SUCROSE BREAKDOWN AND SYNTHESIS IN THE RIPENING GRAPE BERRY* By P. J. HARDyt Sucrse is the majr translcated sugar in the grapevine (Swansn and EIShishiny 1958), but in the berry glucse and fructse tgether make up the bulk f the sugar cntent at all stages f develpment (Kliewer 1965a). The inversin f sucrse t glucse and fructse in ripening berries is suggested by the presence f invertase (Arnld 1965) and by the ccurrence f apprximately equal amunts f glucse and fructse (Kliewer 1965a). During the early stages f berry develpment, glucse and fructse cncentratins are lw, but at the nset f ripening there begins a phase f rapid glucse and fructse accumulatin (Kliewer 1965a). Besides sugar, the ther majr sluble cnstituents f grapes are malate and tartrate. The cncentratins f bth substances increase rapidly in the immature berry, but when ripening cmmences sharp decreases ccur, particularly in the cncentratin f malate (Kliewer 1965b). Sme wrkers believe that malate and tartrate are translcated frm the leaves (Amerine 1956; Peynaud and Maurie 1958) but there is evidence that sme at least f the malate and tartrate is synthesized in the berry (Hale 1962). The mechanisms f the syntheses f malate and tartrate in the berry are nt knwn. In an experiment designed t give infrmatin n the fate f sucrse in the ripening berry, individual excised sultana (cv. Sultanina, Thmpsn Seedless) berries were presented with radiactive sucrse, glucse, r fructse and the distributin f radiactivity in sugars and rganic acids was examined after 7 5 and 24 hr. Each treatment was duplicated. Materials and M etlwds Unifrmly 14C-labelled sucrse (specific activity 22 8 I-'c/I-'mle), glucse (specific activity 87 I-'c/I-'mle), and fructse (specific activity 86 2 I-'c/I-'mle) were btained frm the Radichemical Centre, Amersham, England, and were used withut altering the specific activities. Samples fr cunting were dried n glass planchets and cunted at infinite thinness at 35% efficiency using a thin end-windw gas-flw detectr. A Nuclear Chicag Actigraph III instrument was used, with a 3 mm slit width and set at 1000 cunts/min fr full-scale deflectin. Twelve sultana berries (apprximately 1 1 g fresh weight) were selected frm a bunch f grapes and cut ff leaving a 5-mm length f pedicel attached. 20-mm lengths f Tygn plastic tubing (internal diameter 1 6 mm) were fitted ver the pedicel stumps and 2 I-'c (1 5 X 106 cunts/min) f radiactive sugar in 10 1-'1 distilled water was placed in each tube in cntact with the cut end f the pedicel. The experiment was carried ut at rm temperature. The slutins were cmpletely taken up by the berries in between 1 and 3 hr, after which the plastic tubes were kept full f distilled water. At 7 5 r 24 hr after administratin f the radiactive sugars, the * Manuscript received August 25, 1966. t Hrticultural Research Sectin, CSIRO, Glen Osmnd, Suth Australia. Amt. J. Bil. Sci., 1967, 20, 465-70
466 SHORT OOMMUNICATIONS pedicel stumps were cut ff at the base and the berries placed in biling 80% ethanl. The berries were later extracted in a further fur changes f 5 ml f 80% ethanl fr a ttal f 100 min. The extracts were evaprated dwn t 2 r 3 ml and cleared by centrifugatin. The aqueus extracts were made up t 10 ml with water and 0 5 ml f each was passed dwn a 10 by 1 cm clumn f Dwex 50W, XlO, hydrgen frm, 20-50-mesh resin t remve basic cmpunds which were afterwards eluted with 100 ml4n NH40H. Recvery frm this resin averaged 96 75 ±3 15%. The Dwex 50 effluent, cntaining sugars and rganic acids, was chrmatgraphed n Whatman 3MM paper using as slvent the rganic phase f a 24-hr aged mixture f n-butanl-acetic acid-water (4 : 1 : 5 vjv) until the slvent reached the bttm f the paper. An autmatic strip scanner was emplyed t examine the distributin f radiactivity and the psitins f individual cmpunds were lcated by reference t standards. Aliquts f the riginal extracts were used fr determinatin f ttal reducing sugar (Shallenberger and Mres 1957) and ketse (Ashwell 1957). Sucrse TABLE 1 RADIOACTIVITY IN SUGARS AND ORGANIC ACIDS FROM INDIVIDUAL GRAPE BERRIES SUPPLIED WITH [140]SUCROSE, [140]GLUCOSE, OR [140]FRUCTOSE The values fr bth duplicates are given Duratin f Feeding (hr) Radiactivity (10-3 X cunts/min) in Sugars and Organic Acids Berry Supplied Berry Supplied Berry Supplied with 21 C* with 21 C* with 