Identification and functional expression in yeast of a grape berry sucrose carrier

Identification and functional expression in yeast of a grape berrysucrose carrier§

Agnès Ageorgesa*, Nicolas Issalya, Sarah Picaudb, Serge Delrotb, Charles Romieua

a UR-Biochimie métabolique et technologie, IPV INRA, 2, place Viala, 34060 Montpellier cedex, Franceb Laboratoire de biochimie et physiologie végétales, CNRS (ESA 6161), bâtiment botanique, 40, avenue du Recteur-Pineau,86022 Poitiers cedex, France

(Received September 28, 1999; accepted November 10, 1999)

Abstract — A large increase in hexose accumulation is typical of grape berry ripening. To analyse sugar transport processesduring grape berry (Vitis vinifera L. cv Ugni Blanc) development, a cDNA encoding for a sucrose transporter, designatedVvSUT1 was cloned. Sucrose transport activity of the VvSUT1 protein was demonstrated by heterologous expression in yeast.A study of gene expression along berry development showed thatVvSUT1 transcripts are present at all stages but accumulatewith the onset of ripening. This work shows that sucrose transport at the plasma membrane of grape berry cells may play a rolein the accumulation of sugars during maturation. © 2000 Éditions scientifiques et médicales Elsevier SAS

Development / grape berry / membrane transporter / sucrose / unloading

FCCP, carbonyl cyanide p-trifluoromethyoxyphenyldrazone / FW, fresh weight / RT, reverse transcription

1. INTRODUCTION

Because of the widespread demand for wine, tablegrapes and dried fruit, the common grape,Vitis vin-ifera L., is one of the most economically importantfruit crop in the world. Like a small number of otherfruits (orange, strawberry), the ripening of grapes isnon-climacteric. The onset of ripening has been stud-ied more extensively in climacteric fruits and appearsas a highly regulated developmental process in whichactivation of specific genes takes place [21]. By con-trast, the triggering of ripening in non-climacteric fruitsuch as grapes is still poorly understood, at least at themolecular level [2].

The development of the berry consists of twogrowth periods [2, 14]. During the first phase, theberries increase in size and accumulate organic acidsuntil a plateau is reached. The onset of ripening whichis called veraison, can be detected by an increaseddeformability and by an accumulation of anthocyanins

in red grape varieties. During the second growthperiod, the volume and softness of the berries increase,sugars accumulate and the level of malic aciddecreases. Neoglucogenesis occurs in grape but cannotaccount for the large amounts of sugar accumulated.An exhaustive study based on a C balance at the berrylevel showed that the rate of assimilate influx into theberry rapidly increases by a factor of three at the onsetof ripening [19].

Sucrose is the main carbohydrate used for longdistance transport in grape [12]. Upon its arrival in thephloem of the berry, sucrose can be unloaded into theapoplast, eventually cleaved by an apoplastic inver-tase, and sucrose and/or hexoses can then be taken upby the mesocarp. It is also possible that for quite a longperiod during berry development, symplastic connec-tions between the sieve tubes and the mesocarp cellsremain [19]. Once in the cytoplasm of the mesocarpcells, sucrose and hexoses must be transported bytonoplast carriers since hexoses accumulate in thevacuole. The presence of a very active acid invertasein the vacuole [23, 31] would favour the transport ofthe disaccharide into the vacuole. However, it was

* Author to whom correspondence should be addressed (fax +33 4 99 61 28 57; e-mail [email protected])

§ The accession number for the sequence reported in thispaper is AF182445.

Plant Physiol. Biochem., 2000,38 (3), 177−185 / © 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reservedS0981942800007300/ADD

Plant Physiol. Biochem., 0981-9428/00/3/© 2000 E´ ditions scientifiques et médicales Elsevier SAS. All rights reserved

recently shown that a hexose transporter located at theplasma membrane is expressed during the ripeningprocess, suggesting that at least part of the sucroseimported by the berry is hydrolysed prior to accumu-lation in the flesh cells [7]. Finally, glucose and fruc-tose accumulate in roughly equal amounts in thevacuole, up to 0.5 M each at maturity [8]. Whateverthe route taken, transport of sugars through the differ-ent membranes is a possible regulatory checkpoint forthe accumulation of sugar during berry development.

