In vitro sugar uptake by grapefruit ( Citrus paradisi ) juice-sac cells

CSIRO PUBLISHING

www.publish.csiro.au/journals/fpb Functional Plant Biology, 2005, 32, 357–366

In vitro sugar uptake by grapefruit (Citrus paradisi) juice-sac cells

Moshe HubermanA, Uri ZehaviB, Wilfred D. SteinC, Ed EtxeberriaD and Raphael GorenA,E

ARobert H Smith Institute of Plant Sciences and Genetics in Agriculture, The Kennedy–Leigh Centrefor Horticultural Research, The Hebrew University of Jerusalem, POB 12, Rehovot 76100, Israel.

BInstitute of Biochemistry, Food Science and Nutrition, The Hebrew University of Jerusalem,POB 12, Rehovot 76100, Israel.

CBiological Chemistry Silberman Institute of Life Sciences, The Hebrew University of Jerusalem,POB 1255 Jerusalem 91904, Israel.

DCitrus Research and Education Center, University of Florida, 700 Experiment Station Road,Lake Alfred, FL 33850, USA.

ECorresponding author. Email: [email protected]

Abstract. To further our understanding of the mechanisms of sugar uptake and accumulation into grapefruit(Citrus paradisi Macf. cv. Marsh seedless), the patterns of uptake and utilisation of sucrose, glucose and fructose byCitrus juice cells was investigated. Analyses were conducted on sliced juice sacs that were incubated in radioactive[14C]-sugar solutions with unlabelled sugars, in the presence or absence of metabolic inhibitors. Both hexosesdemonstrated an initial uptake peak in December and a second uptake peak in February–March. From Marchthrough April the rates of sucrose uptake increased to levels comparable to those of glucose and fructose. Sucroseand its moieties fructose and glucose entered the juice cells of Citrus juice fruit by an insaturable, and mostly byan independent, process. However, NaN3 and carbonylcyanide m-chlorophenylhydrazone (CCCP) produced slightinhibition of these processes. Cells took up hexoses at a greater rate than sucrose, with accumulation reaching aplateau by 4–8 h, and then continuing unabated, in the case of glucose, for 42 h. Uptake of all three sugars increasedlinearly in the range of sugar concentrations tested, which extended from 0.01 to 320 mM, denoting an insaturablesystem for sugar uptake. 14CO2 evolution was relatively low in all the experiments, the lowest evolution beingrecorded when the uptake of [14C]-sucrose was studied, while the highest 14CO2 evolution was recorded whenthe uptake of [14C]-glucose was studied. The data demonstrate a preferential utilisation of glucose over fructoseand sucrose. In all the experiments, the two metabolic inhibitors significantly inhibited the decarboxylation of thethree sugars.

Keywords: citric acid, Citrus, fruit quality, fruit respiration, sugar accumulation, sugar transport.

Introduction

The mechanisms of sugar transport into storage cells aremajor factors determining commercial fruit quality and shelflife. In mature citrus fruits, sugar content varies between5 and 12% on a fresh weight basis, while organic acidscontent varies between 0.5 and 2.0% on a fresh weightbasis, depending on variety and environmental conditions(McCready et al. 1950; Sinclair 1972; Ting and Russell1986). Sugars, together with organic acids (mostly citrate),constitute a major element in the determination of fruitquality as expressed by the ratio of total soluble solids / acid.Despite the relatively high concentration of soluble sugars

Abbreviations used: CCCP, carbonylcyanide m-chlorophenylhydrazone; DNP, dinitrophenol; PCMBS, p-chloromercuribenzene sulphonic acid;TCA, tricarboxylic acid.

stored in the vacuole of juice cells (Echeverria and Valich1988; Echeverria and Burns 1989; Garcia-Luis et al. 1991),the less abundant organic acids have been considered as boththe primary energy and the carbon source during later stagesof fruit development. Murata (1977), who studied [1,5-14C]-citric acid metabolism in ‘Satsuma’ mandarin fruit segmentsduring storage, found that citric acid was rapidly respiredas 14CO2. Moreover, Popova et al. (1995), who studied[6-14C]-citrate conversion in leaves of maize and wheat, alsoreported that the main route of citrate metabolism was via therespiration cycle. This distinction between sugars and acids isalso based on several other pieces of evidence demonstrating

© CSIRO 2005 10.1071/FP04125 1445-4408/05/040357

358 Functional Plant Biology M. Huberman et al.

the continuous accumulation of sugars throughout fruitdevelopment and even after harvest (Echeverria and Ismail1987, 1990).

Recent demonstrations of hexokinase activity associatedwith the external mitochondrial membrane of juice cells(Goren et al. 2000) and other tissues (Laterveer et al. 1995),as well as the production of 14CO2 from externally supplied[14C]-sucrose and [14C]-fructose to juice sacs slices, anddecarboxylation via [14C]-pyruvic and [14C]-citric acids(Goren et al. 2000), led us to this investigation of themechanisms of sugar uptake, transport, accumulation, andmetabolism, and their combined relation to fruit qualityin Citrus fruits. We focused on 14C-sucrose, since it isthe translocated sugar in Citrus. We also included glucoseand sucrose, the labelled moieties of sucrose, in thisresearch project.