21 c* [140]Sucrse [14O]Glucse [14O]Fructse 7 5 824 196 516 1117 302 685 24 570 308 292 404 410 449 * 1 5 X 106 cunts/min. determinatins, made after alkaline destructin f fructse (Pntis and Lelir 1962), revealed that the sucrse cncentratin in each berry was less than 0 03 gj100 g fresh weight, and the ketse values were therefre assumed t be due t fructse. Glucse was determined by difference. Results The mean fresh weight f the 12 berries was 1 106 ±0 1O g. The mean glucse and fructse cncentratins were 3 34±0 32 g and 3 31±0 31 g per 100 g fresh weight, respectively. These values place the berries at the stage f develpment when glucse and fructse cncentratins are increasing rapidly, and when malate and tartrate cncentratins are n the decline (Kliewer 1965a, 1965b). The radiactivity in basic cmpunds (amin acids) frm all berries was small and will nt be discussed further. The ttal radiactivity in sugars and rganic acids fr each berry is shwn in Table 1. At 7 5 hr, the radiactivity in sugars and
SHORT COMMUNICATIONS 467 rganic acids frm [14C]sucrse-treated berries represented abut tw-thirds f the ttal supplied radiactivity, but in the berries treated with [14C]glucse r [14C]fructse cnsiderably less radiactivity was present at this time, suggesting that sucrse was the least readily metablized f the three. Mre radiactivity was fund in the sugars and rganic acids f [14C]fructse-treated berries at 7 5 hr than f [14C]glucsetreated berries, suggesting that glucse was the mre readily metablized f the tw exgenusly supplied mnsaccharides. Mter 24 hr, decreases had ccurred in the radiactivity islated frm [14C]sucrse- and [14C]fructse-treated berries, especially in the frmer case. N such decrease ccurred in the berries presented with [14C]glucse. In Table 1 it can be seen that there were cnsiderable differences between duplicates in regard t ttal radiactivity islated. The patterns f distributin f radiactivity [Figs. l(a)-l(c)] in the duplicates were, hwever, remarkably unifrm. In all instances radiactivity was fund in malate shwing that this cmpund can be synthesized frm sugar in the berry. Hwever, n radiactivity was detected in tartrate. When either [14C]glucse r [14C]fructse were supplied, bth mnsaccharides became labelled, demnstrating the presence f a mechanism fr the intercnversin f these sugars. Sucrse synthesis ccurred in all berries presented with radiactive mnsaccharides. 7 5 hr after administratin f [14C]sucrse [Fig. l(a)] fructse ws the predminantly labelled mnsaccharide, suggesting that the glucse miety f the exgenusly supplied sucrse was preferentially metablized. When [14C]glucse was supplied, glucse and fructse were fund t be equally labelled at 7 5 hr [Fig. l(b)] but when [14C]fructse was supplied, the extracted mnsaccharides were labelled mainly in fructse at this time [Fig. l(c)]. At 7 5 hr, a smewhat greater prprtin f the radiactivity was fund in malate frm [14C]glucse- than frm [14C]fructse-treated berries. These bservatins suggest that glucse was the mre readily metablized f the tw exgenusly supplied mnsaccharides. In Figures l(a) and l(b) it can be seen that at 7 5 hr after berries were presented with [14C]sucrse r [14C]fructse, the predminantly labelled mnsaccharide was fructse in bth instances. After 24 hr, fructse remained as the predminantly labelled mnsaccharide in berries treated with [14C]fructse, but in the [14C]sucrsetreated berries, glucse and fructse had becme equally labelled. The ttal radiactivity was als reduced t a greater extent between 7 5 and 24 hr in the [14C]sucrsetreated berries. These findings indicate that at 7 5 hr a prprtin f the exgenusly supplied [14C]fructse had penetrated t a site where it culd nt subsequently be metablized r cnverted t glucse, whereas in the [14C]sucrse-treated berries, much f the fructse which had becme labelled at 7 5 hr was later metablized. Discussin The results indicated that exgenusly supplied glucse was mre readily metablized in the berries than was fructse. This culd result frm the preferential phsphrylatin f glucse r the mre rapid penetratin f glucse t metablic sites in the berry. The predminant labelling f fructse 7 5 hr after administratin f [14C]sucrse culd therefre be the result f the preferential utilizatin f glucse
