The Plant Cell, Vol. 12, 1153–1164, July 2000, www.plantcell.org © 2000 American Society of Plant Physiologists

SUT2, a Putative Sucrose Sensor in Sieve Elements

Laurence Barker,

a

Christina Kühn,

a

Andreas Weise,

a

Alexander Schulz,

b

Christiane Gebhardt,

c

Brigitte Hirner,

a

Hanjo Hellmann,

a

Waltraud Schulze,

a

John M. Ward,

a

and Wolf B. Frommer

a,1

a

Plant Physiology, Zentrum für Molekularbiologie der Pflanzen (ZMBP), Universität Tübingen, Auf der Morgenstelle 1,D-72076 Tübingen, Germany

b

Department of Plant Biology, Royal Veterinary and Agricultural University, KVL, Thorvaldsensvej 40, DK-1871Frederiksberg C, Copenhagen, Denmark

c

Max Planck Institut für Züchtungsforschung, Carl von Linné Weg 10, 50829 Cologne, Germany

In leaves, sucrose uptake kinetics involve high- and low-affinity components. A family of low- and high-affinity sucrosetransporters (SUT) was identified. SUT1 serves as a high-affinity transporter essential for phloem loading and long-dis-tance transport in solanaceous species. SUT4 is a low-affinity transporter with an expression pattern overlapping thatof SUT1. Both SUT1 and SUT4 localize to enucleate sieve elements of tomato. New sucrose transporter–like proteins,named SUT2, from tomato and Arabidopsis contain extended cytoplasmic domains, thus structurally resembling theyeast sugar sensors SNF3 and RGT2. Features common to these sensors are low codon bias, environment of the start

codon, low expression, and lack of detectable transport activity. In contrast to

LeSUT1

, which is induced during thesink-to-source transition of leaves,

SUT2

is more highly expressed in sink than in source leaves and is inducible by su-crose. LeSUT2 protein colocalizes with the low- and high-affinity sucrose transporters in sieve elements of tomato pet-ioles, indicating that multiple SUT mRNAs or proteins travel from companion cells to enucleate sieve elements. The

SUT2

gene maps on chromosome V of potato and is linked to a major quantitative trait locus for tuber starch contentand yield. Thus, the putative sugar sensor identified colocalizes with two other sucrose transporters, differs from themin kinetic properties, and potentially regulates the relative activity of low- and high-affinity sucrose transport into sieveelements.

INTRODUCTION

Sucrose, the major product of photosynthesis in matureleaves, is loaded into the vascular tissue for translocation toheterotrophic tissues to support their growth. In solana-ceous plants, SUT1 is essential for phloem loading intosieve elements (Riesmeier et al., 1994; Kühn et al., 1996;Bürkle et al., 1998). SUT1 serves as a high-affinity trans-porter for sucrose (

K

m

z

1 mM; Riesmeier et al., 1993),whereas SUT4, with a

K

m

of

z

11 mM, is a low-affinity su-crose transporter (Weise et al., 2000). Both proteins colocal-ize in sieve elements (Kühn et al., 1997; Weise et al., 2000).Localization of SUT1 protein in sieve elements and SUT1mRNA at the orifices of plasmodesmata interconnectingcompanion cells and sieve elements, together with the highturnover of both SUT1 mRNA and protein, indicate that traf-ficking of mRNA or protein occurs from companion cells intoenucleate sieve elements by way of plasmodesmata (Kühnet al., 1997).

Sugar transport is highly regulated, and sucrose-specific

signaling pathways are involved in controlling transport ac-tivity (Chiou and Bush, 1998), potentially by using proteinphosphorylation (Roblin et al., 1998). Overexpression ofpyruvate decarboxylase in potato leads to a 10-fold in-crease in sugar export, demonstrating the capacity to regu-late sugar export from leaves within a wide dynamic range(Tadege et al., 1998). This poses the question of how regula-tion is coordinated between sieve elements that contain thetransporters and companion cells in which transcriptiontakes place (Lalonde et al., 1999).

Yeast, for which the study of transporter regulation ismuch further advanced, may provide hints as to the generalmechanism of regulation of sugar transport. Complex regula-tory networks control uptake of nutrients in response tointracellular needs and a rapidly changing external environ-ment. For metabolization of sugars, yeast has developed atwo-step regulatory system to ensure coordination betweenthe external availability of sugars and the enzymatic machin-ery inside cells: (1) the extracellular concentration of sugarsis sensed, and sugar transport activity is regulated accord-ingly; and (2) sugar transport activity determines the flux ofsugars into the cell, subsequently generating intracellular sig-nals for adapting metabolism (Lalonde et al., 1999).

1

To whom correspondence should be addressed. E-mail frommer@uni-tuebingen.de; fax 49-7071-29-3287.

1154 The Plant Cell

The 18 yeast monosaccharide transporters found thus farspan a wide range of affinities, and thus, depending on theexternal sugar availability, the kinetic parameters of uptakecan be adapted accordingly, for instance, by changing fromlow to high affinity if sugar availability drops (Lalonde et al.,1999; Wieczorke et al., 1999). HXT2 and HXT7, for example,which serve as high-affinity glucose transporters, are in-duced by low concentrations of glucose but are repressedat high concentrations, whereas the low-affinity transporterHXT1 is induced by high concentrations of glucose (Özcanand Johnston, 1995; Boles and Hollenberg, 1997). Conse-quently, the sensors for extracellular glucose must respondnot only to the kind of carbon source but also to its concen-tration.

Glucose is sensed by SNF3 and RGT2, which trigger theinduction of hexose transporter genes based on the glucoseconcentration (Liang and Gaber, 1996; Özcan et al., 1996b).These two proteins resemble glucose transporters but, inaddition, possess unusually long C-terminal domains thatreside in the cytoplasm (Özcan et al., 1996a). The large C-ter-minal extension of SNF3 (303 amino acids) contains twonearly identical repeats of 25 amino acids. One of these re-peats is also present in RGT2. The C-terminal domains ofSNF3 and RGT2 mediate the signaling function of the pro-teins (Özcan et al., 1998; Vagnoli et al., 1998). Deletion ofthese extensions leads to a loss of sensor function. Attach-ment of the SNF3 C terminus to glucose transporters HXT1and HXT2 confers the ability to signal glucose availability.Apparently, SNF3 is a sensor of low glucose concentrationsthat mainly regulates expression of high-affinity glucose trans-porters, whereas RGT2 is responsible for sensing high glu-cose concentrations and regulating expression of low-affinity transporters. Consequently, induction of the high-affinity transporter HXT2 by low concentrations of glucose isabsent in

snf3

mutants, and induction of the low-affinitytransporter HXT1 by high concentrations of glucose isstrongly reduced in

rgt2

mutants. In addition, SNF3 is re-quired for repression of the high-affinity transporters HXT2,-6, and -7 at high glucose concentrations (Liang and Gaber,1996; Vagnoli et al., 1998). Despite the high homology ofSNF3 and RGT2 to glucose permeases, neither appears tobe able to mediate substantial glucose transport (Liang andGaber, 1996; Özcan et al., 1998).