Different mechanisms for sugar uptake into plant cells(secondary active, facilitated diffusion or combination ofboth) have been described and characterised (Komor 1977;Komor et al. 1981; Saftner et al. 1983; Damon et al. 1988;Goldschmidt and Koch 1996), depending on the type of tissue(Griffith et al. 1987a, b) and the mode of uptake systeminvestigated (Regen and Tarpley 1974; Forney and Breen1986; Picaud et al. 2003). In general, uptake of sugars byplant tissues or isolated protoplasts is considered an energy-dependent process (Komor 1977; McRae et al. 2002; Koch2004). Presumably, the uptake of sugars occurs either throughsucrose- or hexose-specific transport systems (Miron et al.2002), prior to or after sucrose hydrolysis by invertase(Koch 2004). Hydrolysis of sucrose leads to the uptakeof the respective hexoses (Giaquinta et al. 1983; Cardemiland Varner 1984; McRae et al. 2002; Miron et al.2002) by plasmalemma-bound hexose / H+ transporters. InAnanas comosus (L.) Merr. it has been reported that sucrosecan be transported across the tonoplast membranes withoutthe need for a H+ gradient (McRae et al. 2002). In sugarbeet taproot tissue (Wyse 1979; Wyse et al. 1986), leaftissue of Beta vulgaris (Maynard and Lucas 1982) andin leaf discs of Pisum sativum L. (Aked and Hall 1993),fructose and sucrose uptake is characterised by bothactive and passive biphasic mechanisms. In this regard,plasmalemma-bound active sucrose transporters have beenclearly demonstrated (Lemoine 2000) and indirect evidencesupports their existence in storage cells (Manning et al.2001). Furthermore, the presence of hexose transportershas been firmly established for beetroot cells (Dreyer et al.1999) and in hexose-accumulating tomato fruits (Miron et al.2002), among others.

Post-phloem transport of photoassimilates into Citrusfruits is believed to occur thorough the apoplast (Koch1984; Garcia-Luis et al. 1991) and with apparently verylittle sucrose inversion (Koch and Avingne 1990). From theapoplast, sugars are transported into the juice-sac cells tosupport an entire array of metabolic activities and, during later

stages of development, accumulation into the large centralvacuole. Goren et al. (2000) showed that mature Citrus juicecells are capable of taking up sucrose and hexoses fromthe incubation medium, a result that further supports anapoplastic route for post-phloem transport in Citrus fruits.Interestingly, the results demonstrated a faster metabolicutilisation of fructose than sucrose in the juice-sac cells.

Saftner et al. (1983), who studied the mode of sucroseuptake into root discs of sugar beet, reported thatcarbonylcyanide m-chlorophenylhydrazone (CCCP), themetabolic uncoupler that increases the apparent protonconductance of membranes and reduces the electrochemicalpotential of H+ across membranes and ATP production,inhibited active sucrose uptake. Aked and Hall (1993)demonstrated that CCCP inhibited significantly the uptake ofglucose and fructose but did not substantially inhibit sucroseinto lower epidermis leaf discs of pea.

The objective of the present study was to investigate,in an in vitro system of grapefruit juice-sac cells: (a) themode of sucrose, glucose, and fructose uptake, as well astheir metabolism as expressed by CO2 evolution; and (b) todetermine, with CCCP and NaN3, whether sucrose, glucoseand fructose uptake into the juice-sac slices is an active orpassive process. The results indicate that sucrose, glucose andfructose enter Citrus juice sac cells by a process that is notsaturable, and is a �µH+-independent process. The uptakeof all studied sugars was affected by metabolic inhibitors.Furthermore, externally supplied hexoses are metabolised athigher rates than sucrose, which appears to be transportedinto the cell without prior inversion.

Materials and methodsPlant material

Mature Citrus fruits were obtained from 16-year-old grapefruit trees(Citrus paradisi Macf. cv. ‘Marsh seedless’) grafted on ‘Troyer citrange’[Citrus sinensis (L.) Osbeck × Poncirus trifoliata (L.)] rootstocks grownat the experimental farm at the Faculty of Agriculture, The HebrewUniversity of Jerusalem, Rehovot, on sandy loam soil. Experimentswere conducted between mid-November and mid-April. Fruit werebrought immediately to the laboratory. At every sampling date during thematurity season (mid-December–March), fruits were picked from fourtrees. The fruits were peeled and the endocarp, containing the juice sacs,used for experiments. The locular membranes were carefully removedand segments were randomly selected from each fruit. Slices (2–3 mmthick, 8–10 mm wide) from each segment, and containing the juice cells,were used as experimental material. A replicate consisted of four juice-sac slices, each from a different tree. The data are shown as means ofsix replicates in duplicate experiments (Fig. 1I).

