STUDIES ON THE CHEMISTRY OF THE LIVING BARK OF THE BLACK LOCUSTIN RELATION TO ITS FROST HARDINESS. VIII. POSSIBLE ENZYMATIC

PROCESSES INVOLVED IN STARCH-SUCROSEINTERCONVERSIONS 1, 2, 3

M. H. EWART,4 D. SIMINOVITCH,5 AND D. R. BRIGGSDEPARTMENT OF AGRICULTURAL BIOCHEMISTRY, UNIVERSITY OF MINNESOTA.

ST. PAUL 1, MINNESOTA

In studies on the chemical basis of frost resistancein plants Siminovitch et al (25, 26) observed pro-nounced seasonal changes in the absolute and relativeconcentrations of starch and sucrose in the livingbark of the black locust tree. Their data suggestedthat these reserve carbohydrates are interconvertedby a reversible process which is dependent on tem-perature and season. Low temperatures appeared tofavor conversion of starch to sucrose while high tem-peratures favored the reverse process. The in vitroconversion of starch to sucrose observed in maplesap, by Bois and Nadeau (7, 8) was, like the in vivoprocess, favored by low temperatures. The conver-sion of sucrose to starch in plants is not as wellknown as the conversion of starch to sucrose. It isusually assumed that the synthesis of starch is cata-lyzed by phosphorylase. However, recent demon-strations of the enzymatic synthesis of starch and re-lated polysaccharides from disaccharides without in-termediate phosphorylations (3, 11, 12, 18, 19, 20,21, 22, 23, 24, 28) suggest that the phosphorylase re-action may not always be involved in the synthesis ofstarch in plants. It is the purpose of this paper todiscuss some possible mechanisms for starch-sucroseinterconversions and to present the results of certainexperiments which may have a bearing on themechanism in the living bark tissue of the blacklocust tree.

The known enzymatic processes which may be in-volved in starch-sucrose interconversions are sum-marized in figure 1. The obvious possible route forthe conversion of starch to sucrose involves phos-phorolytic degradation of starch to glucose-l-phos-phate (G-1-P) as catalyzed by phosphorylase (re-action A), followed by combination of G-1-P withfructose to form sucrose as catalyzed by sucrose

1 Received August 24, 1953.2The authors are indebted to the Herman Frasch

Foundation for Chemical Research for a grant in sup-port of studies on the problem of winter hardiness inplants. The present paper is a report on part of thisresearch.

3 Paper No. 3037, Scientific Journal Series, Minne-sota Agricultural Experiment Station. Contents of thispaper constitute a part of a thesis submitted by M. H.Ewart to the Graduate School of the University ofMinnesota in partial fulfillment of the requirements forthe Ph.D. degree, 1951.

4 Present address: Food and Drug Laboratories, De-partment of National Health and Welfare, 35 JohnStreet, Ottawa, Ontario, Canada.

5 Present address: Division of Chemistry, Depart-ment of Agriculture, Science Service, Ottawa, Ontario,Canada.

phosphorylase (reaction B) (10, 16, 17). The re-quired fructose molecule could be produced from asecond molecule of G-1-P by way of glucose-6-phos-phate (G-6-P) and fructose-6-phosphate (F-6-P) asintermediates. These reactions very probably do notrepresent the actual route of the conversion of starchto sucrose because the occurrence of sucrose plhos-phorylase in higher plants has not been established.Furthermore, Hassid and Doudoroff (16) pointed outthat the equilibrium point of the sucrose phos-phorylase reaction,

K = [Sucrose] [H3P04=0]05 at pH 6.6,[Fructose] [G-1-P] 0.5apH6,

favors the breakdown rather than the synthesis ofsucrose. Yet many plants produce high concentra-tions of sucrose while the concentrations of fructoseand G-1-P remain low. Unless plant cells can re-move sucrose from the sphere of influence of the en-zyme, this reaction cannot be involved in the syn-thesis of sucrose.

A more plausible mechanism for the formation ofsucrose is suggested by the work of Calvin and Ben-son (9). They observed that during the photosyn-thetic assimilation of radioactive carbon dioxide byalgae, the hexose phosphates became radioactive be-fore sucrose, and sucrose became radioactive beforeglucose or fructose. This indicated that the immedi-ate precursors to sucrose were two hexose phos-phates. Calvin and Benson specifically suggestedfructose-6-phosphate (F-6-P) and G-1-P. The F-6-P

Z9CS~~1Ct||GLUCOSE* _ dextranrucrmee SUCRCSEj levansucrese +

DEXTRAH (C) (D) LEVAN

(1.K,(E.UI (H

D.ThXX

MALTOSEala.es

- GLUCOSE vMALTOSE amylolltase

4* GLUCOSE(7)

FIG. 1. Summarystarch and sucrose.