Little is known about sugar sensing and signaling withrespect to transport of sucrose and glucose in plants(Hellmann et al., 2000). Yeast thus may serve as a model toimprove our understanding of the role of sugar transportersin sugar sensing. The existence of plasma membrane–bound sensing pathways has been suggested from physio-logic analyses using nonmetabolizable glucose analogs(Martin et al., 1997; Roitsch, 1999). Furthermore, a geneticapproach has led to identification of sugar-regulated signal-ing cascades that modulate sugar uptake (Hellmann et al.,2000).

To investigate whether plants contain multiple sucrosetransporters present in sieve elements and to identify puta-

tive sugar sensors, we initiated a search for additional su-crose transporter–like genes. A new sucrose transporter–like protein, SUT2, was identified that colocalizes with low-and high-affinity sucrose transporters in enucleate sieve ele-ments of tomato and shares features with yeast sugar sen-sors. Most importantly,

SUT2

is a candidate gene for amajor quantitative trait locus (QTL) for tuber starch and yieldin potato, the genome of which is colinear with that of to-mato (Gebhardt et al., 1991).

RESULTS

Isolation of Sucrose Transporter cDNAs

Two novel

SUT

homologs were identified from tomato bylow-stringency hybridization and reverse transcription–poly-merase chain reaction (PCR).

LeSUT1

is highly similar to

StSUT1

from potato (94% similarity of amino acid se-quences) and appears to be the ortholog of

StSUT1

frompotato (Kühn et al., 1997).

LeSUT2,

with a cDNA of 2144 bp,encodes a 65-kD protein with unique features not reportedpreviously for SUT homologs. SUT2 has an N-terminal do-main

z

30 amino acids longer than the sucrose transportersalready identified (see Lalonde et al., 1999), and its ex-tended central cytoplasmic loop is

z

50 amino acids longer(Figure 1A). According to high-stringency DNA gel blot anal-ysis,

LeSUT2

does not have a closely related paralog in thetomato genome (Figure 2B). Screening bacterial artificialchromosome and expressed sequence tag databases led tothe identification of a putative ortholog of

LeSUT2

in Arabi-dopsis,

AtSUT2

, that contains the same two extended do-mains (Figure 1B). The SUT2s are 82% similar (68%identical), and the genes are structurally related, that is, theintron–exon borders and the number of introns are con-served (Figure 2A). Besides the domains that are conservedin all sucrose transporter–like proteins, LeSUT2 and AtSUT2share highly conserved sequence motifs within the ex-tended central loop, suggesting a functional similarity be-tween AtSUT2 and LeSUT2 as well (Figures 1B and 1C).Similar to the yeast hexose sensors, both SUT2 proteins arecharacterized by low codon bias and low homology withKozak consensus (Kozak, 1996), indicating a low translationefficiency (Iraqui et al., 1999; J.M. Ward, unpublished re-sults).

Expression of

SUT

Genes in Yeast

LeSUT2

and

AtSUT2

were cloned into the yeast expressionvector pDR195 (

LeSUT2

) and pDR196 (

AtSUT2

) for func-tional characterization (Rentsch et al., 1995). LeSUT1 com-plemented the yeast mutant SUSY7/ura3 (Riesmeier et al.,1992), consistent with being the ortholog of StSUT1 (Figure3A), whereas LeSUT2 and AtSUT2 were unable to comple-

Putative Sucrose Sensor 1155

ment the mutant. Similar results were obtained when 5

9

and3

9

untranslated regions of

LeSUT2

were removed and a dif-ferent vector (YEPlac112A1NE) was used (Riesmeier et al.,1992). For all constructs, a variety of complementation con-ditions was tested, including sucrose concentrations be-tween 0.5 and 2% and the pH of the medium ranging from4.0 to 7.0.

To verify expression of LeSUT2, we raised polyclonal anti-bodies against synthetic peptides of LeSUT2 and used themfor protein gel blot analysis. Specificity of the antisera wasverified on membrane proteins isolated from yeast cells ex-pressing LeSUT2. LeSUT2 migrates as a larger protein thanLeSUT1, as their sequences would predict, and appears asa doublet at

z

65/67 kD, possibly because of post-trans-lational modifications in yeast (Figure 3B). No signals weredetected in yeast transformed with the empty vector. Anti-bodies raised against StSUT1 located LeSUT1 in plasmamembranes that had been purified from tomato leavesand showed it to migrate at an apparent molecular mass of46 kD (Figure 3B). Sequences chosen for antisera produc-tion were unique. Lack of cross-reactivity with knownLeSUTs was confirmed by immunolocalization in yeast (Fig-ure 4).

Expression of Sucrose Transporter Genes in Tomato

RNA gel blot analysis was performed to investigate organ-specific expression of

LeSUT1

and

LeSUT2

.

LeSUT1

wasmost highly expressed in source leaves, similar to its ortho-log in potato (Riesmeier et al., 1993), with lower expression

Figure 1.

Sucrose Transporter Sequence Analysis.

(A)

Structural comparison of SUT1 and SUT2.

Hydrophobicity plots(Kyte and Doolittle, 1982) (window of 11 amino acids) of LeSUT1 andLeSUT2 were overlayed. Both contain 12 putative transmembranedomains (labeled with roman numerals I to XII), but SUT2 containsseveral hydrophilic domains not present in SUT1.

(B)

Predicted topology of LeSUT2. All amino acids conserved inboth SUT2 orthologs are shown in black. The central loop regioncontains two highly conserved sequence motifs: central conservedbox (CCB) 1 and 2, which are present only in SUT2 orthologs (under-lined).

(C)

Homology of sucrose transporters. The tree is based on maxi-mum parsimony analysis (Swofford, 1998) for protein sequences ofaligned sucrose transporters from tobacco (NtSUT1 [Bürkle et al.,1998] and NtSUT3 [Lemoine et al., 1999]), potato (StSUT1 [Riesmeieret al., 1993]), tomato (LeSUT1, LeSUT2, and LeSUT4), Arabidopsis(AtSUC1, AtSUC2 [Sauer and Stolz, 1994], AtSUT2, and AtSUT4),and

Schizosaccharomyces pombe

(SpSUT1 [CAB16264]). Percent-age bootstrap values of 1000 replicates are given at each branchpoint. Branch lengths (drawn in the horizontal dimension only) areproportional to phylogenetic distance.

1156 The Plant Cell

in all other organs investigated (Figure 5A). Expression of

SUT2

was low in general but was higher in sink leaves andstems than in source leaves.

