Molecular Plant • Volume 3 • Number 6 • Pages 1049–1063 • November 2010 RESEARCH ARTICLE

Functional Characterization and RNAi-MediatedSuppression Reveals Roles for Hexose Transportersin Sugar Accumulation by Tomato Fruit

David W. McCurdy, Stephen Dibley, Ricky Cahyanegara, Antony Martin and John W. Patrick1

School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia

ABSTRACT Hexoses accumulate to high concentrations (;200 mM) in storage parenchyma cells of tomato fruit. Hexoses

are sourced from the fruit apoplasm as hydrolysis products of phloem-imported sucrose. Three hexose transporters

(LeHT1, LeHT2, LeHT3), expressed in fruit storage parenchyma cells, may contribute to hexose uptake by these cells.

An analysis of their full-length sequences demonstrated that all three transporters belong to the STP sub-family of mono-

saccharide transporters that localize to plasma membranes. Heterologous expression of LeHT1 (and previously LeHT2,

Gear et al., 2000), but not LeHT3, rescued a hexose transport-impaired yeast mutant when raised on glucose or fructose

as the sole carbon source. Biochemically, LeHT1, similarly to LeHT2, exhibited transport properties consistent with a high-

affinity glucose/H1 symporter. Significantly, LeHT1 and LeHT2 also functioned as low-affinity fructose/H1 symporterswith

apparentKm values commensuratewith those of fruit tissues. A substantial reduction (80–90%) in fruit expression levels of

all LeHT genes by RNAi-mediated knockdown caused a 55% decrease in fruit hexose accumulation. In contrast, photo-

assimilate production by source leaves and phloem transport capacity to fruit were unaffected by transporter knockdown.

Collectively, these findings demonstrate that LeHTs play key roles in driving accumulation of hexoses into storage paren-

chyma cells during tomato fruit development.

Key words: Fruit; hexose transporter; sink; tomato; RNAi.

INTRODUCTION

Phloem unloading in most sinks follows symplasmic routes

from importing sieve elements to recipient sink cells through

high densities of interconnecting plasmodesmata (Lalonde

et al., 2003). However, hydrostatic pressure gradients driving

phloem import and symplasmic unloading could be compro-

mised in those sinks accumulating soluble sugars to high

concentrations (Lalonde et al., 2003). In these physiological

contexts, hydraulic independence between phloem and sink

cells can be achieved by separating phloem and sink cell sym-

plasmic domains with an obligatory apoplasmic step in the

phloem-unloading pathway. Examples of this unloading strat-

egy are found in fleshy fruits including apple (Zhang et al.,

2004), grape (Zhang et al., 2006), and tomato (Ruan and

Patrick, 1995). Estimates of transmembrane fluxes of sugars

to, and from, the tomato fruit apoplasm along the phloem-

unloading pathway are consistent with these steps being facil-

itated by membrane proteins (Offler and Horder, 1992).

A model of sucrose efflux from importing sieve elements

in tomato fruit is emerging. Fruit-imported sucrose (Ho,

1996) is released from phloem down steep transmembrane

concentration differences (Ruan et al., 1996) generated by ex-

tracellular invertases catalyzing sucrose hydrolysis in the fruit

apoplasm (Godt and Roitsch, 1997). Indeed, up-regulation of

extracellular invertase activity by introgression of LeLIN5

(Baxter et al., 2005), or its down-regulation by RNAi-mediated

suppression (Zanor et al., 2009), demonstrate that extracellular

invertases play a key role in influencing sugar fluxes into, and

within, developing tomato fruit. LIN5, and possibly other ex-

tracellular invertases, are expressed in fruit vascular bundles

(Fridman et al., 2004) and, in particular, vascular parenchyma

cells (Jin et al., 2009). Their activities generate steep sucrose

concentration differences from phloem symplasm to apo-

plasm. Under these conditions, sucrose release across plasma

1 To whom correspondence should be addressed. E-mail John.Patrick@

newcastle.edu.au, fax +61-2-49216923, tel. +61-2-49215712.

ª The Author 2010. Published by the Molecular Plant Shanghai Editorial

Office in association with Oxford University Press on behalf of CSPP and

IPPE, SIBS, CAS.

doi: 10.1093/mp/ssq050, Advance Access publication 10 September 2010

Received 1 June 2010; accepted 2 August 2010

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membranes of phloem cells could be mediated by reversal of

sucrose symporters (Carpaneto et al., 2005) localized to sieve

elements (Hackel et al., 2006). In addition, a low proton motive

force (pmf) resulting from the absence of plasma membrane

H+-ATPases on the phloem (Ruan and Patrick, 1995; Dibley

et al., 2005) would further contribute to symporter reversal

(Carpaneto et al., 2005). Apoplasmic hexoses are accumulated

by hexose/H+ symport into fruit storage parenchyma cells

(Brown et al., 1997; Ruan et al., 1997) for eventual vacuolar

storage (Milner et al., 1995). Significantly, estimates of sugar

accumulation based on kinetic properties of the plasma-

membrane-localized hexose transporter(s) demonstrate that

their transport activities account for 70–80% of the observed

hexose accumulation by fruit storage cells (Brown et al., 1997;

Ruan et al., 1997). Further, differences in maximal transport ac-

tivities of hexose transporters correlate positively with geno-

typic variation in hexose accumulation by tomato fruit

(Ruan et al., 1997).

Based on the above conclusions, we performed a molecular

study of hexose transporters in developing tomato fruit. Com-

plementary DNA sequences of three Lycopersicon esculentum

L. (re-named Solanum lycopersicum) hexose transporters

(LeHT) were cloned from young fruit and flower cDNA libraries

under low stringency, with two partial (LeHT1 and LeHT3) and

one full-length clone (LeHT2) being obtained (Gear et al.,

2000). LeHT1-3 are expressed in storage parenchyma cells of

developing tomato fruit and their temporal expression pro-

files suggest that LeHT3 is the most probable isoform respon-

sible for the bulk of hexose uptake (Dibley et al., 2005). LeHT2

functions as a high-affinity (apparent Km value of 45 lM) hex-

ose/H+ symporter, with a preference for glucose over fructose.

