARATIONS FROMLEMON AND ORANGE FRUITS

SUCROSE SYNTHESIZED

PREPARATION FRUCTOSE FRUCTOSE-6-REACTION PHOSPHATE

REACION REACTION

gmoles imolesOrange Juice

Ammonium sulfate fraction 0.12 0Particulate sediment 0 0

Lemon JuiceAmmonium sulfate fractionspH 2.5 juice 0 0pH 7 juice 0 0

Particulate sedimentpH 2.5 juice 0.17 0pH 7 juice 0.10 0

Small Lemon (15 g size)homogenatesAmmonium sulfate fraction 0 0Particulate sediment 0.15 0

Conditions: 50 .tl Enzynme preparation incubated30 minutes with 0.37 ,umoles UDPG and 2.5 jmoles fruc-tose or fructose-6-phosphate in total of 0.1 ml. Volumesand quantities doubled for analysis of particulate materialand 0.1 ml of supernatant withdrawn for analysis afterincubation.

The data for the juices indicate that the solubleportion of the orange juice contains an active systemfor the synthesis of sucrose from free fructose andUDPG. On the other hand, the lemon has no activityin the soluble fraction; instead, the particulate ma-terial shows a good capacity for sucrose synthesis.Rather than being an integral part of the particulatesystem, the sucrose synthesizing enzyme in lemonsmight be a soluble protein which is adsorbed on theparticles during juicing operations due to the low pH.To test this possibility, a suspension of particles sedi-mented at pH 7 was divided into four portions. Onepart was retained for control; the other parts weredivided into supernatant and sediment fractions bycentrifugation. The sediments were suspended inphosphate-citrate buffer, one at pH 3.5, another atpH 5, and the third at pH 7. Each suspension wasthoroughly rehomogenized in a Potter-Elvehjem ap-paratus, allowed to stand at 40 C for several hours withoccasional shaking, and finally recentrifuged. Allsediments were resuspended in phosphate buffer, pH7, to give the original starting volume. All the finalsuspensions as well as the supernatant fractions werethen analyzed for activity. No significant activitywas found in any of the supernatants and practicallyall the activity (69-81 % of the original) was re-covered on the particles. Thus, although this doesnot completely exclude the possibility that the enzymemight have been irreversibly adsorbed during process-ing, it tends to indicate a rather firm bond betweenthe particles and the enzyme, in contrast to the readily

soluble enzyme of the orange juice. The probabilitythat the enzyme actually is normally associated withthe particles is strengthened by the fact that it is alsofound in the sedimentable fraction in the preparationsfrom the young lemons which do not have an abnor-mally low pH. Other enzymes, normally found inthe soluble form in other organisms, have also beenfound in citrus associated with the particulate ma-terial (3).

Lemon tissue preparations showed an incorpora-tion of activity into sucrose with either glucose-C14or fructose-C'4 at all ages of the fruit. The resultsof pertinent experiments are given in figure 1A, B.With glucose-C'4 as substrate no activity was foundin the free fructose. Similarly, no labeling of the freeglucose occurred when fructose-C'4 was used. De-tectable amounts of activity were always found inthe hexose phosphate although concentrations of thesecompounds in the citrus tissues are very low. It isevident that the albedo had a much higher capacityfor sucrose synthesis per unit of fresh weight thaneither the juice sacs or the flavedo under these condi-tions. The capacity for synthesis by the albedo re-mained surprisingly constant throughout the fruit'sgrowth. Activity in the flavedo and vesicles droppedoff greatly in the larger fruit.

Fruit Size (Diameter in mm)FIG. 1. A. Formation of sucrose from glucose in

isolated tissues from lemons of various ages. B. For-mation of sucrose from fructose in tissue preparationsfrom lemons of two ages. 0, Sucrose formed in albedotissue slices; 0, Sucrose formed in flavedo tissue slices;X, sucrose formed in excised vesicles. No activity wasfound in free fructose with glucose-C'4 as substrate norin free glucose when fructose-C'4 was used. Total freefructose was higher than free glucose in smallest fruitsapproximately equal in larger fruits.

431

PLANT PHYSIOLOGY

When young orange tissues were incubated withlabeled glucose or fructose, no significant labeling ofsucrose occurred. Instead some anomolous reactionswere found (3).

Table II presents the results of the studies withintact fruits in terms of the total activity found ineach sugar of the various tissues of the fruit. Thesedata were also used to calculate the values for specificactivities of the sugars given graphically in figure 2.With glucose as substrate, little activity remained inthe monosaccharides; sucrose had the greatest totalactivity as well as the highest specific activity. Onthe other hand, when fructose was given, it was con-

SPECIFIC ACTIVITY (104 cpff/mg.)