Preparation of the incubation medium

The incubation medium had to be experimentally developed to ensurea stable pH (the natural pH of the grapefruit fruit juice is ∼2.9–3.2),to avoid side effects caused by osmotic changes when incubating thejuice-sac slices for the sugar uptake experiments. Molality of the crudegrapefruit juice-sac homogenate was measured by an osmometer andwas found to be ∼500 mosmol. Given the high acid content by the acid,released through the open cut surfaces of juice sacs (1.8–2.0%), and theneed to maintain 500 mosmol of the incubation medium, pre-incubation

In vitro sugar uptake by grapefruit juice sacs Functional Plant Biology 359

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Fig. 1. Flow system for preparing juice sacs for [14C]-sugar uptake and determination of 14CO2 evolution. I. Grapefruit fruit (A) waspeeled (B), the locular membranes were removed (C), and the segments were cut longitudinal from top and bottom (D). Six slices (2–3 mmthick, 8–10 mm wide) were cut from each segment (E), and each one of the slices (F) was transferred into a 25-mL Erlenmeyer flaskcontaining the pre-incubation buffer. Each treatment consisted of six such replicates. II. The flasks, containing four slices from four differentfruits, were gently agitated and the juice-sac slices were washed three times (5 min each washing). III. The pre-incubation buffer wasreplaced by the same buffer containing antibiotics, labelled and unlabelled sugar and ± inhibitor. The juice sacs slices were incubated forthe required time. IVa. After incubation, the juice-sac slices were transferred into new Erlenmeyer flasks, washed with ice-cold pre-incubationbuffer and extracted for [14C]-sugar uptake. IVb. The Erlenmeyer flask, containing only the incubation buffer, was re-sealed with the sameserum stopper (including the inserted needle holding the filter paper discs), and the buffer was acidified with 20 mL H2SO4 (12 N) to releaseall 14CO2 trapped in the incubation buffer.

buffer included: 250 mM MES buffer (pH 5.6); 150 mM of mannitol; and50 mM CaCl2, to maintain the integrity of the juice sac cell membranes.

Procedure for the sugar uptake experiments

Juice sacs (190–230 mg for each replicate) were separately pre-incubated in 25-mL Erlenmeyer flasks containing 5 mL of pre-incubation buffer. The flasks were gently agitated for 5 min; thebuffer was removed by mild vacuum pressure and replaced witha fresh 5 mL of the same buffer. The flasks were then gentlyagitated again for an additional 5 min (Fig. 1II). After the thirdwash, the pre-incubation buffer was removed and replaced with5 mL of similar buffer (pH 5.6) containing ampicillin, neomycin,mycostatin to avoid microbiological contamination (20 µg mL−1 each),0.5 mM of either 9.15 kBq U-[14C] glucose (10.73 MBq mmol−1)or 9.15 kBq U-[14C]-fructose (10.73 MBq mmol−1) or 9.15 kBq ofsucrose-[14C]-glucose (10.73 MBq mmol−1), or 9.15 kBq U-[14C]-sucrose (22.76 MBq mmol−1). When required, carbonylcyanidem-chlorophenylhydrazone (CCCP), or sodium azide (NaN3) was addedinto the incubation media at the beginning of the experiment. Theflasks were than agitated in a water bath shaker at 25◦C in the dark forthe required time (Fig. 1III).

To determine whether wound ethylene, as well as ethylene due tomannitol in the medium (Riov and Yang 1982) interfered with the

results, juice-sac slices were incubated in the incubation medium for3.5 h and were simultaneously exposed to 10 µL L−1 ethylene. Theresults showed that 10 µL L−1 ethylene did not interfere with themeasurements of 14CO2 evolution during the incubation time period(data not shown).

Determination of 14 CO2 evolution

In order to determine the 14CO2 evolution, a sealed needle wasinserted through a rubber serum stopper to which three WhatmanNo. 1 (Whatman, Kent, UK) filter paper discs (9 mm in diameter)were attached. The filter paper was saturated with 25 µL soluene-350as a 14CO2 trap and sealed into each flask (Fig. 1IVb). At the endof the incubation period, as described above, the serum stopper withthe inserted needle holding the filter paper discs was removed. Theincubation buffer (containing 14CO2 in the form of H14

2 CO3), withoutthe juice-sac sections, was transferred into a new 25-mL flask andresealed with the same serum stopper. Aliquots (2 mL) of 12 N H2SO4

were injected through the attached needle into the incubation solution.The needle was immediately re-sealed and the flasks incubated for30 min in a water-bath shaker at 25◦C in the dark. Following thissecond incubation, the filter paper discs were transferred into a 20-mLscintillation vial containing 10 mL of scintillation solution (Ultimagold, Packard BioScience, Meriden, CT). The vials were shaken


for a minimum of 3 h before the trapped 14CO2 was determined byscintillation spectroscopy (Fig. 1IVb). The original Erlenmeyer flaskscontaining the juice sacs without the incubation media, were keptfor 1–2 min on ice for further analysis of [14C]-sugars uptake. Thedata of 14CO2 evolution were calculated and presented in the figuresas nmol.

Determination of [14 C]-sugar uptake into the juice cells

It was necessary to establish a procedure that would remove [14C]-sugarsfrom the surface of the juice-sac slices and from the cellular free space(apoplast) in order to enable the analysis of only the content of the [14C]-sugars that were taken up by the cells (the cytosol). Figure 2 shows theamount of sugars remaining in juice-sac cell preparations after eachof four successive washings with 10 mL of the sugar free buffer for15–30 s. The tissue had been previously incubated for 3.5 h in either

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Fig. 2. Number of washings required determining the uptake of sugarby juice-sac cells. Juice-sac slices were incubated for 3.5 h witheither 10 mM (A) or 320 mM (B) of the required unlabelled sugarsand either 9.15 kBq of [14C]-glucose (10.73 MBq mmol−1), or [14C]-fructose (10.73 MBq mmol−1), or [14C]-sucrose (22.76 MBq mmol−1).Each experiment consisted of six replications per treatment.