Expressed strongly at fruit maturity, LeHT2 has been conjec-

tured to play a role in retrieving cell wall hydrolysis products

(Gear et al., 2000).

Equimolar concentrations (;15 mM) of glucose and fruc-

tose have been detected in the tomato fruit apoplasm during

their rapid phase of hexose accumulation (Ruan et al.,

1996). This finding indicates a requirement for one or more

low-affinity/high-capacity hexose transporters (e.g. AtSTP3,

Buttner et al., 2000) equally capable of transporting glucose

and fructose into fruit storage parenchyma cells. In this con-

text, the current study set out to obtain full-length sequences

of LeHT1 and LeHT3, functionally characterize these transport-

ers by expression in yeast mutants, compare their functions

with predicted in planta transport properties (see above),

and directly evaluate their physiological role(s) by RNAi-

mediated knockdown.

RESULTS

Cloning and Phylogenetic Analysis of LeHT1 and LeHT3

We used 5’ RACE to obtain full-length sequence information

for LeHT1 and LeHT3 reported previously as partial 3’-cDNA

clones (Gear et al., 2000). Full-length LeHT1 (GenBank ac-

cession number GQ911580) encodes a predicted polypeptide

of 523 amino acids with a calculated molecular mass of 57.9 kD,

while LeHT3 (GenBank accession number GQ911581) encodes

a predicted polypeptide of 513 amino acids corresponding to

a calculated molecular mass of 56.3 kD. Pairwise sequence

comparisons of LeHT1 and LeHT3 with the previously reported

LeHT2 polypeptide (Gear et al., 2000) showed 55–56% identity

(73–74% similarity) between all three sequences. Sequence

alignment of LeHT1, 2 and 3 (Supplemental Figure 1) showed

that their predicted sequences display the characteristic

secondary structure comprising 12 membrane-spanning

domains—a core feature of members of the Major Facilitator

Superfamily (Buttner and Sauer, 2000). Their sequences also

contain a common cytoplasm-exposed loop of approximately

62–66 amino acids between transmembrane helices 6 and 7,

and all are predicted to have their N- and C-termini exposed

to the cytoplasm (data not shown). Conserved sequence motifs

identified in characterized hexose transporters are also pres-

ent in LeHT1-3, including the invariant PETK473–476(LeHT1) mo-

tif positioned immediately after transmembrane helix 12, two

sugar transport signature motifs (Prosite motifs PS00216 and

PS00217, http://ca.expasy.org/cgi-bin/nicedoc.pl?PDOC00190)

(Supplemental Figure 1), and the motif SWGP(M/L)GW(L/T)

(V/I)PSE402–413(LeHT1), which is highly conserved in plant hex-

ose transporters (Sauer and Stadler, 1993). Also present are

amino acid residues known to affect substrate affinity or trans-

port rate of hexoses, such as Q175, Q292, V428 and N431(LeHT1)

(Will et al., 1994, 1998).

Phylogenetic analysis of the predicted LeHT polypeptides

with other hexose transporter sequences from higher plants

is shown in Figure 1. We compared the three LeHT polypepti-

des with the 13 AtSTP (sugar transport protein) sequences

from Arabidopsis (Buttner, 2007), along with STP-like sequen-

ces from other species, and members of the Vacuolar Glucose

Transporter-like (VGT)- and Tonoplast Monosaccharide Trans-

porter (TMT)-class of transporters, founded by AtVGT1 (Aluri

and Buttner, 2007) and AtTMT1 (Wormit et al., 2006), respec-

tively. The LeHT polypeptides cluster in three distinct clades of

the STP-like subfamily, indicating that LeHT sequences are

more closely related to orthologs in other species than with

each other. Significantly, the LeHT-containing clades are clearly

distinct from the vacuolar/tonoplast-associated sequences

(Figure 1). LeHT2, previously characterized as a high-affinity

monosaccharide transporter (Km (Glc) 45 lM, Gear et al.,

2000), clusters with VvHT5 from grape, also characterized as

a high-affinity transporter (Km (Glc) 89 lM, Hayes et al.,

2007). LeHT1 similarly is grouped with other characterized

high-affinity transporters (AtSTP1, Buttner, 2007; VvHT1,

Vignault et al., 2005; LpSTP1, Szenthe et al., 2007), while LeHT3

clusters with VvHT3 (Hayes et al., 2007) that is yet to be func-

tionally characterized.

Functional Characterization of LeHT1 and LeHT3 Expressed

in Yeast

LeHT1 and LeHT3 cDNAs were cloned in the sense orientation

into the yeast vector pDR195 and transformed into the yeast

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mutant strain EBY.VW4000. LeHT2 cDNA was previously cloned

in the sense orientation inpYES2 and transformed into RE700A

(Gear et al., 2000). Both EBY.VW4000 and RE700A lack native

hexose transporters (Wieczorke et al., 1999; Reifenberger

et al., 1995, respectively). Further functional characterization

studies (cf. Gear et al., 2000) were undertaken with LeHT2 as

specified below.

Three independent colonies of LeHT1 and LeHT3 were

tested for complementation of EBY.VW4000 on plates contain-

ing glucose or fructose as the sole carbon source. EBY.VW4000

strain is capable of growth on maltose (Supplemental Figure 2;

for further information, see Wieczorke et al., 1999). Compared

to yeast transformed with empty vector alone (pDR195), ex-

pression of LeHT1 restored growth on a media containing

2% glucose or fructose (Supplemental Figure 2). In contrast,

sequence-verified LeHT3 did not rescue EBY.VW4000 cultured

on media containing 2% glucose or fructose. However, the

transgenic yeast was capable of growth on a medium contain-

ing maltose, indicating that LeHT3 did not compromise cell

function (Supplemental Figure 2). To address the possibility

that LeHT3 is a low-affinity transporter (cf. Zhou et al.,

2007), media hexose concentrations were doubled but this

had no perceivable effect on yeast growth by EBY.VW4000-

LeHT3 (data not shown). We confirmed by RT–PCR that LeHT3

was transcribed in transgenic yeast, as was LeHT1 and LeHT2

(Supplemental Table 1). Furthermore, both LeHT2 and LeHT3

localized to the plasma membrane in transgenic yeast, as de-

termined by carboxy–terminal GFP fusions expressed in

EBY.VW4000 (Supplemental Figure 3). This result implies that

LeHT3 requires post-translational modification to become

transport competent.