0 05 1.0 1-5 0 0.5 10 I5

RATIO, G/F or F/G

FIG. 2 (Top). Specific activity of sugars in the dif-ferent tissues of intact lemons and oranges after uptakeof glucose-C14 or fructose-C14, Solid bars, specific activityof sucrose; cross-hatched bars, specific activity of fructose;speckled bars, specific activity of glucose.

FIG. 3 (Bottont). Ratios of activities of monosac-

charide components of sucrose synthesized from glucose-C14 and fructose-C14 in tissue preparations or intactlemon and orange fruits. Cross-hatched bars, ratio of

activity of glucose to fructose from tissues given glucose-C14, speckled bars, ratio of activity of fructose activity to

glucose in tissues given fructose-C14. Control experi-ments in which the ratios were determined after givinguniformly labeled sucrose indicated variability of 5 percent in final ratios.

verted much less extensively to sucrose in both fruits.

A great deal of activity remained in fructose at the

endl of the experimental period in all tissues. Withthe exception of lemon flavedo, the specific activity offructose exceeded that of sucrose in all tissues.

Sucrose from each of the labeling experimentswas subjected to hydrolysis andl rechromatographedto determine the ratio of activity in glucose to activityin fructose. To determine the accuracy of the methodand to furnish a control, experiments were also per-

formed in which uniformly labeled sucrose was fedto the fruits. The resulting labeled sucrose in thefruit was hydrolyzed and measured in the same man-

ner as the other samples. Results of this work are

included in figure 3. Here it may be seen that activitywas found in both components of the sucrose no mat-

ter which monosaccharide was suppliedl. A ratiowhich closely approaches unity would indicate com-

plete equilibration of the two monosaccharides or de-

rivatives entering into sucrose synthesis. When thesugar supplied enters into sucrose faster than it equi-librates vith the other hexose precursor to sucrose a

ratio greater than unity would be expected. In allthese experiments an excess of the labeled monosac-

charide was present through the duration of the ex-

periment so that one might expect a higher activityin the sucrose moiety corresponding to the sugar fedunless equilibration between the two precursors tosucrose were extremely rapid. The data slhows thatactivity was found in both components of the sucrose

no matter vhich monosaccharide was supplie(l. Illmost cases, the monosaccharides moiety correspond-ing to the sugar supplied still retained a somewhathigher degree of labeling. The close approach ofratios to unity in the orange albedo indicates that a

very rapid equilibration occurred between both sugarsin the tissue. Lemon tissues consistently show a

higher activity in the sugar which was fed than inthe other sugar but even here, the activity ratios are

low enough to indicate a relatively high rate of equi-libration of the sucrose precursors.

DISCuJSSION

Two mechanisms have been established for thesynthesis of sucrose in plant materials (10, 11, 12).One mechanism involves the reaction of UDPG withfree fructose to form sucrose directly while the otherforms sucrose phosphate from the reaction of UDPGwith fructose phosphate. Evidence presented by a

number of workers (5, 6, 13) makes it appear thatphotosynthetic leaves use the latter reaction, exclu-sively. Etiolated wheat seedlings also seem to makeuse largely of the phosphorylated form of fructosein sucrose synthesis. Fresh, green peas have beenshown to contain enzymes for synthesis of sucrose

from free fructose as well as from fructose phosphate(2). In the present study it has been establishedthat cell-free preparations from citrus fruits are

capable of forming sucrose from free fructose andUDPG. No activity could be detected in these sys-tems for the reaction of fructose-6-phosphate with

TISSUE LEMON ORANGE

Flovedo_intitct ' _ _ _ Z _

Albedo,inta ct m =

Ves9icles. _ _ _intaoct _ _

Flavedo _slice ' MG/F (glucose fed)

Altedoe *F/G (fructose fed)slice _ t rcoe.u

Vesicles,exc i sed __l _l _l

432

BEAN-CARBOHYDRATE METABOLISM OF CITRUS FRUITS

TABLE IIDISTRIBUTION OF ACTIVITY IN SUGARS FROM INTACT YOUNG LEMON AND ORANGE FRUITS*

ACTIVITY IN TISSUE/MG OF TISSUE DRY WT

LABELED SUA ORANGE ORANGE ORANGE LEMON LEMON LEMONSl,UBSTRATE IJUGAR FLAVEDO ALBEDO VESICLE FLAVEDO ALBEDO VESICLE

Glucose Glucose 171 536 295 154 293 171Fructose 105 105 47 26 118 54Sucrose 249 1,420 1,470 720 2,810 3,580

Fructose Glucose ... ... ... 204 360 102Fructose 162 1.560 490 692 6,380 1,710Sucrose 388 814 1,150 4,070 4,420 1,870

* Total activity in each sugar per unit of dry weight.Fruits were all in the range of 5 g total weight. Labeled sugar solutions (0.1 ml) containied 1.1 mg of sugar.