Because sucrose transporter activity has been shown tobe regulated by sugars in sugar beet (Chiou and Bush,1998), the regulatory effect of sugars on sucrose transporterexpression was investigated in tomato and potato.

SUT2

was induced in the presence of 100 mM sucrose in sourceleaves from potato.

SUT1

expression remained unaltered,whereas patatin (a storage protein expressed in potato tu-bers) expression was induced primarily by sucrose and to alesser extent by glucose (Figure 5B). In tomato, the sucroseinduction of

LeSUT2

was seen in sink leaves but not insource leaves (data not shown).

Again,

SUT1

expressionwas unaltered in the presence of sucrose.

Colocalization of SUT1 and SUT2 in Tomato Petiolesand Stems

For localization of LeSUT1 and LeSUT2 in tomato, antiserawere affinity purified and used for immunocytochemistry.Both transporters were localized exclusively in sieve ele-ments, as was LeSUT4 (Weise et al., 2000). The presence ofsieve plates, together with the absence of nuclei, allowedunambiguous identification of sieve elements (Figures 6A,6B, and 6D). LeSUT1 was present in sieve elements ofsource-leaf minor veins (Figure 6H), whereas SUT2 was de-tected in the midrib of sink and source leaves but not in mi-nor veins (Figures 6F

and

6G). All three transporters—LeSUT1, LeSUT2 (Figures 6A and 6B), and LeSUT4—colo-calized in mature sieve elements of stem and petiole. Elec-

Figure 2. Genomic Structure of the SUT Loci.

(A) Comparison of the genomic structure of LeSUT2 versus AtSUT2, showing the high conservation of the number of exons (exons 1–14).(B) DNA gel blot analysis of the LeSUTs under high stringency. Genomic DNA from tomato was digested with restriction enzymes, resolved on a1% agarose gel (20 mg/lane), and blotted to Hybond N1. The gel at left shows hybridization with a 32P-labeled 1.3-kb fragment of LeSUT1. Thegel at right shows hybridization with a 32P-labeled LeSUT2 cDNA.

Putative Sucrose Sensor 1157

tron microscopic immunogold labeling clearly showed thatLeSUT2 is present at the plasma membrane (Figure 6J). Pre-immune serum, purified on protein A–Sepharose and usedat the same protein concentration as LeSUT2, caused weakcytoplasmic background labeling, although not at plasmamembranes (Figure 6K).

A series of control experiments was performed to excludeany bias from nonspecific binding. This was necessary be-cause of the preferential binding of immunoglobulins tosieve elements at high concentrations, which was detect-able after long exposures. No immunofluorescence wasdetectable with IgG-enriched purified preimmune serum(Figure 6E), nor after incubation with the secondary antibodyalone (data not shown). Confirmation that purified antiserawere specific resulted from immunolocalization on sphero-plasted yeast cells transformed with transporter genes or anempty expression vector (Figure 4). Localization with a sec-ond antiserum against different domains of SUT1 and SUT2further confirmed results obtained with the first antiserum.Similar results were obtained when tissue was embeddedin acrylic resins after conventional fixation or when tissuewas prepared by high-pressure freezing (data not shown).Antibody–peptide competition experiments also were per-formed to exclude nonspecific antiserum binding. Fluo-rescent signals resulting from SUT2 detection were stronglyreduced or abolished after preincubating the SUT2 antise-rum with its corresponding peptide (Figure 6C). However,preincubation with a peptide corresponding to anotherepitope of the same transporter or to a control peptide hadno effect on SUT2 signals (Figure 6D).

AtSUT2 Expression in Transgenic Plants

Transgenic Arabidopsis plants expressing the reporter genefor

b

-glucuronidase (GUS) under control of a 1.2-kb AtSUT2promoter fragment showed GUS staining in the vascular tis-sue of the petiole and in the major and second-order veinsof source leaves. Among 10 hygromycin-resistant transfor-mant lines, nine showed the same GUS expression. In a fewcases, weak staining of third-order veins was detectable.Staining also was observed in trichomes of source leavesand in hydathodes (Figure 6I). For the longer 2.2-kb pro-moter–GUS construct, only two transformants were ana-lyzed, both showing the same GUS expression pattern asseen for the shorter construct.

Map Positions of

SUT1

,

SUT4,

and

SUT2

Genes in Potato

When used as restriction fragment length polymorphism(RFLP) markers on the potato molecular map, the threecDNA clones each mapped to a separate locus.

StSUT1

identified a locus (

SUT1

) on linkage group XI, whereas

LeSUT4

detected an unlinked locus (

SUT4

) on linkage group

IV.

LeSUT2

identified a single unlinked locus (

SUT2

) on link-age group V (Figure 7).

DISCUSSION

The mechanisms for regulation of sucrose transport duringloading and unloading are not fully understood. Sucrose up-take kinetics determined in plasma membrane vesicles aremultiphasic, suggesting the existence of multiple carrier sys-tems (Lemoine et al., 1996). Theory also predicts additionalcarriers in several different cell types, including sucrose

Figure 3. Expression of LeSUT1 and LeSUT2 in Yeast.

(A) Expression of LeSUT1 and LeSUT2 in SUSY7/ura3. The dish atleft shows that LeSUT1 functionally complements the yeast mutant,SUSY7/ura3 on sucrose (0.5%), whereas LeSUT2 reduced growthbelow that of the empty expression vector. At right, growth on glu-cose (2%).(B) Protein gel blot analysis of LeSUT1 and LeSUT2. At left is shownpurified tomato source-leaf membranes (50 mg) resolved by electro-phoresis. LeSUT1 was detected by using an affinity-purified antise-rum against StSUT1. MM, microsomal membranes; IM, intracellularmembranes; PM, plasma membranes. At right, LeSUT2 was ex-pressed in SUSY7/ura3, and 50 mg of the total membrane proteinwas resolved by electrophoresis. LeSUT2 was detected as a doubletat 66 kD and was absent in yeast transformed with the empty ex-pression vector.

1158 The Plant Cell

import and export systems (Lalonde et al., 1999). In additionto SUT1, three other sucrose transporters have been identi-fied in solanaceous species: LeSUT4, which appears to playa role in low-affinity sucrose transport (Weise et al., 2000);NtSUT3, a carrier involved in pollen nutrition (Lemoine et al.,1999); and SUT2, a putative sucrose sensor. All sucrosetransporters that have been isolated have a predicted topol-ogy of 12 transmembrane domains. SUT2 has not only thispredicted structure but also two additional features: ex-tended N-terminal and central loop domains that average 30and 50 amino acids longer, respectively, than those of otherSUT family members. According to hydrophobicity analysisand experimental evidence for PmSUC2 (Stolz et al., 1999),the N terminus and central loop are localized intracellularly,which allows these extended domains to interact with pro-teins in the cytoplasm. Another characteristic of

SUT2

is thelow similarity to the Kozak consensus for high rates of trans-lation initiation (Kozak, 1996) and a low codon bias, indicat-ing low translatability.