Yeast strains expressing LeHT1 (EBY.VW4000) or LeHT2

(RE700A) (Gear et al., 2000) were shown to be capable of tak-

ing up [14C]glucose or [14C]fructose at rates higher than those

cells transformed with empty vector alone (Figure 2A and 2B,

respectively). In contrast, EBY.VW400-LeHT3 did not accumu-

late either hexose above that found for yeast containing

empty vector alone (data not shown). These findings demon-

strate that LeHT1 and the previously characterized LeHT2

(Gear et al., 2000) encode transporters capable of transport-

ing both hexoses. Interestingly, LeHT2 facilitated higher rates

of fructose uptake than LeHT1 at pH values of less than 6

(Figure 2B and 2D). Each transporter supported constant

rates of [14C]hexose uptake over the first 10 min of exposure

(Figure 2A and 2B). Thus, all subsequent uptake experiments

were conducted over a 4-min period following exposure to

[14C]hexose.

Hexose uptake mediated by LeHT1 and LeHT2 exhibited

pH-dependence with optima at pH 5 for glucose (Figure 2C

and also see Gear et al., 2000) and at pH 6 for fructose

(Figure 2D). Fructose transport by LeHT1 was strikingly influ-

enced by extracellular pH. This transporter supported a 2.6-

fold increase in fructose flux between pH 5 and 6 (Figure

2D) compared to a 1.4-fold increase by LeHT2 across the same

pH range (Figure 2D). The pH profiles displayed by LeHT1 and

LeHT2 informed choice of media pH to examine their kinetic

properties for fructose transport as described below.

Concentration-dependent uptake of both [14C]hexoses by

transformed yeast demonstrated that irrespective of trans-

ported sugar and medium pH, LeHT1 and LeHT2 displayed

saturating transport kinetics typical of a carrier function

(Figure 3). The data were fitted to the Michaelis-Menten rate

Figure 1. Phylogenetic Analysis of LeHTs.

LeHT sequences are indicated by arrows. The tree was constructedusing Phylogeny.fr (Dereeper et al., 2008) as described in Methods.Database accession numbers of the sequences used are: S. lycoper-sicum LeHT1 (GQ911580), LeHT2 (CAB52689), LeHT3 (GQ911581);A. thaliana AtSTP1 (NP_172592), AtSTP2 (AJ001362), AtSTP3(CAA05384), AtSTP4 (BAB01309), AtSTP5 (NM_103182), AtSTP6(AJ001659), AtSTP7 (CAB80698), AtSTP8 (AF077407), AtSTP9(AJ001662), AtSTP10 (DQ056599), AtSTP11 (AJ001664), AtSTP12(NP_193879), AtSTP13 (CAC69074), AtVGT1 (NP_186959), AtTMT1(Z50752), AtTMT2 (AJ532570), AtTMT3 (AJ532571); V. viniferaVvHT1 (AJ001061), VvHT2 (AY663846), VvHT6 (AY861386), VvHT7(AY854146), pGLT (AY608701), VvHT3 (AY538259), VvHT4(AY538260), VvHT5 (AY538261); O. sativa OsMST1 (AB052883),OsMST2 (AB052884), OsMST3 (AB052885), OsMST4 (AY342321),OsMST5 (NM_001067662), OsMST6 (AY342322); L. polyphyllusLpSTP1 (Szenthe et al., 2007); A. comosus AcMST1 (EF460876);H. vulgare HvSTP1 (CAD58958).

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equation using least-squares non-linear regression (GraphPad

Prism 5.0) to derive statistical estimates of apparent Km and

Vmax values for each transporter (Figure 3). The most interest-

ing finding was that LeHT1 and LeHT2 exhibited apparent Km

values for fructose in the mM range and were an order of mag-

nitude higher than those found for glucose (Figure 3A–3D ver-

sus 3E). Moreover, and consistent with their pH profiles (Figure

2D), apparentKm andVmax values of both transporters for fruc-

tose increased significantly when extracellular pH was raised

from 5 to 6 (Figure 3A versus 3C; 3B versus 3D). Both kinetic

parameters were greater for LeHT1 compared with LeHT2

for fructose (Figure 3A, 3C versus 3B, 3D) and glucose (i.e. ap-

parent Km values of 76 lM for LeHT1, Figure 3E versus 45 lM

for LeHT2, Gear et al., 2000, respectively).

Carrier function (Figure 3) was further confirmed by inhibi-

tion of transporter activity when transformed yeast cells were

exposed to phlorizin (Table 1), a non-transported competitive

inhibitor of sucrose and glucose carrier proteins (Lemoine and

Delrot, 1987). The prospect of hexose transport utilizing an

ATPase-generated H+ gradient was explored by manipulating

the pmf with the protonophores, 2,4-dintrophenol (DNP), and

carbonyl cyanide (CCCP). Both protonophores significantly re-

duced [14C]glucose and [14C]fructose transport (Table 1). Col-

lectively, these data demonstrate that an inward-directed

pmf across the yeast plasma membrane is required for glucose

as well as fructose uptake by LeHT1 and LeHT2 (also see Gear

et al., 2000 for glucose uptake by LeHT2).

Substrate specificity of each transporter was evaluated by

determining impacts on [14C]glucose or [14C]fructose uptake

in the presence of specified sugar species supplied to the bath-

ing media at 103 greater concentration. Fructose, glucose,

and the non-metabolizable glucose analog, 3-O-methyl glu-

cose, each competed strongly for the binding site of LeHT1

and LeHT2 with transported glucose or fructose (Table 2

and for glucose transport by LeHT2, see Gear et al., 2000). Sur-

prisingly, sucrose was found to exert a profound competitive

effect on fructose, but not glucose, transport by LeHT1 and

LeHT2 (Table 2).