AMTLOSE

A1(G)a(0)

A'LOPWSTIII

- 3PO4phosrhorylas.

_- H3Fo4(A)

FRUCTOSE

GLUCOSE.1-P

GLYCOLYSIS

of enzymatic reactions involving

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seems, indeed, to be a likely precursor because it pro-vides fructose with the furanose configuration andthe energy release associated with liberation of theinorganic phosphate would decrease the probabilityof the reverse reaction. However, until such a re-action is demonstrated, the conversion of starch tosucrose cannot be explained on the basis of knownenzyme reactions.

The opposite process, namely the conversion ofsucrose to starch, might take place simply by the re-verse of reactions (A) and (B). However, pendingthe identification of sucrose phosphorylase in plants,this explanation cannot be accepted. An alternativeprocess for the formation of starch from sucrosewould involve direct conversion without the forma-tion of phosphorylated intermediates. Several enzymesystems which catalyze direct conversion of sucrose topolysaccharides have been found in bacteria. Theseenzymes belong to the group called transglycosidasesor transglycosylases (20) because the reactions in-volve exchanging the glycosidic linkage of a disac-charide for that of a polysaccharide. Dextransu-crase, an enzyme found in Leuconostoc mesenteroidesby Hehre (18, 19), catalyzes the direct conversion ofsucrose to fructose and dextran, an a-1,6-glucosidiclinked polymer of D-glucose (reaction C). Hestrinand Avineri-Shapiro (22) obtained a levansucrasefrom Aerobacter levanicum. This enzyme catalyzesthe dissimilation of sucrose to glucose and levan, apolyfructoside having 2,6-linkages and retaining thefuranose configuration of the fructose units (reactionD). Amylosucrase, an enzyme which catalyzes thedirect conversion of sucrose to fructose and a poly-saccharide of the starch-glycogen type was obtainedfrom Neisseria perfiava by Hehre and co-workers(21) (reaction E).

The equilibrium points of the levansucrase, dex-transucrase, and amylosucrase reactions stronglyfavor the production of polysaccharide. This iscaused by the higher energy of the glycosidic linkageof sucrose as compared to that of the polysaccha-rides, and possibly by the relatively low concentra-tion of terminal glycosidic linkages in the polysaccha-rides. The high energy of the sucrose linkage may bedue in part to the furanose configuration of the fruc-tose moiety. In the case of the levansucrase reac-tion, the furanose configuration of fructose is re-tained in the polysaccharide and detection of the re-verse reaction has been reported (12).

Transglycosidases which catalyze reactions involv-ing carbohydrates other than sucrose have also beenfound. MIonod and Toriani (23, 27) and Doudoroffand co-workers (11) found an amylomaltase in cul-tures of Escherichia coli. This enzyme catalyzes thereversible interconversion of maltose and starch (re-action F).

The reversibility of this reaction is not surprisingbecause both reactants have the same glycosidic link-age. The conversion of amylose to amylopectin bythe Q-enzyme (4, 5) (reaction G) and the conversionof dextrins to dextran by dextrandextrinase (20) (re-

action H) are transglycosidase reactions as they in-volve the production of 1,6-glycosidic linkages at theexpense of 1,4-glycosidic linkages. The equilibriumpoints of both reactions appear to favor the 1,6-link-ages, indicating that these have a lower energy con-tent than the 1,4-linkages.

Most of these enzyme systems have been foundonly in bacteria. Only the starch phosphorylase, theQ-enzyme, and the amylases have yet been found inhigher plants. Since all of the reactions of sucroselisted here favor the destruction of sucrose it is evi-dent that the conversion of starch to sucrose cannotbe satisfactorily explained on the basis of these re-actions. Whether conversion of sucrose to starchproceeds via phosphorylation or by direct conver-sion, only the glucose moiety appears in the polysac-charide. Both processes would be expected to causean accumulation of fructose. Siminovitch et al (26)did not observe a significant accumulation of fructoseassociated with the production of starch in locustbark tissue. Thus, if either process is involved in theconversion of the black locust tree, the fructose mustbe rapidly metabolized or converted to polysaccha-ride by a different route.