In contrast to other members of the SUT family, SUT2does not complement the yeast mutant that is deficient insucrose uptake, and its expression in yeast appears tocause toxicity. Even in a more sensitive selection system,one deficient in hexose uptake, which thus leads to reducedbackground growth, SUT2 is still nonfunctional. Although anapparently full-length SUT2 protein can be detected by pro-tein gel blot analysis, the data do not exclude the possibilitythat nonfunctionality is the result of insufficient targeting toyeast plasma membranes or that the large central loop actsas an autoinhibitory domain.

Putative Sensor Function of SUT2

In tomato, SUT2 was localized to sieve elements, in which itcolocalizes with the high- and low-affinity sucrose transport-ers SUT1 and SUT4, respectively. SUT2 is regulated by su-crose, adding to other evidence for the sugar regulation ofsucrose transporters at the plasma membrane level (Chiouand Bush, 1998). In general, SUT2, which shows many fea-tures similar to the yeast sugar sensors SNF3 and RGT2,may be involved directly in controlling expression, activity,and turnover of the other two sucrose transporters SUT1and SUT4 and thereby may act to control sucrose fluxesacross the plasma membrane of sieve elements.

Interestingly, the hexose transporter family from Arabi-dopsis also contains several transporter-like members thatcontain extended cytosolic loops, which thus represent po-tential candidates for hexose sensors (Lalonde et al., 1999).An emerging mechanism found in many organisms for vari-ous substrates is the presence of transporters or trans-porter-like proteins involved in sensing and regulating the

Figure 4. Specificity of the LeSUT2 Antiserum Shown by Yeast Im-munofluorescence.

LeSUT1, LeSUT2, or the empty vector pDR195 was expressed inSUSY7/ura3, the cells were fixed, and SUT proteins were detectedwith affinity-purified antisera. No cross-reactivity of the SUT1 antise-rum on SUT2-expressing yeast or SUT2 antiserum on SUT1-expressing yeast was detected. No signals were detected on yeastexpressing pDR195.

Figure 5. Expression of SUT2.

(A) Organ-specific expression of LeSUT1 and LeSUT2 (20 mg/lane)in stringent hybridization conditions. Probes were the full-length cDNAof LeSUT1 and a 1.3-kb fragment of LeSUT2.(B) Comparison of SUT1, SUT2, and patatin expression under differ-ent treatments in potato. SUT2 was specifically induced after treat-ment with sucrose (100 mM), whereas SUT1 expression remainedunchanged. Both sucrose and to a lesser extent glucose (100 mM)induced patatin expression. Sorbitol also was used as an osmoticcontrol at a concentration of 100 mM.

Putative Sucrose Sensor 1159

Figure 6. Immunocytochemical Localization of SUT Proteins.

(A) Immunofluorescent detection of SUT1 in enucleate sieve elements of tomato stems, colocalized with the aniline blue–stained callose in thesieve plate. DAPI-stained nuclei are visible in neighboring phloem cells.(B) SUT2 colocalized with aniline blue–stained sieve plate callose in enucleate sieve elements of stem.(C) and (D) Loss of LeSUT2 signal after incubation of SUT2 antiserum with its corresponding peptide (C). No reduction in signal after incubationof SUT2 antiserum with a control peptide (D).(E) No signals detected with preimmune serum; aniline blue–stained sieve plate callose indicates presence of sieve element.(F) No immunofluorescence labeling detectable in source-leaf minor veins of tomato plants treated with anti-LeSUT2 antiserum.(G) Bright-field view of the same section as in (F), showing minor vein anatomy.(H) LeSUT1 detectable in source-leaf minor veins on a serial section as in (F).(I) Source leaf of a transgenic Arabidopsis plant transformed with the 1.2-kb AtSUT2 promoter–GUS fusion construct. GUS staining is detect-able in first-, second-, and third-order veins but not in minor veins.(J) Immunogold labeling of the sieve element plasma membrane in a petiole cross-section by using anti-LeSUT2 antibodies.(K) Control treated with IgG-enriched preimmune serum on a serial section as in (J).Bar in (A) to (H) 5 40 mm; bar in (J) and (K) 5 500 nm. n, nucleus; pp, phloem parenchyma cell; se, sieve element; sp, sieve plate; w, cell wall.

1160 The Plant Cell

activity of carriers according to substrate requirement, suchas the bacterial iron/citrate transporter/sensor FecA, the yeastamino acid sensor SSY1, and the putative plant metal sen-sor EIN2 (Boles and Hollenberg, 1997; Alonso et al., 1999;Braun and Killmann, 1999; Iraqui et al., 1999).

High conservation of central sequence motifs within theextended cytoplasmic loops of SUT2 proteins (CCB1 andCCB2) suggests an important role for SUT2 function. Ex-tended cytoplasmic domains could represent domains foreffector binding or signal transduction. For example, theCCB2 domain contains a serine residue at a highly con-served position, which is predicted to be the target for regu-

lation by phosphorylation by a serine/threonine kinase.Sucrose transport already has been described to be regu-lated by phosphorylation (Roblin et al., 1998).

SUT2 Trafficking into Sieve Elements

The present study of SUT2 localization together with a par-allel analysis of SUT4 has shown that three SUT proteins,falling into three clusters, colocalize in sieve elements (Fig-ure 1C; Weise et al., 2000). SUT1 is likely to be transportedfrom the companion cell to the sieve element, probably inthe form of mRNA, given that both its mRNA and proteinturn over rapidly (Kühn et al., 1997). Previous studies show-ing that SUT1 mRNA localizes to the orifices of plasmodes-mata both in companion cells and in sieve elements lead tothe suggestion that all three SUTs use similar traffickingmechanisms for their mRNA. The mechanism may involvereceptor-mediated transport of RNA, similar to that occur-ring in Drosophila oocytes (St. Johnston, 1995), possiblyinvolving a CmPP16 homolog that has been shown to in-crease SUT1 mRNA mobility between mesophyll cells(Xoconostle-Cázares et al., 1999). Although the structuralrequirements of the mRNA that allow for plasmodesmatalmovement have not been identified, investigation into the in-volvement of cis elements or a common RNA structure inthe three SUT genes may help reveal such a mechanism.