RNAi Suppresses LeHT Gene Expression in Red Ripe and

20-DAA Fruit

In addition to LeHT1-3 genes (Gear et al., 2000; Dibley et al.,

2005), a search of tomato EST databases on the Sol Genomics

Network (http://solgenomics.net/) showed that other hexose

transporter-like genes are expressed in tomato fruit. These in-

clude STP sequences that are not strongly or differentially

expressed in fruit (data not shown), or cluster more closely

with either plastidic or tonoplast hexose transporters (see Sup-

plemental Table 2). Based on this information, the most likely

contributors to hexose accumulation in tomato fruit are

Figure 2. Time Course and pH-Dependence of Hexose Transport by LeHT1 and LeHT2 Expressed in Yeast.

Time course of (A) [14C]glucose and (B) [14C]fructose uptake from media containing 50 lM sugars buffered at pH 5. pH-dependent uptakerates of (C) [14C]glucose and (D) [14C]fructose from HEPES/MES-buffered media containing 50 lM glucose or 800 lM fructose, respectively.Bars are standard errors of means derived from a minimum of four replicates per treatment.

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LeHT1-3, and particularly LeHT3 due to coincidence of its tem-

poral expression profile with hexose accumulation (Dibley

et al., 2005). To test this role of LeHTs, and particularly LeHT3,

LeHTexpression was targeted by intron-mediated hairpin RNA

interference (ihpRNA; Wesley et al., 2001). Two ihpRNA con-

structs were generated using pHANNIBAL (for details, see

Methods and Wesley et al., 2001). In an attempt to selectively

knockdown LeHT3 expression, one construct (ihpRNA–LeHT3)

contained a 201-bp fragment amplified from the 3’-UTR of

LeHT3 and shared a 47.3 and 48.7% nucleotide sequence iden-

tity with LeHT1 and LeHT2, respectively. The second construct

(ihpRNA–LeHT) contained a 401-bp fragment amplified from

a conserved portion of the coding region of LeHT3 that had

a 60.4 and 62.6% sequence identity with LeHT1 and LeHT2, re-

spectively. Transformation of both ihpRNA constructs into cv.

Money Maker resulted in a 20–27% reduction in T1 seed

weight compared with wild-type accompanied by a complete

loss in germination capacity. Therefore, T0 lines carrying single

copies of either the ihpRNA–LeHT3 or ihpRNA–LeHT construct

were propagated clonally to produce experimental material

for analysis (see Methods for details).

Effects of both ihpRNA constructs on LeHT expression in

pericarp of red ripe fruit were determined by real-time PCR.

For reasons unknown, but possibly due to sequence similarities

in the 3’-UTR of the LeHT genes, selective knockdown of LeHT3

expression was not achieved (Table 3). Rather, expression levels

Figure 3. Concentration-Dependent Uptake of Hexoses by LeHT1 and LeHT2 Expressed in Yeast.

Concentration-dependent uptake of fructose (A–D) and glucose (E) facilitated by LeHT1 (A, C, E) or LeHT2 (B, D) from media buffered atpH 5 (A, B, E) or pH 6 (C, D). Bars are standard errors of means derived from a minimum of four replicates per treatment. Apparent Km andVmax values were derived by fitting non-linear regressions of the Michaelis-Menten rate equation to the data using least-squares. Thedegree of fit is indicated by R2 values.

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of all three LeHT genes were knocked down substantially in

three ihpRNA–LeHT3 single copy lines (i.e. LeHT3i–043, –055,

–021) compared to the regeneration control line (LeHTo–

003). Knockdowns ranged from 73 to 96%, with LeHT3i–021

line displaying the highest knockdown, with LeHT3 expression

marginally more depressed than that of LeHT1 and LeHT2

(Table 3). In contrast, impacts on LeHT expression using the

ihpRNA–LeHT construct were more variable. One line

(LeHTi–036) showed no significant effect on LeHT expression

levels compared to LeHTo–003. The remaining two lines

(LeHTi–007, –005) exhibited a knockdown range for LeHT ex-

pression comparable to that elicited by the LeHT3i lines (Table 3

and Supplemental Table 3). Analyzing impacts of ihpRNA on

LeHT gene expression in fruit, harvested 20 d after anthesis

(DAA), was restricted to LeHT3i–021. Similar to red ripe fruit,

maximum knockdown occurred for LeHT3, while LeHT1 and

LeHT2 expression was proportionately less affected (Table 3).

Hexose Transporter Expression Influences Fruit Hexose

and Biomass Accumulation

We analyzed pericarp hexose levels and dry weights in the

three ihpRNA–LeHT3 lines showing knockdown of LeHT gene

expression. Compared to LeHTo–003, hexose concentrations in

bulk pericarp saps of red ripe fruit were reduced by 22–28% in

all three LeHT3i lines (Table 3). Similar relative reductions were

found in LeHTi–007 and LeHTi–005 in which knockdown of

LeHT gene expression averaged 87% (Supplemental Table

3). Significantly, for LeHTi–036, in which LeHT expression

was unaffected, pericarp hexose concentration did not differ

significantly from that of the regeneration control, LeHTo–003

(Supplemental Table 3). This outcome demonstrates a direct

and causal link between levels of LeHT expression and hexose

concentrations in fruit pericarp.

Focusing on the LeHT3i lines (Table 3), reductions in pericarp

hexose concentrations of red ripe fruit were accentuated fur-

ther in estimates of their pericarp hexose contents. These

ranged from reductions of 25% up to 41% for LeHT3i–

021—an outcome arising from the compounding effect of a re-

duced final fruit size in these lines (Table 3). In contrast, at

20 DAA, pericarp hexose concentrations and contents of

LeHT3i–021 did not differ significantly from LeHTo–003, de-

spite a substantial decrease in LeHT expression levels (Table 3).

Accumulation of hexoses from 20 DAA to red ripe was reduced

by 55% for LeHT3i–021 compared with LeHTo–003 (Table 3).

Pericarp biomass exhibited similar responses to fruit hexo-

ses, being unaffected at 20 DAA but reduced at red ripe in

the LeHT3i lines (Table 3). In absolute terms, biomass differen-

ces between LeHT3i lines and regenerated control, LeHTo–003,

largely are accounted for by differences in their hexose con-

tents (Table 3). This finding is consistent with most fruit

Table 2. Sugar Specificity of LeHT1 and LeHT2 Expressed in Yeast.