The detection of a phosphorylase system in theliving bark of the black locust tree and a study ofsome of the properties of this enzyme system weredescribed in an earlier paper (13). The possibilitythat phosphorylase and glucose-i-phosphate may beinvolved in the production of starch can be tested bydetermining whether or not the ratio of inorganicphosphate to glucose-i-phosphate in the tissue at thetime of starch synthesis is favorable to such synthesisby phosphorylase. The equilibrium point of the re-action catalyzed by the locust tree phosphorylase(reaction A) is, as in the case of other phosphoryl-ases, independent of the concentration of polysac-charide (13, 14, 15). It may be represented by theequation

pG-1-P -K

where P and G-1-P represent the total concentra-tions of inorganic orthophosphate and glucose-l-phosphate, respectively. The equilibrium ratio, K,is a function of the pH, varying from 10.8 at pH 5.0to 3.1 at pH 7.0 (14). Phosphorylase may be ex-pected to cause accumulation of starch only if theratio of P to G-1-P is less than the value of the equi-librium ratio. If chemical analysis of tissue, in whichstarch is accumulating, indicates that the ratio of Pto G-1-P is favorable to starch synthesis it wouldsupport the usual assumption that the starch is syn-thesized by phosphorylase. If, on the other hand,the observed ratio is unfavorable to starch synthesisit would suggest that some other mechanism is in-volved.

It might be argued that the relative concentra-tions of inorganic phosphate and glucose-i-phosphatein the whole tissue are not necessarily the same asthose within the environment of the phosphorylase.

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EWART ET AL-STARCH-SUCROSE INTERCONVERSIONS

The two compounds may not diffuse with equal easebetween the extracellular and intracellular fluids, orthe protoplasm may have a mechanism for maintain-ing local concentrations of glucose-i-phosphate.Phosphorylase has been detected in many plant tis-sues (29, 30) by infiltrating the starch-free tissueswith solutions of glucose-i-phosphate and subse-quently testing for starch. This consists, in effect, ofartificially changing the ratio of inorganic phosphateto glucose-i-phosphate within the cells and is de-pendent on diffusion of glucose-i-phosphate intothem. If inorganic phosphate diffuse into the cellsas readily as glucose-i-phosphate, it would be ex-pected that infiltration with a solution of the twocompounds in equilibrium proportions would notcause starch accumulation. If, however, the cells arecapable of preferentially excluding inorganic phos-phate or of concentrating glucose-i-phosphate, such amixture could stimulate starch synthesis and the ob-served relative concentrations of these materials inthe total tissue would not be significant.

To test the possible role that phosphorylase mightplay in the starch-sucrose fluctuations which occur inthe living bark tissue of the black locust tree, sam-ples were analyzed and the ratios of inorganic phos-phate to glucose-l-phosphate were determined atvarious times during the year or under circumstanceswshere starch accumulation or starch disappearance inthe tissues was known to be taking place. Studies onthe effects of varying the ratios of the concentrationsof inorganic phosphate to glucose-i-phosphate artifi-cially by the infiltration technique were made in cer-tain cases in order to determine whether selectivel)ermeability might constitute a modifying circum-stance in the evaluation of the significance of theseanalyses.A marked increase in the starch content of the

bark tissue of normal locust trees occurs during Mayor with the onset of warm spring weather (26).This quickly reaches a maximum which in the 1950season occurred in the last week of May, followed bya rapid disappearance of starch to a spring minimumby mid-June. The starch content remains lowthroughout July, followed by a slow increase duringAugust, September, and October. With the onset ofcold weather in late October and during November,starch disappears again. If logs are taken from nor-mal trees in late May, when the spring maximum ofstarch content is observed, and are stored at a lowtemperature (30 C), the starch disappears and aconcomitant and equivalent amount of sucrose ap-pears. Analyses obtained on samples from normaltrees at various times during the year or from logsstored under abnormal temperature conditions thusfurnish ample opportunity to determine the possiblerole of phosphorylase in starch synthesis or disap-pearance in the tissue.