Glucose activity, 9.3 X 106 cpm in both experiments. Fructose activity 8.0 X 106 cpm in the orange and 16 X 106cpm in the lemon.

UDPG. The more indirect evidence of the tracerstudies does indicate, however, that a mechanism mustexist for the formation of sucrose without the utiliza-tion of free fructose and this path may be more im-portant in the intact cell than the free fructosemechanism.

As in previous studies in intact tissues (13) theconclusion that a phosphorylated form of fructoseparticipates in sucrose synthesis in citrus must restmainly on the fact that the monosaccharide com-ponents within sucrose become rapidly equilibratedwithout similar equilibration of the free hexoses. Inpeel slices and intact vesicles sucrose was rapidlylabeled by either glucose-C14 or fructose-C14. Ex-amination of the sucrose formed showed that bothhexose moieties of sucrose were labeled (fig 3). Nolabeling of the free fructose pool occurred with glu-cose-C14 as the substrate. It is obvious from thesucrose degradation that rapid isomerization of glu-cose to fructose did occur before synthesis of the su-crose. Since such an isomerization is most likely tooccur through the phosphorylated forms it would besafe to assume that fructose phosphate would be avail-able for sucrose synthesis. This is supported bythe fact that activity was always found in the areacorresponding to the hexose phosphates. It wouldthen seem most likely that the mechanism of synthesisof sucrose in these experiments would be phosphoryla-tion of glucose followed by simultaneous isomeriza-tion and formation of UDPG; condensation of UDPG,and fructose phosphate could then occur with subse-quent dephosphorylation of the sucrose. The ob-servations cannot completely eliminate the possibilitythat free fructose might have participated in the for-mation of sucrose in these isolated tissue experimentsbut several unlikely assumptions would have to bemade to support this possibility. If the free fructoseis participating, the pool involved must be very smallin order to account for the lack of any apparent activi-ty in the free fructose. Since the most likely path ofisomerization of glucose and fructose is through phos-

phorylated intermediates, an added dephosphorylationstep would be required to form the active free fruc-tose pool. This added step would tend to increasethe probability of a wide spread in the activity of thetwo hexose moieties of the sucrose. It should alsobe noted that if formation of sucrose were occurringthrough reaction of UDPG with a small sequesteredpool of free fructose the feeding of fructose-C14 mightbe expected to give sucrose with a much higher ac-tivity in the fructose than the glucose moiety. Thisis not the case since glucose and fructose each givevery similar labeling patterns in the sucrose molecule,indicating very close relationships in the pathwaysfollowed by the two sugars in entering the sucrose.Thus it would appear that the condensation of fruc-tose phosphate with UDPG is the most likely reactionfor the formation of sucrose from sugars supplied ex-ternally to isolated tissues of citrus fruits.

In the intact fruits the free fructose pool doesbecome labeled when glucose-C14 is the substrate sothat evidence for the participation of the fructosephosphate pathway is not so unequivocal. The highspecific activity of sucrose relative to fructose in theglucose utilization experiments does make it clearthat the normally observed main endogenous pool offructose cannot contribute greatly to the synthesis ofsucrose. This does not exclude possible utilizationof isolated small pools of free fructose which couldhave a high specific activity.

Results of the tissue slice experiments indicatethat the albedo is the most active site of sucrose syn-thesis in lemons. Interfering reactions preventedsimilar comparisons in orange tissues. The partici-pation of a transport system makes it difficult toassess the contribution that each tissue makes to su-crose synthesis in the intact fruit. The sugars mustpass first through a vascular network in the albedobefore reaching the other tissues so that many trans-formations could occur during the transport phase.The differences in the distribution of activity in thehexose components of sucrose isolated from the dif-

433

PLANT PHYSIOLOGY

ferent tissues indicates that each tissue must makesome contribution to the synthesis of sucrose. Thefructose to glucose activity ratio in sucrose from thealbedo of oranges fed fructose-C14 was 1.0 (fig 3).This ratio was 1.4 in the flavedo and 1.2 in the vesiclesin the same experiment indicating some dilution ofthe glucose portion of the sucrose may have occurredafter the sugars left the albedo or that independentsynthesis took place in the other tissues. Similarly,when glucose was fed, the glucose to fructose activityratio was 1.1 in the albedo but 1.3 and 1.2, respec-

tively, in the flavedo and juice sacs indicating changesoccurring in these tissues. Intact lemons do notshow the same wide differences between activity ra-

tios in (lifferent tissues but the work with isolatedtissues shows that different tissues may carry out in-dependent synthesis of sucrose.