Hexose transporter Transported sugar

Transport rate (nmol 10�8 cells min�1) in:

Control Fructose Glucose 3-O-methyl glucose Sucrose

LeHT1 Glucose 0.90 0.24 (73) ND 0.01 (99) 0.82 (9)

6 0.01 6 0.01 6 0.06 6 0.06

Fructose 1.00 ND 0.05 (95) 0.06 (94) 0.27 (73)

6 0.10 6 0.01 6 0.01 6 0.02

LeHT2 Fructose 1.00 ND 0.04 (96) 0.12 (88) 0.06 (94)

6 0.05 6 0.01 6 0.05 6 0.01

Effect of competing sugars on rates of [14C]glucose and [14C]fructose uptake facilitated by LeHT1 or LeHT2 expressed in yeast from media buffered atpH 5 and containing 50 lM glucose or 800 lM fructose. Competing sugars were present in the media at concentrations 10-fold higher than thetransported sugar. Mean and standard error of the mean from at least four replicates per treatment. Percentage inhibition is given in parentheses.

Table 1. Metabolic Dependence of Hexose Transport by LeHT1 and LeHT2 Expressed in Yeast.

Hexose transporter Transported sugar

Transport rate (nmol 10�8 cells min�1) in:

Control 1 mM phlorizin 100 lM DNP 50 lM CCCP

LeHT1 Glucose 1.00 0.62 (38) 0.24 (76) 0.57 (43)

6 0.18 6 0.06 6 0.02 6 0.07

Fructose 1.10 0.14 (87) 0.25 (77) 0.11 (90)

6 0.04 6 0.01 6 0.03 6 0.02

LeHT2 Fructose 1.43 1.34 (6) 0.78 (45) 0.67 (53)

6 0.07 6 0.02 6 0.78 6 0.11

Rates of [14C]glucose and [14C]fructose uptake facilitated by LeHT1 or LeHT2 expressed in yeast from media buffered at pH 5 and containing 50 lMglucose or 800 lM fructose with specified metabolic inhibitors. Mean and standard error of the mean from at least four replicates per treatment.Percentage inhibition is given in parentheses.

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biomass being accrued as stored hexoses across this phase of

fruit development (Dibley et al., 2005).

Reduced Hexose Transporter Expression Does Not Alter

Photoassimilate Production or Phloem Transport

Since ihpRNAi was under control of the constitutive CaMV 35S

promoter, reduced hexose accumulation in fruit pericarp (see

Table 3) may have arisen partially or fully from affects on pho-

toassimilate production in leaves or subsequent phloem trans-

port of photoassimilates (sucrose) to developing fruits. These

possibilities were examined by obtaining estimates of photo-

assimilate production and phloem transport delivery to fruit at

the mid-phase of fruit hexose accumulation (i.e. 30 DAA). For

these experiments, we compared the regenerated control line,

LeHTo–003, with the LeHT3i–021 line that showed greatest

knockdown of LeHT expression in red ripe fruit (see Table 3).

Net photosynthetic rates and areas of leaves positioned imme-

diately either side of the test truss were determined to com-

ment on photoassimilate availability to developing fruit.

Coincidentally, rates of phloem delivery to a fruit within each

test truss were obtained by measuring rates of sucrose exuda-

tion from cut pedicel stumps collected by an EDTA bleeding

technique (see Methods). Transcript levels of LeHT1, LeHT2,

and LeHT3 were determined from total RNA extracted from

tissue discs of the test leaves upon completion of phloem

sap collection.

In source leaves of cv. Money Maker, LeHT2 was the most

strongly expressed LeHT gene, with LeHT1 and LeHT3 showing

comparable but 3.2-fold lower levels of expression compared

to LeHT2 (Table 4). Despite lowered LeHT expression in leaves

of LeHT3i–021, leaf areas and light-saturated rates of leaf pho-

tosynthesis were found to be identical to those of LeHTo–003

(Table 4). This result indicates photoassimilate levels available

for fruit hexose accumulation had not been compromised by

ihpRNAi. The recorded rates of light-saturated photosynthesis

are in the reported range for tomato leaves (e.g. Nunes-Nesi

et al., 2007).

Rates of phloem transport could be affected by the sugar

retrieval function of hexose transporters during symplasmic

transit from mesophyll cells to leaf minor veins (Buttner

et al., 2000; Schofield et al., 2009) and competing for sugars

leaked from the phloem translocation stream within stems

(Hafke et al., 2005). Impacts of RNAi knockdown on hexose

transporter expression in stems were not examined where

Table 3. RNAi-Mediated Knockdown of LeHT Expression and Hexose Accumulation in Fruit.

Stage of fruit development and parameter measuredControl

Transgenic (ihpRNA–LeHT3)

LeHTo–003 LeHT3i–043 LeHT3i–055 LeHT3i–021

Red ripe fruit

RNAi knockdown (relative expression)

LeHT1 7.2 1.6 (78) 1.1 (85) 1.1 (85)

6 0.2 6 0.8 6 0.2 6 0.5

LeHT2 10.5 2.0 (81) 1.8 (83) 1.1 (90)

6 1.1 6 0.7 6 0.7 6 0.5

LeHT3 19.3 4.0 (79) 5.3 (73) 0.7 (96)

6 2.6 6 1.3 6 0.8 6 0.3

Hexose concentration (mM) 226.0 6 4.4 177.1 6 5.11 164.6 6 13.41 163.8 6 8.2

Hexose content (g) 1.62 6 0.11 1.21 6 0.18 1.04 6 0.16 0.96 6 0.10

Dry weight (g) 2.82 6 0.20 2.43 6 0.35 2.13 6 0.28 1.95 6 0.11

20-DAA fruit

RNAi knockdown (relative expression)

LeHT1 26.1 nd nd 15.3 (41)

6 4.3 6 2.4

LeHT2 12.2 nd nd 4.0 (67)

6 2.4 6 1.1

LeHT3 41.8 nd nd 3.0 (93)

6 8.3 6 0.2

Hexose concentration (mM) 160.0 6 7.8 nd nd 158.4 6 6.1

Hexose content (g) 0.27 6 0.02 nd nd 0.25 6 0.01

Dry weight (g) 0.84 6 0.09 nd nd 0.83 6 0.04

Analysis of LeHT gene expression, pericarp hexose concentration, hexose content, and dry weight in S. lycopersicum (cv. Money Maker) fruit at redripe and 20 DAA in three lines of transgenic plants expressing the ihpRNA–LeHT3 construct (LeHT3i–043, –055, –021) and a control line (LeHTo–003).Data are presented as mean and standard error of the mean from at least four replicates per treatment. The percentage reduction in LeHT relativeexpression compared to the control is indicated in brackets.