EXPERIMENTALMATERIALS: Tissue samples weighing approxi-

mately 1.0 gm were removed from the living bark ofblack locust trees by a technique similar to that de-

scribed by Siminovitch, Wilson and Briggs (26).Samples were obtained from a tree, B, at varioustimes throughout the 1950 season from early May tomid-October. In addition, two neighboring trees, Aand C, were felled May 1 and May 22 respectively,and were sampled at the time of cutting. Logs fromthese trees were stored at 3° C and sampled againJuly 5 and June 28, respectively. Tests for starch withiodine-potassium iodide reagent made when the sam-ples were taken indicated qualitatively the changeswhich were taking place in the content of starch.These changes were more quantitatively indicated,however, by analyses which were made on tissuefrom neighboring trees at the same time (26).

TISSUE EXTRACTS: Distilled water extracts of thetissue samples were used in the analyses for phos-phorus compounds. This was necessary because ex-tracts obtained with trichloracetic acid solutions in-variably became cloudy when later mixed with am-monium molybdate. All equipment used in the ex-traction procedure was previously chilled at -15° C,and the extractions were carried out in a cold roomat approximately 3° C. The tissue samples werefrozen by placing them on a block of dry ice for 1/2hour. They were then macerated vigorously in amortar with 10 ml of cold (0.0 to 3.00 C) distilledwater, and the liquid was transferred to a smallcentrifuge tube and centrifuged. The supernatantliquid was decanted into a 50-ml volumetric flask.The residue in the mortar was triturated with 3 ad-ditional portions of 7 to 8 ml of the cold distilledwater. Each extract was then mixed in successionwith the accumulated residue in the centrifuge tubeand centrifuged separately. The combined extractsin the 50-ml volumetric flask were mixed with 15 mlof cold 16 % (by weight) trichloracetic acid and di-luted to volume with cold distilled water. The pre-cipitate was separated by centrifugation and thesupernatant used in the analyses for inorganic phos-phate and for inorganic plus 7-minute acid-hydrolyz-able phosphate. This extraction procedure gavesatisfactory recovery of inorganic phosphate addedto tissue .samples in a mortar prior to the macera-tion treatment.

Aliquots to be analyzed for inorganic phosphatewere immediately neutralized to pH 4.0 with 1 Nsodium acetate to minimize the danger of hydrolysisof the labile phosphate esters. They were thenplaced in a boiling water bath for 7 minutes, cooled,diluted to 25 ml, and centrifuged. Aliquots to beanalyzed for the sum of inorganic plus acid-hydrolyz-able phosphate were heated in boiling water for 7minutes prior to the addition of sodium acetate.They were then cooled, mixed with the same volumeof sodium acetate as that used for the previous sam-ples, diluted to 25 ml, and centrifuged. Because ofevaporation and destruction of trichloracetate duringthe heating, the pH of samples exceeded 4.0 at thisstage.

ANALYTICAL PROCEDURES: The 7-minute acid-hydrolyzable phosphate is reported as glucose-i-phosphate. Actually other acid labile phosphate

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compounds may have been present in the extracts,causing the reported values to be too high. Twoprocedures were used to estimate the concentrationsof phosphate in the extracts. Procedure I was de-scribed in an earlier paper (13). Procedure II wasbased on the isobutyl alcohol extraction proceduresof Berenblum and Chain (6) and Allen (1) modifiedto avoid errors caused by the turbidity which some-times developed in the aqueous solutions. Aliquotsof 5 to 10 ml from the supernatant fluid of neu-tralized extracts were transferred to glass-stopperedseparatory funnels. Each was mixed with sufficient10 % trichloracetic acid to readjust the pH to 4.0,then with 5 ml of 0.1 Mi acetate buffer, pH 4.0, 2.5ml of 1 % ascorbic acid in acetate buffer, and 2 mlof 2.5 % ammonium molybdate. The color was al-lowed to develop for 10 minutes and then 10 ml ofisobutyl alcohol followed by 1 ml of 0.35M oxalate(0.35 M oxalic acid neutralized to pH 4.0 with0.35 M dipotassium oxalate) were added. The funnelwas swirled to assure thorough mixing of the oxalate.Then 3 ml of 10N sulfuric acid were added and thefunnel was inverted several times. The alcohol layer,containing most of the molybdenum blue, was sepa-rated and the aqueous phase was re-extracted with 5ml of isobutyl alcohol. The two extracts were com-

bined in a 25-ml volumetric flask to which 0.5 ml ofthe 1 % ascorbic acid solution had been added pre-viously. The solution was diluted to 25 ml with95 % ethanol and the optical density determined at670 m,u using a Coleman Model II spectrophoto-meter. The blank samples contained all of the re-agents but not the tissue extract.