The molecular events associated with the control ofripening are just beginning to be studied in grapeberries. Transcripts encoding for two putative vacuolarinvertases are present early in the development of theberries, and their expression decreases rapidly afterveraison [3]. In vitro acid invertase activities exceedby 100–200-fold the rate of hexoses accumulationduring ripening [31], and considerable invertase activ-ity can be detected largely before sugar accumulationbegins [6]. More recently, we have shown that twoputative hexose transporters display an increasedexpression during the sugar accumulation period [7].This induction may be part of a controlling pathwayinvolved in the onset of ripening. The present paper isa follow-up study of previous work on sugar transport-ers [7] and addresses the characterisation and theexpression pattern of sucrose carrier in grape berriesduring their development.

2. RESULTS

2.1. Molecular cloning of sucrose transportercDNA

Two highly conserved motifs, FGWALQL andDTDWMG, were identified by amino acid sequencealignment of sucrose/H+ symporters already cloned inplants. Degenerated primers deduced from these pep-tide sequences and first-strand cDNA made frommature berries were used to amplify a DNA fragmentof about 820 bp, which was subcloned and sequenced.A comparison of the deduced amino acid sequence ofthis PCR product with the corresponding fragments ofplant Suc/H+ symporters revealed 52–73 % similarityat the protein level. The complete cDNA, namedVvSUT1 was then produced using 5’ - and 3’ -RACEPCR techniques. The entire sequence of the SUT1clone is shown in figure 1. The open reading frame forVvSUT1 is 1 503 bp long encoding for a protein of501 amino acid residues with a predicted molecularmass of 54 kDa and an isoelectric point of 9.3. Theclone starts 92 bp upstream from the first ATG. DNA

sequence upstream of the start ATG does not containother ATG codons, and possesses a TAG stop codonwithin the VvSUT1 translation frame. This confirmsthe start ATG given in figure 1. The sequence ofVvSUT1 ends with 15A, which could be the start of apoly(A) tail. The deduced amino acid sequence ofVvSUT1 shares 81.2, 55 and 52.3 % identical aminoacids with DcSUT1 [28], RsCR1 [33] and PmSUC1[10], respectively. No significant homology was foundto other proteins in the database, except with one clonecalled ‘ fruit-ripening-related gene’ found in straw-berry (accession No. AF041408 [17]).

Hydropathy analysis of the deduced polypeptidesuggested the presence of twelve transmembrane-spanning domains, which could be resolved into twoparts of six hydrophobic loops each, separated by alarge central hydrophilic segment of about forty aminoacids (figure 1). This model is consistent with thestructure proposed for the mono- and disaccharidetransporters identified so far [18, 26]. The regions ofhighest conservation are the membrane-spanningdomains, whereas the major differences are located inthe N-terminal sequence preceding the first potentialmembrane-spanning domain and in the large hydro-philic loop in the centre of the protein. The motif(R/K) X2/3 (R/K) was found in the hydrophilic loops 3(RFGRR) and 9 (KLCKK). This motif is a conservedstructural feature among members of the major super-family of transmembrane facilitators [18]. There is oneconsensus sequence for N-glycosylation found inVvSUT1 (Asn-151), but the location of this Asn-X-Thr consensus sequence within a putative transmem-brane region suggests that this asparagine is notglycosylated in the mature protein. A phylogenetic treewas constructed based on the complete amino acidsequences of VvSUT1 and twelve other sucrose trans-porters. The thirteen sequences appear divided intoseveral subclasses that may relate to either functionaldifferences or differences in localisation (figure 2).VvSUT1 was found to be very close to DcSUT1(accession No. Y16766 [28]) and these two sequencesare slightly different from the other plant Suc/H+

symporters sequences.