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each of the three LeHT isoforms is expressed equally (Gear

et al., 2000). Comparable rates of sucrose exudation between

genotypes from cut stumps of their fruit pedicels indicated

that phloem loading and longitudinal transport were not com-

promised by reduced hexose transporter expression (Table 4).

Estimated rates of sucrose exudation exceeded (by ;20%) in

planta rates of phloem import estimated from dry matter gains

by fruit (data not shown). Together with the absence of hexo-

ses in collected sap exudates (data not shown), this observation

provides increased certainty that sucrose exudation rates from

cut pedicel stumps provided representative measures of the

potential capacity of the phloem to support import rates into

developing fruit.

DISCUSSION

Most hexose transporters functionally characterized to date

are those from Arabidopsis (eight, Buttner, 2007) and rice

(four, Wang et al., 2008 and publications cited therein). Based

on their expression patterns, these transporters serve to re-

trieve hexoses lost to apoplasms of source or sink tissues

and in fueling metabolic requirements of specialized cell types

such as guard cells and pollen grains (Buttner, 2007). Hexose

concentrations accumulated by tissues/cells in these physiolog-

ical contexts rarely exceed 10 mM. In contrast, less is known

about hexose transporters expressed in organs that accumu-

late these sugars to high concentrations (.100 mM), such as

tap roots (e.g. carrot, Tang et al., 1999) and fleshy fruits (apple,

Zhang et al., 2004; grape, Vignault et al., 2005; Hayes et al.,

2007; tomato, Gear et al., 2000; Dibley et al., 2005). The current

study builds on our knowledge of hexose transporters

expressed in tomato fruit (Gear et al., 2000; Dibley et al.,

2005) where hexoses accumulate to concentrations of up to

200 mM in their storage parenchyma cells (Ruan et al.,

1996). During the storage phase of sugar accumulation,

phloem-imported sucrose released to the fruit apoplasm (Ruan

and Patrick, 1995) is hydrolyzed by extracellular invertases (Ho,

1996). The resulting hexose moieties are transported from the

fruit apoplasm into the storage parenchyma cells (Brown et al.,

1997; Ruan et al., 1997). Thus, the biochemical properties of

hexose transporters active in fruit storage parenchyma cells

(i.e. LeHT1, LeHT2, and LeHT3, Dibley et al., 2005) are central

to the sugar accumulation process.

Phylogenetic analysis (Figure 1) demonstrated that LeHT1,

LeHT2, and LeHT3 share amino acid sequence identity with

the STP sub-family of monosaccharide transporters (Buttner,

2007). Transporters that have been characterized functionally

in this sub-family mediate active membrane transport of hexo-

ses in symport with protons returning down their electrochem-

ical gradient. For example, eight STPs from Arabidopsis

(Buttner, 2007), four from grape (Vignault et al., 2005; Hayes

et al., 2007) and four from rice (Wang et al., 2008), are known

to function as hexose/H+ symporters. Added to this growing list

are LeHT2 (Gear et al., 2000) and now LeHT1 (see Figures 2 and

3, and Table 1). The presence of hexose/H+ symporters in the

storage parenchyma cells of tomato fruit is a prerequisite to

account for the four-fold concentrating step for glucose and

fructose between the fruit apoplasm and protoplast compart-

ments of storage parenchyma cells (Ruan et al., 1996).

The amino acid sequence of LeHT3 (Supplemental Figure 1)

exhibited all the features of known plant hexose transporters,

including the highly conserved motif SWGP(M/L)GW(L/T) (V/

I)PSE403–414LeHT1 (Sauer and Stadler, 1993) as well as Q178,

Q296, V429, and V432 residues known to affect hexose trans-

port kinetics (Will et al., 1994, 1998). However, transformation

of the hexose transporter null mutant of yeast, EBY.VW4000

(Wieczorke et al., 1999), with sequence-verified clones of

LeHT3, did not result in the transgenic yeast cells supporting

hexose transport. This outcome occurred despite expression

of LeHT3 message in EBY.VW4000, as verified by RT–PCR (Sup-

plemental Table 1), and trafficking of LeHT3 protein to the

yeast plasma membrane as determined by a LeHT3–GFP fusion

protein (Supplemental Figure 3). Assuming the LeHT3–GFP fu-

sion protein is properly folded in the yeast plasma membrane,

this latter observation raises the intriguing possibility that

LeHT3 requires post-translational modification for activity in

yeast. Failures to express functional membrane proteins, in-

cluding hexose transporters (e.g. see Toyofuka et al., 2000;

Hayes et al., 2007), are not uncommon and the underlying

causes remain obscure. Expression profiles of grape (VvHT3,

Hayes et al., 2007) and tomato (LeHT3, Dibley et al., 2005) hex-

ose transporters indicate these are the most likely candidates

to support sugar uptake in berry and fruit tissues, respectively,

during the major stage of storage product accumulation. In-

triguingly, these hexose transporters, which share high amino

acid sequence identity (75% and see Figure 1), were both

Table 4. Photoassimilate Production and Phloem Transport areUnaffected by RNAi-Knockdown.

Parameter measured

Genetic line

LeHTo–003 LeHT3i–021

RNAi knockdown (relative expression)

LeHT1 34.7 14.7 (58)

6 4.8 6 1.7

LeHT2 111.2 74.2 (33)

6 3.2 6 11.6

LeHT3 33.9 5.3 (84)

6 4.9 6 1.6

Leaf area (3 10�2 m2) 2.37 6 0.14 2.41 6 0.16

Leaf photosynthesis(lmol CO2 m�2 s�1)

13.4 6 0.3 12.5 6 0.8

Rate of phloem delivery(lmol sucrose h�1)

10.9 6 1.5 10.8 6 2.2

Analysis of LeHT gene expression, leaf area, leaf photosynthesis, andrate of phloem delivery in source leaves of S. lycopersicum (cv. MoneyMaker) transformed with the ihpRNA–LeHT3 construct (021) anda control line (003). Data are presented as mean and standard error ofthe mean from at least four replicates per treatment. The percentagereduction in LeHT relative expression compared to control is indicatedin parentheses.