10 mM (Fig. 2A) or 320 mM unlabelled sucrose (Fig. 2B), into which thelabelled sugars were added at the beginning of the incubation period.Based on these data, the juice-sac slices were routinely washed threetimes before the extraction of the labelled sugars that were taken upby the cytosol. After collecting the third washing buffer, the remainingjuice sac slices were extracted with 10 mL of 100% ethanol by grindingwith a mortar and pestle (Fig. 1IVa). The mortar and pestle were rinsedwith additional 5 mL of 100% ethanol and combined with the extractionsolution. The combined solutions were incubated in 50-mL flasks for30 min in an ice bath and shaken for a few seconds every 5 min.The solutions were then filtered through Miracloth and centrifuged(10 min, 10 000 g, 4◦C, Sorvall RC-5C plus). Aliquots (0.5 mL) of thesupernatant were transferred into scintillation vials for the determinationof total radioactivity uptake by the juice sacs (Fig. 1IVa). Care wastaken that each replicate, out of the six replicates for each sugar at eachtime, was incubated for the same period of time to enable calculationof the average value for each measurement. Sugar uptake and 14CO2

accumulation are reported as nmol, based on the specific activity of the14C-labelled sugars.

Results

Sugar uptake and CO2 evolution

In grapefruit, storage of sucrose and hexoses in the juicecells occurs at accelerated rates during the last stages offruit development (Purvis and Yelenosky 1983). In thepresent study, the capacity of the juice-sac slices to take up[14C]-sucrose, as well as [14C]-fructose and [14C]-glucose,and their metabolic decarboxylation to 14CO2 during thesedevelopmental stages was investigated. Figure 3A–C showsthat uptake rates for both glucose and fructose, duringthe early maturity period (mid December through to thebeginning of February) were significantly higher than thatof sucrose. Similar uptake results were reported for Prunuspersico fruit tissue (Vizzotto et al. 1996) from an externalsolution of 0.5 mM sucrose. Both hexoses had an initialuptake peak around December and a second uptake peak inFebruary–March. From March through to April, the rates ofsucrose uptake increased to levels comparable to those ofglucose and fructose.

The utilisation of sugars in mature Citrus fruits, asexpressed by CO2 evolution, was relatively low (compareespecially Fig. 3C with Fig. 3A, B), probably as a result of lowrespiratory rates (Aharoni 1968; Koch and Avingne 1990).The rates of CO2 evolution were significantly higher withglucose as a substrate, while comparatively little CO2 wasproduced when juice-sac slices were supplied with eitherfructose or sucrose (Fig. 3B, C).

The time-course analyses of sugar uptake by juice-sacslice, harvested between December and January (Fig. 4A)indicated that glucose accumulation was significantly higherthan that of fructose and sucrose. Uptake of sucrose andfructose levelled off after ∼6 h, while glucose accumulationwas biphasic, with a second uptake phase starting after 18 hof incubation. Evolution of 14CO2 was similar during the first4–6 h for all three sugars (Fig. 4B). In parallel to sugar uptake,14CO2 evolution from fructose and sucrose reached a steady


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Fig. 3. Rate of sugar uptake and 14CO2 evolution during differentstages of grapefruit maturation. Juice-sac slices were incubated for4–15 h with 0.5 mM of the required unlabelled sugars and either9.15 kBq of [14C]-glucose (10.73 MBq mmol−1) (A), [14C]-fructose(10.73 MBq mmol−1) (B), or [14C]-sucrose (22.76 MBq mmol−1) (C).Values are means ± SE (P<0.05) of six replicates.

state by 6 h of incubation, but that for glucose continued torise up to 42 h of incubation.

Juice-sac sections were incubated with increasing sugarconcentrations for 4.5 h (close to the initial plateau level).Over the entire range of the concentrations from 0.5to 320 mM, uptake of the radiolabel sugars into thecytosol remained constant (decreasing specific activityas concentration of the unlabelled sugars in the mediaincreased). The horizontal regression lines (Fig. 5A) indicatea linear increase in uptake, suggesting that transport isnot saturable, and hence, perhaps, not carrier-mediated. Iftransport is carrier-mediated, the affinity of carrier for sugarsmust be quite low, given that linearity was maintained upto 320 mM external sugar. Figure 5A shows the results for

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Fig. 4. Effect of incubation time on sugar accumulation into grapefruitjuice-sac slices (A) and CO2 production from the sac slices (B). Sliceswere obtained from grapefruit picked during December and January,and incubated for the required time as indicated on the x-axis withglucose (�), fructose (•), or sucrose (�). For other experimental detailssee legend of Fig. 3. Solid lines are fit to logarithmic or polynomialcurves in either second- or third-order. Values are means ± SE (P<0.05)of six replicates.

glucose, fructose, and sucrose accumulation by juice sacsfrom fruit collected in March. Similar results were found forglucose and fructose in fruits collected during mid-December(data not shown). The fact that 14CO2 evolution was alsolinear (Fig. 5B) supports the interpretation that sugar entryinto the juice sacs is insaturable.