HISTOCHEMICAL STUDIES: The black locust barktissues selected for histochemical studies of the ef-fects of artificially varying the ratio of inorganicphosphate to glucose-i-phosphate contained verylittle or no starch detectable by qualitative tests withiodine-potassium iodide reagent. Thin tangentialsections of the living bark were cut free-hand with arazor blade, washed by suspending in tap water forapproximately one hour, and then transferred to thetest solutions containing glucose-i-phosphate. Solu-tions of dipotassium glucose-l-phosphate had an al-kaline pH and did not give positive histochemicaltests for phosphorylase in viable cells unless theywere adjusted to a weakly acid pH. Citric acid wasused to acidify these solutions. Comparisons weremade, at pH 6, of the rates of starch synthesis bythe cells in the presence of equal concentrations ofglucose-i-phosphate with and without added inor-ganic phosphate. If the accumulation of starch in

TABLE ITHE CONCENTRATION OF WATER-SOLUBLE INORGANIC PHOSPHATE AND 7-MINUTE ACID HYDROLYZABLE PHOSPHATE

IN THE LIVING BARK OF BLACK LOCUST TREEsPROBABLE CHANGES P/GM DRY MATTER RATIODATE TREE IN STARCH PROCEDURE RACIOCONCENTRATION INORGANIC ACID LABILE INOEG. P/ACID LABILE P

mg mgMay 1 A Increasing I 0.360 0.0096 37.5

it 'i II 0.335 0.0117 28.6"c it I 0.326 0.0133 24.5it it II 0.310 0.010 31.0

May 11 B Increasing I 0.350 0.0098 35.7it(4 II 0.340 0.012 28.3

it "I 0.395 0.0098 40.3it "II 0.376 0.012 31.3

May 22 C Increasing I 0.647 0.0214 30.2""II 0.640 0.0129 49.5"'I 0.681 0.0171 39.8

II 0.658 0.0171 38.5Mav 24 B Near maximum I 0.462 0.0294 15.7"" " II 0.470 0.0181 26.0

I 0.556 0.0135 41.2II 0.547 0.0158 34.6

June 15 B Near minimum I 0.425 0.0168 25.3it it" " II 0.435 0.0056 77.5cc ic" " I 0.455 0.0197 23.1it it It II 0.453 0.0113 40.0

July 5 B Near minimum I 0.516 0.0165 312It I I I 4 II 0.516 0.011 47.0"" " I 0.453 0.013 34.8

II 0.473 0.009 52.5Sept. 8 B Increasing 1 0.624 0.033 18.9

"g it I 0.641 0.006 107.0Oct. 12 B Increasing I 0.295 0.010 29.5it "c I 0.307 0.033 9.3

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TABLE IITHE CONCENTRATION OF WATER-SOLUBLE INORGANIC PHOSPHATE AND 7-MINUTE AcID HYDROLYZABLE PHOSPHATE IN THELIVING BARK OF BLACK LOCUST TREES AT THE TIME WHEN TREES WERE CUT AND AFTER STORAGE FOB SEVERAL WEEKS

AT 30 C. (STORAGE WAS ASSOCIATED WITH A DISAPPEARANCE OF STARCH AND ACCUMULATiON OF SUCROSE)

DATE DATE STORAGE ANAL. P/GM DRY MATTER RATIOTREE, CUT SAMPLED TIME PROC. IN\ORGANICACI LABILE INORG. P/ACID LABILE P

mg mgA May 1 May 1 ....... I 0.360 0.0096 37.5

II 0.335 0.0117 28.6(Starch increasing) 1 0.326 0.0133 24.5

II 0.310 0.010 31.0A May 1 July 5 65 days I 0.658 0.0068 97.0

II 0.688 0.0052 132.0(Starch depleted) I 0.615 0.0203 30.3

II 0.656 0.0058 113.0C May 22 May 22 ....... I 0.647 0.0214 30.2

II 0.640 0.0129 49.5(Starch near maximum) I 0.681 0.0171 39.8

II 0.658 0.0171 38.5C May 22 June 28 37 days I 0.716 0.0235 30.0

II 0.726 0.0043 169.0(Starch depleted) I 0.735 0.0235 31.2

II 0.742 nil ....