2.2. Functional expression of VvSUT1 in yeast

The entire VvSUT1 cDNA was cloned into the yeastexpression vector NEV-N [27] in both the sense andantisense orientation, downstream of the Saccharomy-ces cerevisiae PMA1 promoter, giving the plasmidsNEV-6S (sense construct) or NEV-10AS (antisenseconstruct). NEV-6S and NEV-10AS (which was usedas control for sugar uptake experiments) were trans

178 A. Ageorges et al.

Plant Physiol. Biochem.

Figure 1. Nucleotide and deduced amino acid sequence of VvSUT1 DNA. Nucleotides are numbered on the right. The translational stop codon ismarked by an asterisk (*). Putative transmembrane segments are underlined. The restriction sites for EcoR1, BamHI, XhoI and HindIII are indicatedon the sequence.

Sucrose transporter from grape berry 179

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ferred into S. cerevisiae strain Σ22574d [13]. Trans-port studies were performed in the presence of high(10 mM) glucose concentration in order to inactivatethe yeast extracellular invertase [27]. Figure 3 Ashows that the yeast strain Σ22574d-NEV-6S was ableto transport sucrose at a high rate across the yeastplasma membrane, whereas only a residual uptake ratewas seen in the Σ22574d-NEV-10AS antisense strain.The addition of the proton gradient uncoupler FCCPimmediately stopped the sucrose transport activity ofthe transgenic yeast (figure 3 B), suggesting thatsucrose uptake is linked to the proton electrochemicalgradient across the plasma membrane.

2.3. Genomic analysisTotal genomic DNA was digested with six different

restriction enzymes and probed with the full-lengthSUT1 clone. DNA gel blot analysis performed underhigh-stringency conditions (0.2 × SSC, 65 °C)revealed a simple pattern of few bands per lane(figure 4). There was no restriction sites for SalI andXbaI in the cDNA probe, and only a single band wasdetected from these digestions. The other restrictionsenzymes, BamHI, XhoI and HindIII, which cut onlyonce within the probe, produced two or three frag-ments hybridising to the cDNA probe. Digestion withEcoRI, which cuts twice in VvSUT1, resulted in fivebands, two of which were poorly labelled. This South-ern blot analysis suggests that VvSUT1 is present inlow copy number in the V. vinifera genome and that itdoes not constitute part of a large multigenic family.

2.4. Grape berry ripening: sugar accumulationand accumulation of VvSUT1 transcripts

Glucose and fructose accumulation started at7 weeks after anthesis (figure 5 A). The concentrationof both hexoses increased from 15 to 81 mg⋅g–1 FWbetween week 7 and 15 in the berries, whereas thesucrose concentration remained lower than 12 mg⋅g–1

FW. There was three to four times more glucose thanfructose present in green berries (before veraison) asalready documented in Findlay et al. [8]. By contrast,

Figure 2. Comparison of sucrose transporters based on the degrees ofsimilarity of their amino acid sequences. The phylogenetic tree wasconstructed with the program PHYLIP. VvSUT1 was aligned with thecorresponding sucrose transporters as follows: Arabidopsis(AtSUC1S, No. X75365; AtSUC2S, No. X75382), Ricinus communis(RcSCR1, No. Z31561), potato (StSUCTR, No. X69165), tobacco(NtSUT1A, No. X82276), Plantago major (PmSUC1, No. X84389;PmSUC2, No. X75764), spinach (SOs21, No. X67125), fava bean(VfSUT1, No. Z93774), sugar beet (BvSUT1, No. X83850), carrot(DcSUT1, No. Y16766; DcSUT2, No. Y16768).

Figure 3. Uptake of sucrose by S. cerevisiae cells transformed withVvSUT1 in sense (● ) respectively antisense (·) orientation. Labelledsubstrate was added to the yeast suspension at time-point zero. A,Incubation in 0.25 mM sucrose to the yeast suspension; B, sameincubation with addition of 0.1 mM FCCP (final concentration) at10 min to the yeast suspension transformed with sense construct (▲).For all experiments, D-glucose (10 mM) was added in the incorpora-tion medium 5 min prior to the radiolabelled sucrose.