SUT2 Is Expressed in the Transport but Not in the Loading Phloem

Expression of the AtSUT2 promoter reporter gene fusion wasfound in the vascular tissue of leaf major veins in trichomesand in hydathodes but not in minor veins, the places ofphloem loading. These data are consistent with the immu-nolocalization data of LeSUT2. Different parts of the phloemwere classified according to their different functions into thecollection phloem, the transport phloem, and the deliveryphloem (van Bel, 1996). Anatomic characteristics reflect thedifferent tasks of the phloem (i.e., the size relationship be-tween the companion cells and the sieve elements: sieve ele-ments of the transport phloem, to which a retrieval functionwas assigned, are bigger than the neighboring companioncells). Thus, differences in the localization of SUT1, which isstrongly expressed in source-leaf minor veins (Riesmeier etal., 1993; Kühn et al., 1997), and SUT2, which is localized inthe transport phloem, indicate a strikingly different function ofthe two sucrose transporter–like proteins.

Linkage of SUT Genes to QTLs for Tuber Starch Content and Tuber Yield

Transport of sucrose from source leaves to sink tubers isessential for starch accumulation in developing tubers.

Figure 7. Mapping of SUT Loci.

cDNA clones StSUT1, LeSUT4, and LeSUT2 were used as RFLPmarkers in mapping population BC9162 and identified SUT1(StSUT1) on linkage group XI, SUT4 (LeSUT4) on linkage group IV,and SUT2 (LeSUT2) on linkage group V. Centimorgan distances be-tween SUT loci and anchor RFLP markers (Gebhardt et al., 1999) areindicated to the left of the linkage groups. GP and CP markers areanonymous potato genomic and cDNA fragments, respectively.Small letters in parentheses indicate that more than one locus wasidentified by the marker probe. Functional gene markers (Gebhardtet al., 1999) are as follows: BE, branching enzyme; MBF, mas bind-ing factor; StPto, Solanum tuberosum homolog of tomato resistancegene Pto; UGPase, UDP–glucose pyrophosphorylase. Positions ofQTLs for tuber starch content [ts(i), ts(k), ts(n), ts(a)] and tuber yield[yi(a)], shown to the left of the linkage groups, are derived fromSchäfer-Pregl et al. (1998), based on anchor RFLP markers withknown genetic distance to QTL and SUT loci.

Putative Sucrose Sensor 1161

Genetic variability of the genes controlling sucrose transporttherefore may have a quantitative effect on the starch con-tent of mature tubers. QTLs for tuber starch content and tu-ber yield have been positioned on the potato RFLP map.QTLs for tuber yield and tuber starch content are linked,suggesting that at least some factors controlling tuberstarch content have pleiotropic effects on tuber yield(Schäfer-Pregl et al., 1998). Given their essential physiologicrole, genes controlling sucrose transport are candidates forQTLs controlling tuber starch content. If alleles at one ormore transporter or sensor loci indeed are responsible for aQTL effect on tuber starch content, tight genetic linkage willbe observed between the QTL and the candidate gene lo-cus. Therefore, the positions of SUT loci were compared withthe positions of QTL for starch content and tuber yield.

Three unlinked SUT loci were identified in the potato mo-lecular map: SUT4 on linkage group IV, SUT1 on linkagegroup XI, and SUT2 on linkage group V. On linkage group IV,QTLs for tuber starch content are located in the intervalflanked by RFLP loci GP180(a) and GP261(a) and are closelylinked to the branching enzyme locus BE (Schäfer-Pregl etal., 1998). SUT4, however, maps in between the two QTLs(Figure 7). Similarly, a minor QTL for tuber starch content islinked to marker loci GP125 and UGPase (UDP–glucose py-rophosphorylase) on linkage group XI (Schäfer-Pregl et al.,1998), whereas SUT1 occupies a more proximal position onthe same linkage group (Figure 7). On linkage group V, amajor QTL for tuber starch content and tuber yield is linkedto markers GP179 and GP291(a) (Schäfer-Pregl et al., 1998).The SUT2 locus is tightly linked to the same markers (Figure7) and therefore to this QTL. This linkage suggests that (1)SUT2 alleles may play an important role in controlling tuberstarch content in the field and (2) SUT2 alleles may havepleiotropic effects on tuber starch content and tuber yield.Further work is required to confirm this important role, be-cause linkage analysis cannot exclude the possibility thatthe gene controlling the QTL is linked, but is functionally un-related to, the candidate gene locus SUT2.

METHODS

LeSUT Genes and Expression Analysis

Screening a flower cDNA library from Lycopersicon esculentum cvUC82b with an NtSUT3 genomic fragment (Lemoine et al., 1999) un-der low stringency led to the isolation of 15 LeSUT1 cDNA clones.The partial genomic sequence of LeSUT2 was isolated by screeninga genomic library from L. esculentum cv VFN8 in EMBL-3 (Clontech,Palo Alto, CA). By hybridization with StSUT1 under nonstringent con-ditions, 11 positive l phages were obtained. One of the more weaklyhybridizing l phages (17/2) represented LeSUT2. A 2.1-kb EcoRIfragment was used to identify the LeSUT2 cDNA from a leaf cDNA li-brary from L. esculentum VC82b, in l-ZAPXR (Stratagene).

The partial genomic LeSUT2 clone was characterized further byusing a 2.1-kb XhoI fragment, overlapping the LeSUT2 cDNA from

positions 274 to 604 upstream of the ATG, and a 4-kb genomic XhoIfragment, containing sequences from position 802 to the end of theLeSUT2 cDNA. The gap in the genomic sequence between the twofragments was filled by polymerase chain reaction (PCR) of the DNAof the l-phage isolate 17/2 with the use of appropriate primers.

RNA was isolated from different organs of greenhouse grown L. es-culentum Moneymaker, as described (Schwacke et al., 1999). RNA gelblots were hybridized in 50% formamide at 428C and washed with 0.2 3SSC/0.1% SDS at 658C (1 3 SSC is 0.15 M NaCl and 0.015 M sodiumcitrate). For DNA gel blot analysis, genomic DNA from L. esculentumwas digested with restriction enzymes, resolved on 1% agarose gels,and blotted to Hybond N1 (Amersham Pharmacia Biotech). Hybridiza-tion was performed with 32P-labeled probes under high stringency, fol-lowed by washing at 658C in 0.2 3 SSC/0.1% SDS.

The LeSUT2 sequence was deposited in GenBank under the ac-cession number AF166498.

AtSUT2 Analysis

The cDNA of AtSUT2 was cloned by reverse transcription–PCR fromArabidopsis thaliana Columbia (Col-0) ecotype leaves. After reversetranscription of 5 mg of total leaf RNA, PCR was performed by usingspecific primers with the cloning sites EcoRI (f) and XhoI (r), and theproduct was cloned into the yeast expression vector pDR196. Thegenomic sequence of AtSUT2 is in GenBank under the accessionnumber AC004138.