the cells was retarded or prevented by the added in-organic phosphate in amounts approaching the equi-librium ratio, it would indicate that the cells did notpreferentially admit or concentrate the phosphateester. The experiments were carried out both withand without vacuum infiltration, but there was no

apparent difference in the results obtained.To be confident that starch accumulation oc-

curred in viable cells, tissue sections were stainedwith neutral red and examined microscopically.Cells which were stained by the neutral red were

considered viable. The tissue sections were thenwashed with 95 % ethanol to remove the stain,washed with water to remove the ethanol, andstained with iodine-potassium iodide reagent. Thetissue sections were again examined microscopicallyand, because of the characteristic shapes of differentsections, it was possible to say with confidencewhether starch accumulation had occurred in viablecells.

RESULTSThe results of the analyses for phosphorus com-

pounds in bark samples at the time of sampling inthe field are shown in table I. The probable natureof changes takingf place in the concentrations ofstarch in the samples, based on the observations ofSiminovitch, Wilson and Briggs (26), are also indi-cated. The results of the analyses of samples fromtrees A and C before and after storage at 3 C are

shown in table II. When the final analyses were

carried out on the samples stored at 30 C, most ofthe starch had disappeared, and there had been a

pronounced increase in the concentration of sucrose.

Therefore it is assumed that these samples repre-

sented tissue in a condition favoring the conversionof starch to sucrose.

The lowest ratio of inorganic to acid labile phos-phate (glucose-l-phosphate) obtained in these anal-yses was 9.3. Since the equilibrium ratios vary from3.1 to pH 7.0 to 9.0 at pH 5.5 (13, 14) and the pHof the tissue was never less than 6.0, these results in-dicate that the conditions were unfavorable for theproduction of starch by a phosphorylase mechanismin all of the tissues analyzed. However, theystrongly suggest that phosphorolysis might be in-

TABLE IIITHE RESULTS OF INFILTRATION OF TISSUE SECTIONS FROMLIVING BARK OF THE BLACK LOCUST TREE WITH SOLU-TIONS OF GLUCOSE-1-PHOSPHATE AND GLUCOSE-1-PHOS-

PHATE PLUS I-NORGAN IC PHOSPHATE

TEST TEMP. TIME STARCH CONDITIONSOLUTION PRODUCTION OF CELLS

° C hr1.0o G-1-PK2* 3-4 10 nil Viablewith no pH 23-25 5 nil Viableadjustment

1.0 %o G-1-PK2*acidified to 3-4 10 ++, granular ViablepH 6.0 with 23-25 5 -H-+, Viablecitric acid

1.0 % G-1-PK2* Somedissolved in 23-25 50-60 ±,granular still0.1 M phos- viablephate, pH 6.0

[inorganic P][G-1-PK2] *

* G-1-PK2 represents the dihydrate of dipotassiumglucose-l-phosphate.

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volved in the disappearance -of starch in those tissuesin which its concentration was decreasing.

The results of the histochemical experiments aresummarized in table III. With weakly acid test solu-tions (pH 6.0) containing glucose-i-phosphate, butno added inorganic phosphate, there was definite ac-cumulation of starch within viable cells. The pro-duction of starch in the tissues was definitely inhib-ited by addition of inorganic phosphate to yield aratio of inorganic phosphate to G-1-P of 3.7. Theequilibrium ratio at this pH is approximately 6.7(14). This observation suggests that the inorganicphosphate had diffused into the viable cells as readilyas glucose-i-phosphate. In those cells which werenot alive, as indicated by the neutral red stainingtest, the starch which accumulated was diffusedthroughout the protoplasm while that which ap-peared in viable cells was granular.

DISCUSSIONIn these experiments a considerable variation in

the analytical values for the concentrations of acid-hydrolyzable phosphate was observed. This ischiefly owing to the fact that these values representthe differences between the observed inorganic phos-phate concentrations before and after acid-hydroly-sis. They are subject to the same absolute errors asthe much higher concentrations of inorganic phos-phate. However, the observed ratios were consist-ently unfavorable to the production of starch. Inmost instances this would be true even after allow-ance for a 100 % error in the estimation of acidlabile phosphate.