Figure 4. Southern hybridisation of genomic DNA isolated from theleaves of V. vinifera (cv Ugni blanc) plants. The DNA (10 µg per lane)was restricted with: lanes (1) EcoRI, (2) BamHI, (3) SalI, (4) XhoI, (5)XbaI, (6) HindIII, prior to separation in a 0.8 % agarose gel, and[32P]-labelled VvSUT1 cDNA was used for hybridisation.



the glucose/fructose ratio rapidly approached the oneafter the onset of ripening, when hexoses are accumu-lated at a high rate, which is typical for sucrolysis byan invertase. The sugar concentration reached 1 M perberry at the end of ripening, and sucrose accounted foronly 7 % of the total sugars of mature grape berry(figure 5 A).

The expression level of VvSUT1 was too low to bedetected by direct northern blotting. We used quanti-tative PCR as an alternative method to determine therelative amount of VvSUT1 mRNA. Control experi-ments showed that only a single band of the expectedsize was amplified when using a cDNA-templatetranscribed from total RNA and sequence-specificprimers for SUT1. Moreover, only a single DNA band

of the expected size was also obtained when usingsequence-specific primers for �-tubulin, ADH andhexoses transporters whereas the correspondinggenomic sequences contained introns (not shown). Therelevance of RT-PCR for quantitative determinationwas assessed with �-tubulin mRNA. Its relative abun-dance was estimated either with northern blot or afterRT-PCR using �-tubulin-specific primers. �-TubulinmRNA accumulated throughout the development ofthe berry and this accumulation was equally quantifiedby both methods (not shown). The VvSUT1 geneturned out to be expressed at any stage of the berrydevelopment (figure 5 C), but the transcripts showedhigher accumulation from the veraison onward (fig-ure 5 B). Three independent RT-PCR reactions wereperformed using two different batches of RNA andyielded similar results.

3. DISCUSSION

The results presented above provide the first evi-dence for the presence of a sucrose transporterexpressed in a sink tissue of a lignous plant. Thesucrose transporter described in this report was iso-lated from grape berries, an organ that accumulates upto 1 M of hexoses in the vacuole of the pericarp duringthe ripening process. The VvSUT1 cDNA sequenceshares high homologies with other plant sucrose trans-porters identified so far [10, 22, 27, 33]. Among thevarious sucrose transporters, VvSUT1 was found to bevery close to DcSUT1 (81.2 % homology) [28]. Thesetwo sequences are slightly different from other plantsucrose/H+ symporters sequences identified so far.However, transporters with similar biochemical char-acteristics (e.g. transporters with a neutral pH opti-mum) were not grouped together in the phylogenetictree (figure 2). Therefore, it is difficult to draw aconclusion from the fact that VvSUT1 (found in grapeberries, a sink organ) and DcSUT1 (expressed mainlyin carrot mature leaves, a source organ) are closetogether.

When the cDNA was expressed in yeast, it con-ferred the ability to accumulate [14C]-sucrose, provid-ing evidence that this gene encodes for a functionalsugar transporter in plants. The protonophore FCCPinhibited sucrose transport completely, suggesting thatVvSUT1 mediates active transport and probablyH+/sucrose symport. This is in accordance with func-tional data from other studies indicating that sucrose istransported together with protons [1, 5].

VvSUT1 may not be the sole sucrose transporterexpressed in the grape berry. However, Southern plot

Figure 5. Quantitative PCR analysis of sucrose transporter expres-sion during grape berry development. Berries were sampled from 1 to15 weeks post-flowering. A, Weight and sugar content of the berries;B, corresponding autoradiograms showing the signals obtained withthe VvSUT1 probe; C, signal intensity obtained with sucrose trans-porter probe expressed in arbitrary units (AU) on a per microgram ofRNA basis. Intensities are expressed as percentages of the maximalvalue detected on the hybridisation membrane. The time of veraison isindicated by a dashed line.


vol. 38 (3) 2000

analysis revealed that the VvSUT1 gene was present inlow copy number and that under our hybridisationconditions, the full-length probe is specific for only asingle transcript. Accordingly, in other species inwhich gene families of sugar transporters have beenidentified, high-stringency hybridisation using full-length cDNA probes has also been shown to bespecific for only one member [34].