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found to be incapable of complementing EBY.VW4000 (Hayes

et al., 2007; this study). Further work is required to determine

whether these transporters require post-translational modifi-

cation in planta to achieve transport capacity (e.g. see Conde

et al., 2006; Krugel et al., 2008), and whether lack of this post-

translational machinery in yeast explains the inability of these

two transporters to complement hexose transport-deficient

yeast mutants.

Flow of hexoses, generated by extracellular hydrolysis of

phloem-imported sucrose, from the fruit apoplasm to ulti-

mate vacuolar compartmentation in storage parenchyma

cells of tomato fruit relies on hexose transporters to facilitate

their inward movement across the plasma and tonoplast

membranes. Based on findings for STP homologs in Arabi-

dopsis (Buttner, 2007), grape (Vignault et al., 2005; Hayes

et al., 2007), and rice (Wang et al., 2008), an expectation

would be that LeHT1, LeHT2, and LeHT3 localize to cell

plasma membranes—an expectation supported by the obser-

vation that LeHT2 and LeHT3 are targeted to the plasma

membrane in yeast. Transport from cytoplasm to vacuole

across the tonoplast membrane is facilitated in tomato fruit

(Milner et al., 1995) but genes encoding these transporters

are yet to be identified. These genes could be homologs of

tonoplast-localized transporters thus far cloned from Arabi-

dopsis (AtTMT1-3, Carter et al., 2004; Wormit et al., 2006;

AtVGT1, Aluri and Buttner, 2007) and pineapple (AcMST1,

Antony et al., 2008).

Equimolar concentrations of glucose and fructose are lo-

cated in both apoplasmic and protoplasmic compartments

of developing tomato fruit (Ruan et al., 1996), indicating a re-

quirement for comparable capacity for membrane transport of

each hexose. Heterologous expression in yeast demonstrated

that LeHT1 and LeHT2 were capable of transporting glucose

and fructose (Figures 2 and 3) with evidence that both hexoses

competed for the same binding site on each transporter

(Table 2; also see Gear et al., 2000). Indeed, LeHT1 was found

capable of supporting yeast growth on a fructose medium

(Supplemental Figure 2). This contrasts with most members

of the STP sub-family of monosaccharide transporters that ex-

hibit a strong to exclusive preference for glucose over fructose

(Buttner, 2007)—a property shared by all four of the grape

hexose transporters functionally characterized to date

(Vignault et al., 2005; Hayes et al., 2007). However, expression

profiles of these grape hexose transporters indicate they are

unlikely to contribute significantly to hexose accumulation

in developing berries (Vignault et al., 2005; Hayes et al.,

2007). This physiological role is conjectured to be the preserve

of VvHT3 that is yet to be characterized functionally (Hayes

et al., 2007). The notable exceptions to the glucose specificity

of STP-sub-family members (Buttner, 2007) are the sub-group

of transporters phylogenetically linked with LeHT2 (Figure 1).

In all cases, fructose competed with glucose for their trans-

porter binding sites (AtSTP13, Nørholm et al., 2006; LeHT2,

Table 2; OsMST4, Wang et al., 2008; VvHT5, Hayes et al.,

2007) and where tested, these transporters were found to

be capable of facilitating fructose transport (AtSTP13,

Nørholm et al., 2006; LeHT2, Figures 2 and 3; OsMST4, Wang

et al., 2007, 2008). The rice hexose transporter, OsMST4, exhib-

its the greatest capacity for fructose transport (Wang et al.,

2007, 2008) and a comparable pH optimum to that of LeHT2

(Figure 2D). These properties might be attributed to a change

in one amino acid at the highly conserved sugar binding site of

plant hexose transporters as shown by the selective shift in

glucose affinity and pH optima (from 4.5 to 7) of the HUP1

transporter of Chlorella by the amino acid substitution D44E

(Will et al., 1998). The conundrum to the above argument is

to reconcile this with the observed close identity of LeHT1 with

a sub-group of STP transporters (Figure 1) that under the test

conditions applied, commonly exhibit low or non-existent af-

finities for fructose (e.g. AtSTP1, Boorer et al., 1994; AtSTP4,

Truernit et al., 1996; VvHT1, Vignault et al., 2005 but cf. LpSTP1,

Szenthe et al., 2007). However, it is pertinent to point out that

these past studies employed bathing media set at pH values

optimal for glucose uptake (i.e. ,pH 5) that are well below

the more basic pH optima observed for fructose transport

(Wang et al., 2007, 2008; Figure 2C and 2D). This could account

for findings of low or non-detectable capacities of these mem-

brane proteins to facilitate fructose transport. Indeed, pH-

dependent changes in tertiary structures of LeHT1 and LeHT2

hexose-binding sites clearly allow sucrose to become a potent

competitor with fructose but not glucose transport (Table 2).

Such competition has been observed in the reverse direction

for a sucrose transporter from Phaseolus vulgaris, PvSUF1

(Zhou et al., 2007). These findings build a case that some iso-

forms of hexose and sucrose transporters do not discriminate

strongly between sucrose and hexoses.