Effects of NaN3 and CCCP on sugaruptake and CO2 evolution

To determine whether sugar uptake into Citrus juice cellsis energy dependent, sugar uptake from a 100 mM solution


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Fig. 5. Rates of [14C]-sugar uptake (A) and 14CO2 evolution(B) as a function of concentration of added sugar. For otherexperimental details see the legend of Fig. 3, except that fruitwere picked at the end of December, and incubated for 4.5 h withunlabelled sugars as indicated on the x-axis, with the requiredlabelled sugar. Solid lines are fit to linear curves. The fittedregression equations are (A): glucose (�) y = 0.10x + 34.5, r2=0.041,n = 6, P<0.05; fructose (•) y = 0.094x + 33.6, r2=0.017, n = 6,P<0.05; sucrose (�) y = −0.405x + 10.3, r2=0.65, n = 6, P<0.05.(B): glucose (�) y = −0.01x + 0.59, r2=0.23, n = 6, P<0.05; fructose(•) y = −0.027x + 0.55, r2=0.07, n = 6, P<0.05; sucrose (�)y = −0.064x + 0.23, r2=0.41, n = 6, P<0.05.

was studied in the presence of metabolic inhibitors inthe incubation medium for 4 h. The effect of NaN3 wasstudied (Fig. 6) in an attempt to confirm whether sugars, andespecially sucrose, might serve as energy source in additionto the organic acids. At a concentration of 0.01 mM NaN3,a statistically significant decrease of sucrose uptake wasobserved (Fig. 6). When the juice-sac slices were treated witheither glucose or fructose in the presence of NaN3, a slightbut significant decrease of their uptake was recorded up tothe concentration of 0.05 mM. At higher concentrations ofNaN3, fructose uptake continued to decrease while glucose

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Fig. 6. Effect of NaN3 concentrations on the rates of sugar uptake (A)and CO2 production (B). Experimental details as in Fig. 3, except thatfruits were picked during December and January, and incubated for 4 hwith 100 mM unlabelled sugars. Solid lines are fit to polynomial curvesof second- or third-order. * Represent level of significance of differencebetween sugar uptake in control and treatment by t-test (*, P<0.05;**, P<0.001; ***, P<0.0001). Values are means ± SE of six replicates.

uptake increased even above the level of the untreated control(Fig. 6A). However, evolution of CO2 was markedly reducedby all the concentrations of NaN3 with the all three sugars assubstrates (Fig. 6B).

The effect of the metabolic uncoupler, CCCP, was similarto that of NaN3 (Fig. 7). Hexoses and sucrose uptake wassignificantly reduced by CCCP. Sucrose and fructose uptakesdecreased slightly but significantly by 0.01 mM of CCCP andlevelled off at higher concentrations of the uncoupler. Theuptake of glucose continued to decrease also in the presenceof 1.0 mM of CCCP followed by an increase in uptake whentreated with 10 mM of CCCP (Fig. 7A). CO2 evolution in


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Fig. 7. Effects of CCCP concentrations on the rates of sugar uptake (A)and CO2 production (B). For other details see the legend of Fig. 3, exceptthat fruits were picked during December and January, and incubated for4 h with 100 mM unlabelled sugars. Solid lines are fit to polynomialcurves of second- or third-order. * Represent level of significanceof difference between sugar uptake control and treatment by t-test(*, P<0.05; **, P<0.001; ***, P<0.0001). Values are means ± SE ofsix replicates.

the presence of [14C]-sucrose and [14C]-fructose decreasedsteadily as a result of increasing concentration of CCCP(Fig. 7B).

We investigated further the possibility that sucrose servesas a substrate for respiration by incubating juice-sac sectionsin asymmetrically labelled sucrose and sucrose labelleduniformly only in the [14C]-glucose moiety. The experimentswere conducted in the absence and presence of CCCP.There was an indication that 0.1 mM CCCP caused a slightinhibition of both [14C]-sucrose and [14C]-glucose uptake(Fig. 8A) consistent with the data of Fig. 7A. The effectswere not significant, possibly because of the small sample

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Fig. 8. Effect of CCCP on rates of sugar uptakes (A) and CO2

evolution (B) by juice sacs slices. Juice-sac slices were obtained fromgrapefruit picked at the end of February. Slices were incubated for 4 hin the required unlabelled 0.5 mM sugar and with either 9.15 kBq ofasymmetrically-labelled sucrose–[14C]-glucose (22.76 MBq mmol−1),or symmetrically-labelled [14C]-sucrose (22.76 MBq mmol−1) or [14C]-glucose (10.73 MBq mmol−1). Open bars represent sugars alone, greybars contain additional 10−4 mM CCCP. Values are means ± SE of sixreplicates.

size. In contrast, under similar conditions, 14CO2 evolutionfrom all three labelled substances was strongly inhibited inthe presence of CCCP (Fig. 8B). In the absence of CCCP,the level of CO2 evolution was much higher, when [14C]-glucose was used as substrate, than when the label was in theasymmetrically labelled sucrose.