VvSUT1 encodes for a sucrose transporter expressedat all stages of berry development. Nevertheless,quantitative PCR analysis revealed an increased accu-mulation of VvSUT1 transcripts at the onset of ripen-ing, which remained until fruit maturity. RT-PCR canbe used for quantitative purpose first because (a) theRNA source was not contaminated by genomic DNA,and (b) the quantitative information obtained matchedthat obtained with northern blots when working withother probes such as �-tubulin or ADH. The increasedexpression of VvSUT1 occurs a few weeks before themaximal expression of VvHT1, a grape berry hexosetransporter recently cloned [7]. The fact that autorad-iographic signals can be detected only after RT-PCRindicates that the VvSUT1 transcripts represent only asmall proportion of total berry RNA and suggests thatthey are present only in a limited number of cells.Increase in VvSUT1 transcript level at veraison wouldsuggest a transcriptional control (but see [30]). It isnow crucial to analyse the VvSUT1 promoter in orderto see whether it contains sugar responsive boxes asfound in the promoting regions of many genesinvolved in sucrose metabolism [15] and shares com-mon cis-sequences with VvHT1 [7] and the berryalcohol dehydrogenase gene [25].

Assimilates can follow different routes to the vacu-oles of storing cells. The presence in the grape berry ofa sucrose transporter is consistent with the low activityof the cell wall invertase [23] and the considerableactivity of vacuolar invertase [3, 6]. This suggests thatat least part of the sucrose imported by phloem is nothydrolysed prior to accumulation in the flesh cells.Moreover, because sucrose is much more concentratedin the phloem cells than in the vacuoles of the fleshcells, its transport may be easier from a thermody-namical standpoint.

An alternate hypothesis for hexose accumulation ingrape berries has been proposed that involves thebreakdown of apoplast/symplast compartmentation atthe veraison. In this proposal, the plasma membrane ofthe pericarp cells becomes leaky and phloem sapmoves more rapidly into the berry due to the differencein water potential between the source and thesink [16]. Our results do not support this hypothesis

since a sucrose transporter (this report) and a hexosetransporter [7] are still expressed at the plasma mem-brane throughout the ripening process. A spatial analy-sis of the expression of these transporter genes wouldfurther clarify the picture.

During the final editorial processing of this manu-script, Davies et al. published the characterisation ofthree putative sucrose transporters in grape berry (cvShiraz) [4]. Among the three clones presented, one isidentical (at the amino acid level) to VvSUT1. Incomplement to our analysis, they showed that tran-scripts for the three clones identified (includingVvSUT1) are present in the berry throughout develop-ment and that expression of these clones is notrestricted to the berry.

4. METHODS

4.1. Plant material

Berries from Vitis vinifera L. cv Ugni Blanc werecollected from the Domaine du Chapître, Inra(Villeneuve-les-Maguelone, France). Their diameterand weight were measured weekly. Diameter classeswere defined at each harvest and berries falling into amedian class (comprising 50 % of the berries) wereselected. They were frozen in liquid nitrogen andstored at –80 °C.

4.2. Sugar extraction and analysis

The frozen powder was homogenised in EtOH/H2O(80 %, v/v) and boiled for 10 min at 80 °C to inacti-vate invertase activity. The solution was centrifuged at15 000 × g for 5 min, and sugars in the supernatantwere measured by high performance anion-exchangechromatography as described in Ollé et al. [20].