The observed inhibition of starch production byinorganic phosphate in the histochemical tests sug-gests that the over-all ratio of inorganic phosphate toglucose-i-phosphate in the bark tissue is closely simi-lar to the ratio within the environment of the phos-phorylase enzyme. Thus, the evidence indicates thatnormal accumulation of starch in the black locusttree is not catalyzed by phosphorylase. This impliesthat the production of starch which is stimulatedartificially by infiltration with glucose-i-phosphatemay be an abnormal process. Stocking (27) has ar-rived at the same conclusion after studying the in-tracellular location of phosphorylase in leaves. Hereported that although in vivo starch synthesis inthe leaves was initiated with the chloroplasts, phos-phorylase could not be detected in the plastids witheither histochemical or biochemical techniques. Thepossible role of phosphorylase in the degradation ofstarch in plants is not questioned, however.

The conclusion that phosphorylase is not involvedin the synthesis of starch or the conversion of sucroseto starch in the black locust tree is entirely compati-ble with the known reactions for the interconversionof sucrose and polysaccharides (fig 1). Assumingthat conversion of sucrose to starch does not involvephosphorylase, a possible route would appear to be adirect conversion catalyzed by amylosucrase or somesimilar transglycosidase. It may be of significance

that Siminovitch, Wilson and Briggs (26) observedproduction of starch in the black locust tree onlywhen considerable sucrose (3 %) was present. Thedirect conversion would liberate one molecule offructose for each molecule of glucose incorporated inthe polysaccharide. The fact that they did not ob-serve an accumulation of fructose associated with theconversion of sucrose to starch does not necessarilydiscredit the hypothesis. It is possible that duringproduction of polysaccharide from sucrose the fruc-tose moiety is rapidly metabolized.

The reactions shown in figure 1 do not provide adirect route for conversion of starch to sucrose andsuch a reaction is thermodynamically improbable. Arole of phosphorylase in the low temperature break-down of starch and its conversion of sucrose in thenormal metabolic breakdown of starch is likely be-cause the observed ratio of inorganic phosphate toglucose-i-phosphate was at all times favorable forphosphorolysis. The probability that two phos-phorylated hexoses are required for the synthesis ofsucrose (9) lends further support to the assumptionthat phosphorylase is involved in the degradation ofstarch.

SUMMARY1. The possible role of phosphorylase in the

interconversion of sucrose and starch in the livingbark tissue of the black locust tree has been investi-gated.

2. Samples of the living bark tissue were ana-lyzed for inorganic phosphate and for acid labilephosphate (glucose-i-phosphate) at various timesthroughout the growing season. The observed ratiosof inorganic phosphate to glucose-i-phosphate wereunfavorable to the production of starch in all sam-ples, including those in which starch was actually ac-cumulating. This result suggested that some reac-tion other than the phosphorylase reaction was re-sponsible for the synthesis of starch in this tissue.

3. Starch synthesis was demonstrated histo-chemically in sections of the living bark tissue by in-filtrating them with a solution of glucose-l-phos-phate.

4. The artificially stimulated*starch synthesis wasstrongly inhibited by including inorganic phosphatewith the glucose-i-phosphate in the infiltration solu-tion.

5. It is suggested that the artificially stimulatedstarch production is an abnormal reaction.

6. The evidence indicates that phosphorylase isnot involved in the synthesis of starch or the con-version of sucrose to starch, but a role of phos-phorylase in the normal metabolic breakdown ofstarch or in the low temperature conversion of starchto sucrose is not questioned.

LITERATURE CITED1. ALLEN, R. J. L. The estimation of phosphorus.

Biochem. Jour. 34: 858-865. 1940.2. ARREGuIN-LOZANO, B. and BONNER, J. Experiments

on sucrose formation by potato tubers as influ-

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EWART ET AL-STARCH-SUCROSE INTERCONVERSIONS

enced by temperature. Plant Physiol. 24: 720-738. 1949.

3. AVINERI-SHAPIRO, S. and HESTRIN, S. The mecha-nism of polysaccharide production from sucrose.Biochem. Jour. 39: 167-172. 1945.

4. BARKER, S. A., BOURNE, E. J., and PEAT, S. Enzymicsynthesis and degradation of starch. V. The actionof Q-enzyme on starch and its components. Jour.Chem. Soc. 1949: 1712-1717.

5. BARKER, S. A., BOURNE, E. J., WILKINSON, I. A., andPEAT, S. Enzymic synthesis and degradation ofstarch. VII. The mechanism of Q-enzyme action.Jour. Chem. Soc. 1950: 93-99.

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