Discussion

Our results indicate that in juice sacs from mature grapefruitfruit the uptake of sucrose into the cells is insaturable in vitro.Garcia-Luis et al. (1991) also reported that in albedo (thewhite portion of the peel) and juice sacs of developing‘Satsuma’ mandarin sucrose uptake was insaturable upto 300 mM The authors concluded that the uptake wasalmost entirely metabolically independent, and thus passive,


or that the metabolic inhibitors p-chloromercuribenzenesulphonic acid (PCMBS), dinitrophenol (DNP), erythrosineand vanadate were unable to reach the site of any activeaccumulation. In contrast, the uptake of sucrose moieties,fructose and glucose, into the juice sacs of grapefruit fruitwere insaturable, and occur mostly by a �µH+-independentprocess, which was affected by metabolic inhibitors.(Figs 6A, 7A, 8A). The insaturable nature is in agreementwith an endocytosis uptake mechanism recently describedfor ‘sycamore’-cultured cells (Etxeberria et al. 2005a) andfor ‘Murcott’ mandarin juice cells (Etxeberria et al. 2005b).

The uncoupler CCCP, which, at similar concentrationsde-energises electrogenic sugar transport in numerousplant systems (Getz 1991; Echeverria et al. 1997), reducedsugar uptake by the juice cells slightly but significantly,(Figs 6A, 7A, 8A). Inhibitors such as NaN3, had been reportedto reduce sugar uptake in cell suspensions of sugarcane(Maretzki and Thom 1972) and to reduce respirationin Avena fatua seeds (Tilsner and Upadhyaya 1989). InAraucaria araucana, it was suggested that the inhibitionof sucrose uptake induced by NaN3 and other metabolicinhibitors indicates that energy dependent mechanisms areinvolved (directly or indirectly) in sucrose transport (Lozadaand Cardemil 1990). Sucrose uptake by sugar beet andsugarcane cells from solutions containing CCCP was notaffected by the uncoupler and was concluded to occur byfacilitated diffusion (Saftner and Wyse 1980; Wyse et al.1986; Thom and Maretzki 1992), while uptake of sugarsby pecan tissue in the presence of CCCP was consideredto be non-carrier-mediated (Wood 1987). Regen andTarpley (1974) showed that CCCP partly inhibited bothglucose and fructose uptake in peach tissue. Our results(Figs 6A, 7A, 8A) showed a slight but significant reductionin sucrose uptake, when treated with either NaN3 or CCCP(0.01 mM), suggesting some evidence for an active transportsystem of sucrose into the juice-sac cells.

Figure 8A suggests that sucrose is taken up as an intactmolecule, since the uptake of asymmetrically labelledsucrose-[14C]-glucose is much less than with labelled [14C]-glucose alone. This shows that sucrose is not inverted duringthe uptake process (Koch and Avingne 1990), as also reflectedby the difference in CO2 evolution by the two sugars (Fig. 4).Had sucrose-[14C]-glucose or [14C]-sucrose been inverted, itwas expected that the liberated CO2 from the sucrose moietieswould be similar (even higher) to levels obtained when [14C]-glucose alone was supplied. Moreover, the CO2 evolutionthat accompanied sugar uptake suggests they accumulatein the vacuole (Garcia-Luis et al. 1991; Miron et al. 2002),as earlier implied by results of Koch and Avingne (1990),and determined for sugar beet root (Giaquinta 1977; Wyse1979), leaf tissue of pecan (Wood 1987) and sugarcane (Thomand Maretzki 1992). The fact that sucrose uptake levelledoff after 6 h is also an indication that there is no symplasticsucrose hydrolysis before sucrose uptake. If there was sucrose

inversion in the symplast of the juice cells, the resultingglucose would have entered the juice cells, resulting in acontinuous accumulation as with [14C]-glucose (Fig. 4A).This result is supported by the finding that enzyme activities,like those of sucrose synthase and of acid invertase, that arereported to be high in the juice sacs of grapefruit during earlyfruit development, decline markedly during the phase offruit expansion (Lowell et al. 1989). Uptake and metabolismof glucose does not appear to be highly regulated, bothprocesses continuing unabated over the 42 h of incubation.However, the continuous uptake and utilisation of glucosethat was observed in our experiments (Fig. 4A) may bepassive. This is in agreement with Koch and Avingne(1990), since in vivo, sugars arrived into the juice-sac cellsas mostly as sucrose, with very little prior inversion asexplained above.

A portion of each of these sugars is metabolised toCO2 in a process that is strongly affected by NaN3 andCCCP (Figs. 6A, 7A, 8A). The data further indicate that,at least partially, CO2 originated from the TCA cycle aspreviously reported (Goren et al. 2000) after the inversionof sucrose. This is also in agreement with the conclusion ofMurata (1977), who reported that 50% of the radioactivityof injected [14C]-citrate was found in the respired 14CO2 ofcitrus fruit. The possibility that some of CO2 originates fromother metabolic sources, such as amino acids (Murata 1977),the γ -aminobutyric acid pathway (Popova et al. 1995), thepentose phosphate pathway, or the anaerobic conversionof citric acid to ethanol (Davis et al. 1973; Purvis andYelenosky 1983) cannot be ignored. In addition, the data alsodemonstrated that sucrose serves as a respiratory substratein mature Citrus juice-sac cells and establishes a preferencefor glucose as carbon source, as also previously shown(Goren et al. 2000). This explains the consistently higherlevel of fructose in Citrus fruits after internal inversion inthe vacuole (Echeverria and Burns 1989).

Acknowledgments

We acknowledge Professor S Wolf and Dr A Sadka forcritical reading of the manuscript, and Dr P Smirnoff forher suggestions and to Dr H Fut for her statistical assistance.This research was supported by the Israel–USA BinationalAgricultural Research and Development (BARD Project No.IS-2248–93).