4.3. Isolation of cDNA clones for Suc/H+

symporters

Total RNA were extracted from grape berry tissuesas described in Fillion et al. [7]. First strand cDNAswere generated with RNase H-reverse transcriptase(Superscript II, Life Technologies) according to themanufacturer’s instructions in a volume of 20 µL,using 4 µg of post-veraison total RNA as template. A3’ -adapt primer (Life Technologies) was used in thepreparation of first strand cDNA for cloning theinternal cDNA (nucleotides 201–1018, figure 1), andthe 3’ -RACE-PCR product (nucleotides 894–1750,figure 1). The primer S-5 (described below) was used



to prepare the first strand cDNA used to generate the5’ -RACE-PCR product (nucleotides 1–539, figure 1).

4.3.1. Internal cDNA

Based on the alignment (ClustalW [32]) of aminoacid sequences of various plant sucrose transporters,degenerated primers were designed in conservedregions. The forward primer used was 5’ -GCNGC(C/T)GG(G/A/T)(A/G)TNCA(A/G)TT(C/T)GG(G/T)TGGGC-3’ and the reverse primer was 5’ -CCCATCCA(A/G)TC(A/T)GT(A/G)TC-3’ ; these correspond to theconserved amino acid sequences AAG(I/M/V)Q(F/L)GWA (residues 201–226 on the VvSUT1 sequence,figure 1), and DTDWMG (residues 1002–1018),respectively. PCR reactions contained 2 µL first strandcDNA product as a template in a final volume of25 µL, buffered according to the manufacturer’sinstructions. The thermocycling regime used was asfollows: 1 cycle 96 °C 5 min, 48 °C 1 min, 72 °C1 min 30 s, followed by 36 cycles of 92 °C 1 min,48 °C 1 min, 72 °C 1 min 30 s. The reaction was thenterminated by a final extension reaction at 72 °C for8 min.

4.3.2. RACE products

RACE-PCR was used to obtain the 5’ - and 3’ -endsof the final cDNA. 5’ -RACE was conducted using a5’ -RACE System kit (Life Technologies), and3’ -RACE was done as described by Frohman et al. [9].The primers were as follows: SUT1 5’ -RACE primer,5’ -AGCCACATCCAATAACC-3’ (nucleotides 523–539); 3’ -RACE primer, 5’ -TTTCTTTGGGAACTATTTGGG-3’ (nucleotides 894–914).

4.3.3. Total cDNA

The total cDNA sequence of a grapevine sucrosetransporter (VvSUT1) was obtained by combination ofthe information contained in the 5’ - and the 3’ -RACEproducts. The forward primer (defined on the5’ -RACE product) and the reverse primer (defined onthe 3’ -RACE product) were respectively 5’ -CCCGCAAGGCCGCAACTCACC-3’ and 5’ -TACTTGTTAACATTTTACCACCC-3’ (nucleotides 61–81 and1642–1664, figure 1). The PCR reaction was done asdescribed above. The thermocycling regime used wasthe same as the one used to amplify the internalfragment except that the annealing temperature was51 °C. PCR products were purified from agarose gels,ligated into pGEM-Teasy vector, and transferred intoDH5α cells. Double-stranded plasmid templates weresequenced using the Sanger method [24]. Cycle

sequencing was performed by dye deoxyterminatortechniques with the kit protocols from Applied Bio-systems. Sequences were analysed with the automatedApplied Biosystems model 373A DNA sequencingmodel.

4.4. DNA extraction and Southern blot analysis

For Southern blot analysis, genomic DNA wasextracted from young green leaves of Vitis vinifera L.(cv Ugni Blanc) according to Steenkamp et al. [29].Genomic DNA was totally digested with the restrictionenzymes, separated on a 0.8 % agarose gel containingTBE buffer, transferred onto a hybond-N membrane in20× SSC, and hybridised with the radiolabelled cDNAfragment (fragment 89–1605, figure 1). Blots wereperformed in 5× SSPE, 0.5 % SDS, 5× Denhardt’ssolution, 100 µg⋅mL–1 salmon sperm DNA for 16 h at65 °C. Blots were washed for 10 min at room tempera-ture in 2× SSC, 0.1 % SDS, then twice with 0.2× SSCplus 0.1 % SDS at 65 °C for 20 min.