The AtSUT2 promoter fragment was isolated by PCR on A.thaliana Col-0 ecotype genomic DNA by using Pfu-Polymerase(Stratagene). A transcriptional fusion was generated by cloning 2.2-and 1.2-kb promoter fragments into pGPTV-HPT (Becker et al.,1992). Arabidopsis was transformed by vacuum infiltration of trans-formed Agrobacterium GV2260 in 0.53 Murashige and Skoog me-dium (Murashige and Skoog, 1962) containing 5% sucrose, 0.005%Silwet, and 0.044 mM benzylaminopurine (Clough and Bent, 1998).Selection was performed on Murashige and Skoog medium contain-ing 50 mg/L hygromycin. Plant material was infiltrated under vacuumwith 2 mM 5-bromo-4-chloro-3-indolyl-glucuronide, 50 mM sodiumphosphate buffer, pH 7.2, and 0.5% Triton X-100 and was incubatedat 378C for 12 to 16 hr (Jefferson et al., 1987). Destaining was per-formed in 70% ethanol.

Sugar Induction

Plants were grown in the greenhouse with a 16-hr-light/8-hr-dark cy-cle, and leaves were harvested from 3-month-old plants. Petioles ofdetached leaves were re-cut while submerged in water, EDTA (2.5mM) was added to inhibit callose formation, and the cut petioleswere transferred to solutions containing 100 mM sugar, where theywere kept for 24 hr under greenhouse conditions. All experimentswere repeated independently.

Yeast Strain Construction

To construct SUSY7/ura3, we digested Yep24 (Biolabs, Schwal-bach, Germany) with NcoI and ApaI (which deleted an internal 173-bp fragment of the URA3 gene), and then Yep24 was treated with T4polymerase and blunt-ligated, which yielded pDura3 (W.N. Fischerand W.B. Frommer, unpublished results). The truncated Dura3 gene

1162 The Plant Cell

was excised with EcoRI and SmaI, and the linearized Dura3 fragmentwas used for transformation of SUSY7 (Riesmeier et al., 1992). TheDura3 mutants were selected on 5-fluoroorotic acid. Deletion of theinternal URA3 fragment was controlled by PCR (data not shown).

Membrane Isolation and Protein Gel Blot Analysis

Yeast cells were grown in minimal medium (200 mL) to an OD600 ofz1.1. Cells were harvested at 1000g, washed in distilled water, andthen washed in 25 mM Tris-HCl, pH 7.5, containing 5 mM EDTA.Cells were resuspended in 100 mL of the same buffer. In a small glasstube, 1 g of glass beads (0.5 mm in diameter) was added, mixed with3 mL of protease inhibitor mix (0.1 M phenylmethylsulfonyl fluorideand 0.25 M p-aminobenzamidine in DMSO), and vortex-mixed fourtimes for 30 sec each. The cell homogenate was centrifuged for 5min at 1000g, and the membranes were pelleted from the superna-tant by ultracentrifugation at 100,000g for 60 min. The sediment wasresuspended in 480 mL of a solution of 100 mM sodium phosphate,pH 8.0, 50 mM NaCl, 2.5% b-D-dodecylmaltoside containing 1:100(v/v) protease inhibitor mix, and the membranes were solubilized for5 min on ice. Plant plasma membranes were purified as described(Larsson, 1985). SDS-PAGE and protein gel blotting have been de-scribed previously (Lemoine et al., 1996). Immunodetection was byelectrochemiluminescence (Amersham).

Immunocytochemistry

Rabbits were immunized with synthetic peptides coupled to keyholelimpet hemocyanin corresponding to the N terminus or central loopof LeSUT2 (N terminus: MDAVSIRVPYKNLKC; central loop: PKN-EEQRPDKDQGDS). Affinity purification was performed as described(Lemoine et al., 1996). Preimmune serum was purified as above, ex-cept that protein A–Sepharose (Bio-Rad) was used instead of pep-tide affinity. Antiserum for detection of LeSUT1 prepared againstStSUT1 cross-reacts with tomato (Kühn et al., 1997).

Yeast cells prepared for immunolabeling were grown to OD600

z0.75 and fixed in 3.7% formaldehyde for 15 min at 288C. Cultures (2mL) were harvested and resuspended for 30 min at 288C in 1 mL of0.1 M potassium phosphate, pH 6.4, containing 3.7% formaldehyde.Cells were washed three times in 0.1 M potassium phosphate, pH6.4, and once in 1.2 M sorbitol in 0.1 M phosphate citrate buffer, pH5.9. Cells were incubated in 200 mL of 1.2 M sorbitol/phosphate ci-trate with 7 mL of zymolase 100T (ICN Biomedicals Research, Eschwege,Germany) (3 mg/mL in 10% glucose) for 60 min at 288C, which wasfollowed by washing in 1.2 M sorbitol/phosphate citrate, resuspen-sion in 300 mL of 1% BSA in PBS (100 mM sodium phosphate, pH7.5, and 100 mM NaCl), and incubated overnight at 48C with primaryantibody on an orbital shaker. After three washes in BSA–PBS, Cy3-coupled secondary antibody diluted 1:100 was added in BSA–PBS,and the mixture was incubated for 1 hr on an orbital shaker in dark-ness. After four washes in BSA–PBS, cells were analyzed by confo-cal laser scanning microscopy TCP-SP (Leica, Bensheim, Germany).

Fluorescent immunodetection of LeSUTs was performed withmodifications according to Kühn et al. (1997). Hand-cut fragments (1mm2) from tomato leaves, stems, and petioles were fixed overnightunder light vacuum in MOPS buffer (50 mM MOPS/NaOH, pH 6.9, 5mM EGTA, and 2 mM MgCl2) containing 0.1% glutaraldehyde and6% formaldehyde. After three washes with MOPS buffer on ice, frag-ments were dehydrated in an ethanol series. After overnight incuba-

tion in an equal volume solution of ethanol and methacrylate (75% [v/v]butylmethacrylate, 25% [v/v] methylmethacrylate, 0.5% benzoineethyl ether, and 10 mM DTT), the material was embedded in 100%methacrylate. Polymerization took place overnight under UV light(365 nm) at 48C. Semithin sections (1 mm thick) were mounted onHistobond slides (Camon, Weisbaden, Germany) and dried at 508C.To remove the methacrylate, we incubated the slides for 30 sec inacetone; they were then rehydrated by an ethanol series and blockedfor 1 hr with 2% BSA in PBS. After overnight incubation with affinity-purified antibodies, the slides were washed twice in PBS containing0.1% Tween (PBS-T) and once with PBS, followed by 1 hr incubationwith anti-rabbit IgG–fluorescein isothiocyanate conjugate. After threesequential washes with PBS-T, PBS, and distilled water, photo-graphs were taken with a fluorescence microscope (Axiophot; Zeiss)using fluorescein isothiocyanate filters. For double-staining of trans-porters and nuclei with 49,6-diamidino-2-phenylindole (DAPI), sec-tions were treated as described above. After three washes, slideswere incubated for 1 hr in 0.2 mg/mL DAPI. DAPI fluorescence wasdetected at 365 nm. For double staining of transporters and sieveplate callose with aniline blue, slides were incubated for 10 min in0.05% aniline blue in 50 mM sodium phosphate, pH 7.2. Aniline bluefluorescence was detected at 365 nm. For antibody–peptide competi-tion experiments, affinity-purified antiserum was incubated, before im-munolocalization, for z16 hr at 48C, either with its correspondingpeptide or with a peptide mimicking another epitope of the same trans-porter or control peptide (CAFGDPIDSKLSR), at 103 molar excess.