References

Aharoni Y (1968) Respiration of oranges and grapefruit harvested atdifferent stages of development. Journal of Plant Physiology 43,99–102.

Aked J, Hall JL (1993) The uptake of glucose, fructose and sucroseinto the lower epidermis of leaf discs of pea (Pisum sativum L.cv. Argenteum). New Phytologist 123, 271–276.

Cardemil L, Varner JE (1984) Starch degradation metabolism towardssucrose synthesis in germinating Araucaria araucana seeds.Journal of Plant Physiology 76, 1047–1054.


Damon S, Hewitt J, Nieder M, Bennett AB (1988) Sink metabolismin tomato fruit. II. Phloem unloading and sugar uptake. Journal ofPlant Physiology 87, 731–736.

Davis PL, Roe B, Bruemmer JH (1973) Biochemical changesin citrus fruits during controlled atmosphere storage. Journalof Food Science 38, 225–229.

Dreyer I, Horeau C, Lemaillet G, Zimmermann S, Bush D,Rodriguez-Navarro A, Schachtman D, Spalding E, Sentenac H,Gaber R (1999) Identification and characterization of planttransporters using heterologous expression systems. Journal ofExperimental Botany 50, 1073–1087. doi: 10.1093/jexbot/50.suppl 1.1073

Echeverria E, Ismail M (1987) Changes in sugars and acids of citrusfruits during storage. Proceedings of the Florida State HorticulturalSociety 100, 50–52.

Echeverria E, Valich J (1988) Carbohydrate and enzyme distributionin protoplasts from Valencia orange juice sacs. Phytochemistry 27,73–76. doi: 10.1016/0031-9422(88)80593-4

Echeverria E, Burns JK (1989) Vacuolar acid hydrolysis as aphysiological mechanism for sucrose breakdown. Journal of PlantPhysiology 90, 530–533.

Echeverria E, Ismail M (1990) Sugars unrelated to Brix changes instored citrus fruits. HortScience 25, 710.

Echeverria E, Gonzalez PC, Brune A (1997) Characterization ofproton and sugar transport at the tonoplast of sweet lime (Citruslimmetioides) juice cells. Journal of Plant Physiology 101, 291–300.doi: 10.1034/j.1399-3054.1997.1010206.x

Etxeberria E, Baroja-Fernandez E, Munoz FJ, Pozueta-Romero J(2005a) Sucrose inducible endocytosis as a mechanism for nutrientuptake in heterotrophic plant cells. Plant and Cell Physiology(In press).

Etxeberria E, Gonzalez P, Pozueta-Romero J (2005b) Sucrose transportinto citrus juice cells: evidence for an endocytic transportsystem. Journal of the American Society for Horticultural Science(In press).

Forney CF, Breen PJ (1986) Sugar content and uptake in the strawberryfruit. Journal of the American Society for Horticultural Science 111,241–247.

Garcia-Luis A, Didehvar F, Guardiola JL, Baker DA (1991) Thetransport of sugars in developing fruits of satsuma mandarin. Annalsof Botany 68, 649–657.

Getz HP (1991) Sucrose transport in tonoplast vesicles of redbeet roots is linked to ATP hydrolysis. Planta 185, 261–268.doi: 10.1007/BF00194069

Giaquinta RT (1977) Sucrose hydrolysis in relation to phloemtranslocation in Beta vulgaris. Journal of Plant Physiology 60,339–343.

Giaquinta RT, Sadler LW, Franceschi VR (1983) Pathway of phloemunloading of sucrose in corn roots. Journal of Plant Physiology 72,362–367.

Goldschmidt E, Koch K (1996) Citrus. In ‘Photo assimilate distributionin plants and crops’. (Eds E Zamski, A Schaffer) pp. 797–823.(Marcel Dekker: New York)

Goren R, Huberman M, Zehavi U, Chen-Zion M, Echeverria E (2000)Sugar utilization by citrus juice cells as determined by [14C]sucrose and [14C] fructose feeding analyses. Plant Physiology andBiochemistry 38, 507–515. doi: 10.1016/S0981-9428(00)00767-1

Griffith SM, Jones RJ, Brenner ML (1987a) In vitro sugar transportin Zea mays L. kernels. I. Characteristics of sugar absorptionand metabolism by developing maize endosperm. Journal of PlantPhysiology 84, 467–471.

Griffith SM, Jones RJ, Brenner ML (1987b) In vitro sugar transportin Zea mays L. kernels. II. Characteristics of sugar absorptionand metabolism by isolated developing embryos. Journal of PlantPhysiology 84, 472–475.

Koch KE (1984) The path of photosynthate translocation into citrusfruits. Plant, Cell and Environment 7, 647–653.

Koch KE (2004) Sucrose metabolism: regulatory mechanismsand pivotal roles in sugar sensing and plant development.Current Opinion in Plant Biology 7, 235–246. doi: 10.1016/j.pbi.2004.03.014

Koch KE, Avingne T (1990) Postphloem, nonvascular transport incitrus. Kinetics, metabolism, and sugar gradients. Journal of PlantPhysiology 93, 1405–1416.