Axelrod and Seegmiller (1) have investigated theformation of sucrose in apple tissues and found thatsucrose became labeled in both hexose moieties al-though not to the same extent as has been observedhere.

SUMT MIARY

Fornmation of sucrose in oranges and lemons hasbeen studie(d using tracer techniques with isolatedtissues and with intact snmall fruit. Sucrose synthesishas also been followed by normal analytical proce(lureswitlh extracts and pal-ticulate preparations.

Soluble preparations from the juice of matureoranges catalyze the reaction between uridine diphos-phate glucose (UDPG) and free fructose to formsucrose. Soluble material from the juice of lemons isinactive, but particulate preparations were found toactivate the same reaction. No activity could be

found in these systems for catalyzing the reaction be-

tween fructose-6-phosphate and UDPG to form su-crose phosphate.

In isolated tissues (peel slices and excised vesicles)from lemons of all ages, label from glucose-C14 or

fructose-C1" was incorporated into sucrose. Sucrose

was labeled in both hexose components no matterwhich monosaccharide was supplied. No labeling of

the free fructose occurred when glucose was given.This suggests formation of sucrose from a phos-plhorylated form of fructose.

Albedo tissue from lemons carried out a relativelyconstant rate of incorporation of glucose activity intosucrose throughout the growth of the fruit, but the

rate of incorporation in the flavedo and juice sacs

fell off rapidly with increase in age.In the intact fruit, glucose is incorporated into

sucrose much more rapidly than fructose. When

glucose is fed, the free fructose pool also becomeslabeled in both lemons and oranges. When fructoseis the labeled sugar imbibed, a small amount of labelappears in the free glucose of lemons but no conver-

sion to free glucose appears in oranges.

LITERATURE CITED

1. AXELROD, B., and C. G. SEEGAIILLER. 1954. Conver-sion of glucose to sucrose in apple tissue. Jour.

Agr. Food Chem. 2: 1329.2. BEAN, R. C., and W. Z. HASSID. 1955. Synthesis

of disaccharides with pea preparations. Jour.

Amer. Chem. Soc. 77: 5737.3. BEAN, R. C. 1959. Glucose aerodehydro-enase

from young oranges. Fed. Proc. 18: 188.4. BEAN, R. C. and G. G. PORTER. 1959. Detection

and differentiation of sugars and polvols oni singlepaper chromatograms. Anal. Chem. 31: 1929.

5. BUCHANAN, J. G. 1953. Path of carboni in photo-

synthesis. XIX. The identification of sucrose

phosphate in sugar beet leaves. Arch. Biochem.Biophys. 44: 140.

6. BURMA, D. P. and D. C. MORTIMER. 1956. Thebiosynthesis of uridine dipliosphate glucose and su-

crose in sugar beet leaves. Arch. Biochem. Bio-phys. 62: 16.

7. CABIB, E., L. F. LELOIR, aInd C. E. CARDINI. 1953.Uridine diphosphate acetylglucosamine. Jour. Biol.Chem. 203: 1055.

8. DISCHE, Z., and E. BORENFREUND. 1951. A new

method for the detection and determination of ketosugars. Jour. Biol. Chem. 192: 583.

9. EDELMAN, J., Vr. GINSBURG, and WV. Z. HASSID.1955. Conversion of monosaccharides to sucrose

and cellulose in wheat seedlings. Jour. Biol. Chem.213: 843.

10. LELOIR, L. F. and C. E. CARDINI. 1953. Biosvn-thesis of sucrose. Jour. Amer. Chem. Soc. 75:6084.

11. LELOIR, L. F. and C. E. CARDINI 1955. The bio-synthesis of sucrose phosphate. Jour. Biol. Chem.214: 157.

12. LELOIR, L. F., C. E. CARDINI, and J. CHIRIBOGA.1955. The biosynthesis of sucrose. Jour. Biol.Chem. 214: 149.

13. PUTMAN, E. W. and W. Z. HASSID. 1954. Sugartransformations in leaves of Canna indica. I. Syn-thesis and inversion of sucrose. Jour. Biol. Chem.207: 885.

14. PUTMAN, E. W. and W. Z. HASSID. 1954. Isola-lation and purification of radioactive sugars bymeans of paper chromatography. Jour. Biol. Chem.196: 749.

15. PUTMAN, E. W., W. Z. HASSID. G. KROTKOV, andH. A. BARKER. 1948. Preparation of radioactivecarbon-labeled sugars by photosynthesis. Tour.Biol. Chem. 173: 785.

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