For electron microscopic immunogold staining, petiole material oftomato plants was fixed under vacuum for 12 hr with 4% formalde-hyde/0.1% glutaraldehyde in 100 mM Pipes, pH 7.2. After rinsing in100 mM Pipes, tissue was dehydrated in ethanol and embedded inacrylic resins (Unicryl; Plano, Wetzlas, Germany; or LR White; Lon-don Resin Co., Berkshire, UK). Polymerization took place at 608Cover 2 days. Ultrathin sections (100 nm) were stretched with chloro-form and mounted on gold grids (Gilder-Grids 400 Hex; Plano), afterwhich the grids were incubated for 10 min in 0.1 M HCl and washedwith distilled water. After blocking for 1 hr in 2% (v/v) BSA in PBS,grids were incubated with affinity-purified LeSUT2 antibodies diluted1:4 in PBS in a humid chamber at 48C overnight. Sections werewashed with PBS-T and PBS and were incubated for 1 hr with 10-nmgold-coupled goat anti–rabbit IgG (Amersham) diluted 1:50 in PBS.After three washes in PBS-T and PBS, the sections were postfixedfor 10 min in 2% glutaraldehyde in PBS and rinsed with distilled wa-ter. Ultrathin sections were poststained with uranyl acetate and leadcitrate for better tissue contrast. Gold labeling was detected with aPhilips CM 100 microscope (Fellbach, Germany).

Mapping

Restriction fragment length polymorphism (RFLP) analysis and map-ping of cDNA clones StSUT1, LeSUT4, and LeSUT2 were performedin the diploid potato mapping population BC9162 of 67 individuals asdescribed (Gebhardt et al., 1991).

ACKNOWLEDGMENTS

We thank W.N. Fischer for pDura and SUSY7/ura3 and Lukas Bürklefor help with LeSUT characterization. We are very grateful to Nicole

Putative Sucrose Sensor 1163

Thiele and Katrin Herm for excellent technical assistance. This workwas supported by grants from Deutsche Forschungsgemeinschaft(SFB446 B9).

Received February 10, 2000; accepted April 28, 2000.

REFERENCES

Alonso, J.M., Hirayama, T., Roman, G., Nourizadeh, S., andEcker, J.R. (1999). EIN2, a bifunctional transducer of ethyleneand stress responses in Arabidopsis. Science 284, 2148–2152.

Becker, D., Kemper, E., Schell, J., and Masterson, R. (1992). Newplant binary vectors with selectable markers located proximal tothe left T-DNA border. Plant Mol. Biol. 20, 1195–1197.

Boles, E., and Hollenberg, C.P. (1997). The molecular genetics ofhexose transport in yeasts. FEMS Microbiol. Rev. 21, 85–111.

Braun, V., and Killmann, H. (1999). Bacterial solutions to the iron-supply problem. Trends Biochem. Sci., 24, 104–109.

Bürkle, L., Hibberd, J.M., Quick, W.P., Kühn, C., Hirner, B., andFrommer, W.B. (1998). The H1-sucrose co-transporter NtSUT1 isessential for sugar export from tobacco leaves. Plant Physiol. 118,59–68.

Chiou, T.J., and Bush, D.R. (1998). Sucrose is a signal molecule inassimilate partitioning. Proc. Natl. Acad. Sci. USA 95, 4784–4788.

Clough, S.J., and Bent, A.F. (1998). Floral dip: A simplified methodfor Agrobacterium-mediated transformation of Arabidopsis thal-iana. Plant J. 16, 735–743.

Gebhardt, C., Ritter, E., Barone, A., Debener, T., Walkemeier, B.,Schachtschabel, U., Kaufmann, H., Thompson, R.D., Bonierbale,M.W., Ganal, M.W., Tanksley, S.D., and Salamini, F. (1991).RFLP maps of potato and their alignment with the homeologoustomato genome. Theor. Appl. Genet. 83, 49–57.

Gebhardt, C., Ritter, E., and Salamini, F. (1999). RFLP map of thepotato. In DNA-Based Markers in Plants, 2nd ed. R.L.V. Phillips,I.K., ed (New York: Kluwer Academic Publishers).

Hellmann, H., Barker, L., Funck, D., and Frommer, W.B. (2000).The regulation of assimilate allocation and transport. Aust. J.Plant Physiol., in press.

Iraqui, I., Vissers, S., Bernard, F., De Craene, J.-O., Boles, E.,Urrestarazu, A., and Andre, B. (1999). Amino acid signaling inSaccharomyces cerevisiae: A permease-like sensor of externalamino acids and F-box protein Grr1p are required for transcrip-tional induction of the AGP1 gene, which encodes a broad-speci-ficity amino acid permease. Mol. Cell. Biol. 19, 989–1001.

Jefferson, R.A., Kavanagh, T.A., and Bevan, M.W. (1987). GUSfusions: b-Glucuronidase as a sensitive and versatile gene fusionmarker in higher plants. EMBO J. 6, 3901–3907.

Kozak, M. (1996). Interpreting cDNA sequences: Some insightsfrom studies on translation. Mamm. Genome 7, 563–574.

Kühn, C., Quick, W.P., Schulz, A., Sonnewald, U., and Frommer,W.B. (1996). Companion cell–specific inhibition of the potatosucrose transporter SUT1. Plant Cell Environ. 19, 1115–1123.

Kühn, C., Franceschi, V.R., Schulz, A., Lemoine, R., and Frommer,W.B. (1997). Localization and turnover of sucrose transporters in

enucleate sieve elements indicate macromolecular trafficking.Science 275, 1298–1300.

Kyte, J., and Doolittle, F.R. (1982). A simple method for displayingthe hydropathic character of a protein. J. Mol. Biol. 157, 693–698.

Lalonde, S., Boles, E., Hellmann, H., Barker, L., Patrick, J.W.,Frommer, W.B., and Ward, J.M. (1999). A dual function of sugarcarriers: Transport and sugar sensing. Plant Cell 11, 707–726.

Larsson, C. (1985). Plasma Membranes. Vol. 1. H.F. Linskens andJ.F. Jackson, eds (Berlin: Springer Verlag).