4.5. Quantitative PCR analysis

Total RNA (5 µg) extracted from berries harvestedat different stages of development was reverse tran-scribed and amplified as described previously [7]. Theprimers used to amplify the SUT1 fragment were S1(5’ -TGGATTGGATGGTTTCC located in the codingregion of VvSUT1) and S2 (5’ -TACTTGTTAACATTTTACCACC located in the 3’ -non-coding region ofVvSUT1). Sixteen cycles of amplification were run inthe following conditions: 92 °C for 60 s, 50 °C for 40 sand 72 °C for 90 s. With this number of cycles,amplification occurs in the linear range and allowsgood amplification of amplified products. As control,�-tubulin-specific primers were designed to amplify a659-bp DNA fragment from cDNA. The conditions forquantitative PCR were the same as described forSUT1, except that only nine cycles of amplificationwere carried out. Control experiments demonstratedthat the amplification reactions were still in the expo-nential phase. The amplification products were sepa-rated on an agarose gel and blotted onto nylon mem-branes. The membranes were subsequently hybridisedunder stringent conditions (final wash in 0.1× SSC,0.1 % SDS at 65 °C), with a radiolabelled SUT1-cDNA probe (fragment 1349–1656, figure 1) and a�-tubulin probe. Signals on the hybridisation mem-branes were quantified with a phosphor imager (Storm860, Molecular Dynamics) and Imagequant software(Molecular Dynamics).


vol. 38 (3) 2000

4.6. Functional expression of cDNA in yeast

For the expression of VvSUT1 cDNA in yeast cells,a DNA fragment corresponding to the open readingframe of the VvSUT1 clone was isolated by PCR. PCRprimers for the sense construct were designed byintroducing a NotI site upstream of the translationinitiation codon ATG and a NotI site beyond the stopcodon. The following primers were used5’ -TAGCGGCCGCATGGCGGTCCCTGGGGGCCATCG-3’ and 5’ -AAGCGGCCGCAATATAATACCTCATGTGTGG-3’ . Amplification was achieved withthe following conditions: 30 cycles of denaturation at93 °C for 1 min, annealing at 54 °C for 1 min, andelongation at 72 °C for 2 min. The PCR fragmentsobtained were cleaved with NotI and ligated into thecorresponding restriction sites of the E. coli/S. cerevi-siae shuttle vector NEV-N [27]. The resulting plas-mids NEV-6S and NEV-10AS carry their inserts insense orientation and in the antisense orientation,respectively. The yeast strain Σ22574d [13] whichlacks URA3 gene for selection of recombinant cells,was transformed with the cDNAs in sense or antisenseorientation according to the method of Gietz andSchiestl [11]. Yeast cells were plated on SD mediumcontaining 2 % glucose. After 3 d of incubation at30 °C, colonies were picked and replated on solidifiedminimal medium with 2 % sucrose.

4.7. [14C]-Suc uptake studies

Yeast cells were grown in minimal medium to thelogarithmic phase, harvested by centrifugation,washed with 50 mM sodium phosphate buffer(pH 5.0), and resuspended in the same buffer. Sucroseuptake was determined as described by Riesmeieret al. [22]. Briefly, 200 µL cell suspension(108 cells⋅mL–1) were incubated at 30 °C in 0.25 mMsucrose containing 1.85⋅104 Bq [U-14C]-sucrose.Samples were loaded on glassfibre filters and washedthree times with 5 mL ice-cold phosphate buffer. Theradioactivity taken up by the cells was determined byliquid scintillation counting. All transport experimentswere performed at least twice with similar results.

Acknowledgments

We thank Dr Rémi Lemoine (University of Poitiers,France) for the yeast expression vector (NEV-N), theyeast strain Σ22574d, and helpful comments on theSuc uptake studies. This work was supported by‘Action Incitative sur Programme Institut National de

la Recherche Agronomique Aptitude au Développe-ment des Grains et des Fruits’ .

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vol. 38 (3) 2000

Documents

Identification and functional expression in yeast of a grape berry sucrose carrier