7-5 HR / t \ \ 11 (a) Iii \ ""_,.) v. \ ilwl '\ I \ ; I I ",--,/'V L (b) -.JJ"'-- t... '... _L..... -. (c)...,... 'I r-jjl_ ---1JL -/,--... -......- 24 HR J\.. ". t I-+--i f--i 1--------1 5 G F T M... _..- J\ t-.j f---i 1-+-11--------1 1---1 5 G F T M 5 G F T 1---1 M Fig. I.-Distributin f radiactivity (in duplicate) in sugars and rganic acids frm individual grape berries 7 5 r 24 hr after administratin f [l4c]sucrse (a), [14C]glucse (b), r [14C]fructse (c). 0, rigin; S, G, F, T, M: psitins f sucrse, glucse, fructse, tartaric acid, and malic acid, respectively.... 0;, 00 rn p;j.., Q @.., H Z rn I i
SHORT OOMMUNICATIONS 469 fllwing inversin f sucrse. The synthesis f sucrse frm exgenusly supplied mnsaccharides, hwever, indicates the activity f ne r bth f the tw sucrse (r sucrse phsphate) synthesizing enzymes knwn t ccur in plants: UDPglucsefructse transglycsylase r UDPglucse-fructse-6-phsphate transglycsylase (UDP = uridine diphsphate). The frmer enzyme catalyses a freely reversible reactin (Cardini, Lelir, and Chiribga 1955): UDPglucse+fructse UDP+sucrse, and it culd cnceivably participate in sucrse breakdwn prvided that UDPglucse can be utilized. This reactin culd therefre accunt fr the predminant labelling f fructse n administratin f [14C]sucrse [Fig. l(a)]. The results f the present wrk prbably apply nly t sultana berries at the stage f rapid sugar accumulatin: at earlier stages f develpment, the cncentratin f glucse may be up t five times that f fructse (Kliewer 1965a). When 14C02 was presented t the leaves f sultana vines bearing clusters f immature grapes, the mnsaccharides afterwards extracted frm the berries were fund t be labelled predminantly in glucse (Kliewer 1964). These results suggest that the changes in sugar cntent at different stages f berry develpment may be assciated with changing mechanisms f sucrse utilizatin. 7 5 hr fllwing the administratin f [14C]glucse and [14C]fructse, the isla ted radiactivity represented less than ne-half f the ttal supplied radiactivity. Mst f the radiactivity remaining at 7 5 hr was still present at 24 hr. The patterns f distributin f radiactivity in sugars and rganic acids were the same at 24 hr as at 7 5 hr. This indicates that a substantial prprtin f the exgenusly supplied glucse and fructse was rapidly metablized whilst the remainder, after underging intercnversin, was transprted t a metablically inactive site. That the exgenusly supplied mnsaccharides which were metablized did nt first equilibrate with the endgenus glucse and fructse is suggested by the apparent preferential utilizatin f glucse whilst the ttal cntents f glucse and fructse in the berries remained equal. When either [14C]glucse r [14C]fructse was supplied, radiactivity appeared in sucrse. Since the ttal cntent f either glucse r fructse was at least 100 times that f sucrse, the specific activity f the extracted sucrse must have been cnsiderably higher than that f either mnsaccharide in all cases. Thus the bulk f the endgenus glucse and fructse did nt. take part in sucrse synthesis. These results suggest that glucse and fructse accumulate in pls separate frm the sites where sugar intercnversin and breakdwn take place, and that the sugar utilized in metablic prcesses is prbably derived primarily frm sugar transprted int the berry. The present results d nt shw whether these separate regins f sugar metablism and sugar strage represent different regins f the berry r separate intracellular cmpartments. Further studies are presently being undertaken t examine the fate f sucrse in berries at different stages f develpment, emplying sucrse labelled nly in the fructse miety.
470 SHORT COMMUNICATIONS References AMERINE, M. A. (1956).-Wines & Vines 37(10),27-38; 37(11), 53-5. ARNOLD, W. N. (1965).-Bichim. Biphys. Acta 110, 134-47. ASHWELL, G. (1957).-Meth. Enzym. 3, 73-105. CARDINI, C. E., LELOIR, L. F., and CHIRIBOGA, J. (1955).-J. BiI. Ohem. 214, 149-55. HALE, C. R. (1962).-Nature, Lnd. 195, 917. KLIEWER, W. M. (1964).-Plant Physil. 39, 869-80. KLIEWER, W. M. (1965a).-Am. J. Enl. Vitic. 16, 101-10. KLIEWER, W. M. (1965b).-Am. J. Enl. Vitic. 16, 92-100. PEYNAUD, E., and MAURIE, A. (1958).-Am. J. Enl. Vitic. 9, 32-6. PONTIS, H. G., and LELOIR, L. F. (1962).-Meth. Bichem. Analysis 10, 107-36. SHALLENBERGER, R. S., and MOORES, R. G. (1957).-Analyt. Ohem. 29, 27-9. SWANSON, C. A., and ELSHISHlNY, E. D. H. (1958).-Plant Physil. 33, 33-7.