Lemoine, R., Kühn, C., Thiele, N., Delrot, S., and Frommer, W.B.(1996). Antisense inhibition of the sucrose transporter: Effects onamount of carrier and sucrose transport activity. Plant Cell Envi-ron. 19, 1124–1131.

Lemoine, R., Bürkle, L., Barker, L., Sakr, S., Kühn, C., Regnacq,M., Gaillard, C., Delrot, S., and Frommer, W.B. (1999). Identifi-cation of a pollen-specific sucrose transporter–like proteinNtSUT3 from tobacco. FEBS Lett. 454, 325–330.

Liang, H., and Gaber, R.F. (1996). A novel signal transduction path-way in Saccharomyces cerevisiae defined by SNF3-regulatedexpression of HXT6. Mol. Biol. Cell 7, 1953–1966.

Martin, T., Hellmann, H., Schmidt, R., Willmitzer, L., and Frommer,W.B. (1997). Identification of mutants in metabolically regulatedgene expression. Plant J. 11, 53–62.

Murashige, T., and Skoog, F. (1962). A revised medium for rapidgrowth and bioassays with tobacco tissue culture. Physiol. Plant.15, 473–497.

Özcan, S., and Johnston, M. (1995). Three different regulatorymechanisms enable yeast hexose transporter (HXT) genes to beinduced by different levels of glucose. Mol. Cell. Biol. 15, 1564–1572.

Özcan, S., Dover, J., Rosenwald, A.G., Woelfl, S., and Johnston,M. (1996a). Two glucose transporters in S. cerevisiae are glucosesensors that generate a signal for induction of gene expression.Proc. Natl. Acad. Sci. USA 93, 12428–12432.

Özcan, S., Leong, T., and Johnston, M. (1996b). Rgt1p of Saccha-romyces cerevisiae, a key regulator of glucose-induced genes, isboth an activator and a repressor of transcription. Mol. Cell. Biol.16, 6419–6426.

Özcan, S., Dover, J., and Johnston, M. (1998). Glucose sensingand signaling by two glucose receptors in the yeast Saccharomy-ces cerevisiae. EMBO J. 17, 2566–2573.

Rentsch, D., Laloi, M., Rouhara, I., Schmelzer, E., Delrot, S., andFrommer, W. (1995). NTR1 encodes a high affinity oligopeptidetransporter in Arabidopsis. FEBS Lett. 370, 264–268.

Riesmeier, J.W., Willmitzer, L., and Frommer, W.B. (1992). Isola-tion and characterization of a sucrose carrier cDNA from spinachby functional expression in yeast. EMBO J. 11, 4705–4713.

Riesmeier, J., Hirner, B., and Frommer, W.B. (1993). Potatosucrose transporter expression in minor veins indicates a role inphloem loading. Plant Cell 5, 1591–1598.

Riesmeier, J.W., Willmitzer, L., and Frommer, W.B. (1994). Evi-dence for an essential role of the sucrose transporter in phloemloading and assimilate partitioning. EMBO J. 13, 1–7.

Roblin, G., Sakr, S., Bonmort, J., and Delrot, S. (1998). Regulationof a plant plasma membrane sucrose transporter by phosphoryla-tion. FEBS Lett. 424, 165–168.

1164 The Plant Cell

Roitsch, T. (1999). Source–sink regulation by sugar and stress.Curr. Opin. Plant Biol. 2, 198–206.

Sauer, N., and Stolz, J. (1994). SUC1 and SUC2: Two sucrosetransporters from Arabidopsis thaliana; expression and character-ization in baker’s yeast and identification of the histidine-taggedprotein. Plant J. 6, 67–77.

Schäfer-Pregl, R., Ritter, E., Concilio, L., Hesselbach, J., Lovatti,L., Walkemeier, B., Thelen, H., Salamini, F., and Gebhardt, C.(1998). Analysis of quantitative trait loci (QTL) and quantitative traitalleles (QTA) for potato tuber yield and starch content. Theor.Appl. Genet. 97, 834–846.

Schwacke, R., Grallath, S., Breitkreuz, K.E., Stransky, E., Stransky,H., Frommer, W.B., and Rentsch, D. (1999). LeProT1, a trans-porter for proline, glycine betaine, and g-butyric acid in tomatopollen. Plant Cell 11, 377–392.

St. Johnston, D. (1995). The intracellular localization of messengerRNAs. Cell 81, 161–170.

Stolz, J., Ludwig, A., Stadler, R., Biesgen, C., Hagemann, K., andSauer, N. (1999). Structural analysis of a plant sucrose carrierusing monoclonal antibodies and bacteriophage lambda surfacedisplay. FEBS Lett. 453, 375–379.

Swofford, D.L. (1998). PAUP*. Phylogenetic Analysis Using Parsi-mony (*and Other Methods). (Sunderland, MA: Sinauer Associates).

Tadege, M., Bucher, M., Stähli, W., Suter, M., Dupuis, I., and

Kuhlemeier, C. (1998). Activation of plant defense responses andsugar efflux by expression of pyruvate decarboxylase in potatoleaves. Plant J. 16, 661–671.

Vagnoli, P., Coons, D.M., and Bisson, L.F. (1998). The C-terminaldomain of Snf3p mediates glucose-responsive signal transductionin Saccharomyces cerevisiae. FEMS Microbiol. Rev. 160, 31–36.

van Bel, A.J.E. (1996). Interaction between sieve element and com-panion cell and the consequences for photoassimilate distri-bution. Two structural hardware frames with associatedphysiological software packages in dicotyledons. J. Exp. Bot. 47,1129–1140.

Weise, A., Barker, L., Kühn, C., Lalonde, S., Buschmann, H.,Frommer, W.B., and Ward, J.M. (2000). A new subfamily ofsucrose transporters SUT4 with low affinity/high capacity local-ized in enucleate sieve elements of plants. Plant Cell (in press).

Wieczorke, R., Krampe, S., Weierstall, T., Freidel, K., Hollenberg,C., and Boles, E. (1999). Concurrent knock-out of at least 21transporter genes is required to block uptake of hexoses in yeast.FEBS Lett. 464, 123–128.

Xoconostle-Cázares, B., Xiang, Y., Ruiz-Medrano, R., Wang,H.L., Monzer, J., Yoo, B.C., McFarland, K.C., Franceschi, V.R.,and Lucas, W.J. (1999). Plant paralog to viral movement proteinthat potentiates transport of mRNA into the phloem. Science 283,94–98.

DOI 10.1105/tpc.12.7.1153 2000;12;1153-1164Plant Cell

Hirner, Hanjo Hellmann, Waltraud Schulze, John M. Ward and Wolf B. FrommerLaurence Barker, Christina Kühn, Andreas Weise, Alexander Schulz, Christiane Gebhardt, Brigitte

SUT2, a Putative Sucrose Sensor in Sieve Elements

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