Komor E (1977) Sucrose uptake by cotyledons of Ricinus communis L.:characteristics, mechanism and regulation. Planta 137, 119–131.doi: 10.1007/BF00387548

Komor E, Thom M, Maretzki A (1981) The mechanism of sugaruptake by sugarcane suspension cells. Planta 153, 181–192.doi: 10.1007/BF00384100

Lemoine R (2000) Sucrose transporters in plants: update on functionand structure. Biochimica et Biophysica Acta 1465, 246–262.

Laterveer FD, Gellerich FN, Nicolay K (1995) Macromolecules increasethe channeling of ADP from externally associated hexokinase to thematrix of the mitochondria. European Journal of Biochemistry 232,569–577.

Lowell CA, Tomlinson PT, Koch KE (1989) Sucrose-metabolizingenzymes in transport tissues and adjacent sink structures indeveloping citrus fruit. Plant Physiology 90, 1394–1402.

Lozada R, Cardemil L (1990) Further characterization of sucrose uptakeby cotyledons of Araucaria araucana. Energy requirements andspecificity of the uptake system. Plant Physiology and Biochemistry28, 773–778.

Manning K, Davies C, Bowen HC, White PJ (2001) Functionalcharacterization of two ripening-related sucrose transporters fromgrape berries. Annals of Botany 87, 125–129. doi: 10.1006/anbo.2000.1316

Maretzki A, Thom M (1972) Membrane transport of sugars in cellsuspensions of sugarcane. I. Evidence of sites and specificity.Journal of Plant Physiology 49, 172–182.

Maynard JW, Lucas WJ (1982) Sucrose and glucose uptake into Betavulgaris leaf tissue. Journal of Plant Physiology 70, 1436–1443.

McCready RM, Walter ED, Maclay WD (1950) Sugars of citrus juices.Food Technology 4, 19–20.

McRae SR, Christopher JT, Smith JAC, Holtum JAM (2002) Sucrosetransport across the vacuolar membrane of Ananas comosus.Functional Plant Biology 29, 717–724. doi: 10.1071/PP01227

Miron D, Petreikov M, Carmi N, Shen S, Levin H, Granot D, Zamski E,Schaffer AA (2002) Sucrose uptake, invertase localization and geneexpression in developing fruit of Lycopersicon esculentum andthe sucrose-accumulating Lycopersicon hirsutum. Journal of PlantPhysiology 115, 35–47. doi: 10.1034/j.1399-3054.2002.1150104.x

Murata T (1977) Studies on the post harvest physiology and storage ofcitrus fruit. Journal of the Japanese Society for Horticultural Science46, 283–287.

Picaud S, Becq F, Dedaldechamp F, Ageorges A, Delrot S (2003)Cloning and expression of two plasma membrane aquaporinsexpressed during the ripening of grape berry. Functional PlantBiology 30, 621–630. doi: 10.1071/FP02116

Popova TM, Igamberdiev AU, Velichko YI (1995) Effect of electrontransport inhibitors and some metabolites on [6-14C]-citrateconversions in plants. Russian Journal of Plant Physiology 42,680–686.

Purvis AC, Yelenosky G (1983) Translocation of carbohydrates andproline in young grapefruit trees at low temperatures. Journal ofPlant Physiology 73, 877–880.

Regen DM, Tarpley HL (1974) Anomalous transport kinetics and theglucose carrier hypothesis. Biochimica et Biophysica Acta 339,218–233.


Riov J, Yang SF (1982) Stimulation of ethylene evolution incitrus leaf discs by mannitol. Journal of Plant Physiology 710,142–146.

Saftner RA, Wyse RE (1980) Alkali cation / sucrose co-transport inthe root sink of sugar beet. Journal of Plant Physiology 66,884–889.

Saftner RA, Daie J, Wyse RE (1983) Sucrose uptake andcompartmentation in sugar beet taproot tissue. Journal of PlantPhysiology 72, 1–6.

Sinclair WB (1972) ‘The grapefruit: its composition, physiology,and products’. University of California, Division of Agriculture.Science Press p. 660. Library of Congress Catalog Card Number72–619646.

Thom M, Maretzki A (1992) Evidence for direct uptake of sucrose bysugarcane stalk tissue. Journal of Plant Physiology 139, 555–559.

Tilsner HR, Upadhyaya MK (1989) The effect of pH on the action ofrespiratory inhibitors in Avena fatua seeds. Annals of Botany 64,707–711.

Ting SV, Russell LR (1986) ‘Citrus fruits and their products. Analysis,technology.’ (Marcel Dekker Inc.: New York)

Vizzotto G, Pinton R, Varanini Z, Costa G (1996) Sucrose accumulationin developing peach fruit. Physiologia Plantarum 96, 225–230.doi: 10.1034/j.1399-3054.1996.960209.x

Wood BW (1987) Carbohydrate composition of vascular systemexudates and characterization of their uptake by leaf tissue of pecan.Journal of the American Society for Horticultural Science 112,346–351.

Wyse RE (1979) Sucrose uptake by sugar beet tap root tissue. Journalof Plant Physiology 64, 837–841.

Wyse RE, Zamski E, Tomos D (1986) Turgor regulation of sucrosetransport in sugar beet taproot tissue. Journal of Plant Physiology81, 478–481.

Manuscript received 13 July 2004,, received in revised form 9 February2005, accepted 10 March 2005

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In vitro sugar uptake by grapefruit ( Citrus paradisi ) juice-sac cells

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