Carrier-mediated uptake of hexoses by tissue slices of to-

mato fruit pericarp exhibit apparent Km values of 15 mM

(Ruan et al., 1997). The extended diffusive pathway for sugar

movement to plasma membranes of storage cells in tissue sli-

ces, compared to that encountered in yeast, likely contributes

to elevating apparent Km values of plasma membrane trans-

porters by an order of magnitude (see Zhou et al., 2007 for su-

crose transporter comparisons). Working on this assumption,

plant hexose transporters likely to contribute to hexose uptake

in fruit would be expected to exhibit apparentKm values in the

range of 1–10 mM when expressed in yeast. This criterion pre-

cludes LeHT1 and LeHT2 making significant contributions to

the in planta glucose flux, as their apparent Km values are

70 (Figure 3E) and 45 (Gear et al., 2000) lM, respectively. These

values are in the range recorded for Arabidopsis (Buttner,

2007) and grape (Vignault et al., 2005; Hayes et al., 2007)

STP sub-family members but an order of magnitude lower than

those reported for rice STPs (i.e. 0.2–0.3 mM, Wang et al., 2007,

2008 and references cited therein) along with AtSTP3 (Buttner

et al., 2000). Rice hexose transporters exhibit identical affini-

ties for glucose and fructose (Wang et al., 2008). In contrast,

LeHT1 and LeHT2 apparent Km values for fructose exceeded

those for glucose by 65 and 26-fold, respectively (Figure 3A–

3D), and fall in the expected mM range of in planta affinities

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for hexose transport (Ruan et al., 1997). Similar to AtSTP3

(Buttner et al., 2000), these high apparent Km values were

not caused by the transporters functioning as low-affinity

facilitators due to deprotonation (Komor and Tanner, 1975).

Rather, both LeHT1 and LeHT2 function as proton-coupled

symporters as demonstrated by slowed fructose uptake in

the presence of metabolic inhibitors (Table 1). The observed

increases (2.13 and 1.83, respectively) in maximal transport

velocities by LeHT1 and LeHT2 for fructose in response to

a pH shift from 5 to 6 (see Figure 3A–3D) indicate that mem-

brane potential differences rather than transmembrane pro-

ton gradients (?pH) are the principal driver of fructose

transport. Under these conditions, membrane potential differ-

ences are known to increase whilst ?pH decreases (Boxman

et al., 1984). In this context, for in planta transporter perfor-

mance, it is of relevance to note that apoplasmic saps collected

from developing tomato fruit have pH values of 6 (Ruan et al.,

1996). This characteristic provides optimal conditions for fruc-

tose uptake by fruit storage parenchyma cells facilitated by

LeHT1 and LeHT2 (Figure 2C and 2D, and Figure 3A–3D) at

the beginning and conclusion of the major phase of hexose

accumulation (Dibley et al., 2005), respectively.

An appraisal of tomato EST databases (see Supplemental

Figure 2 and associated text in Results) led to the conclusion

that LeHT1-3, reported previously by Gear et al. (2000) and

Dibley et al. (2005), are most likely the main contributors to

hexose accumulation in fruit. An opportunity of testing the

proposed central role of LeHTs in driving hexose accumulation

was afforded by our ihpRNA approach that caused a substan-

tial reduction in expression levels of all three LeHT genes

(Table 3 and Supplemental Table 3). Under these conditions,

fruit hexose concentrations and contents were reduced in

red ripe fruit by up to 40% in the three ihpRNA–LeHT3 trans-

genic lines (Table 3) and two of the three ihpRNA–LeHT lines

analyzed (Supplemental Table 3). Moreover, quantities of hex-

ose accumulated from 20 DAA to red ripe were reduced by

55% (Table 3). This reduction compares favorably with esti-

mates, based on in planta transport kinetic properties, of

transporter activity accounting for 70–80% of hexoses accu-

mulated by fruit storage cells (Brown et al., 1997; Ruan

et al., 1997). The large quantitative decline in hexose accumu-

lation by developing fruit occurred in the absence of any neg-

ative effect on photoassimilate production in their source

leaves and on the capacity of phloem to deliver sucrose as sub-

strate for hexose accumulation (Table 4). Together, these

observations indicate that hexose transporters expressed in

fruit alone, and possibly LeHT3 in particular, play a primary role

in hexose accumulation by developing fruit. This conclusion is

re-enforced by the finding that prior to switching to an apo-

plasmic step in the phloem-unloading pathway (20 DAA and

see Ruan and Patrick, 1995), fruit hexose concentrations and

contents were unaffected despite transporter expression be-

ing repressed in ihpRNA–LeHT3 fruit (Table 3).

Membrane transporter activity prior to the pathway switch

from symplasmic to an apoplasmic phloem-unloading route

would be restricted to serving a retrieval role for symplasmic

delivery of phloem-imported sucrose to fruit storage paren-

chyma cells (Ruan and Patrick, 1995). The absence of a pheno-

type in fruit up to 20 DAA also extended to the vegetative

plant body (data not shown) despite the considerable

down-regulation of LeHT expression in leaves of the LeHT3i–

021 line (Table 4). This finding is consistent with published

reports for T-DNA knockouts of AtSTP1 (Sherson et al., 2000)

and AtSTP13 (Schofield et al., 2009) as well as co-suppression

of MST1 in tobacco transformed with VvHT1 (Leterrier et al.,

2003). However, a depleted growth response was evinced

on exposing VvHT1 transgenic tobacco to low carbon condi-

tions (Leterrier et al., 2003). Similarly, overexpression of

AtSTP13 only elicited increased biomass accumulation when

transgenic plants were raised on media containing a supple-

ment of glucose or sucrose (Schofield et al., 2009). Collectively,

these observations suggest that under optimal conditions for

vegetative growth, an excess capacity and redundancy in

hexose transporter activity exist to meet low-level retrieval

functions. In contrast, accumulating hexoses to high concen-

trations across plasma membranes (Ruan et al., 1996, 1997)

is crucially dependent upon hexose transporter capacity as

demonstrated in this study for tomato fruit.

The LeHT transporters function to concentrate hexoses in

storage parenchyma cells (Ruan et al., 1997) by mediating

an energy-coupled hexose uptake (Table 1) across storage cell

plasma membranes from the fruit apoplasm following hydro-

lysis of phloem-unloaded sucrose by an extracellular invertase

(Jin et al., 2009; Zanor et al., 2009). The extracellular invertases

and hexose transporters are arranged in series and form a func-

tional unit to deliver hexoses as developmental signals as well

as substrate for cell metabolism and storage (Jin et al., 2009;

Zanor et al., 2009).

METHODS

Cloning of Hexose Transporter cDNAs

Our earlier study (Gear et al., 2000) reported the isolation of

a full-length (LeHT2) and two partial (LeHT1 and LeHT3) cDNA

clones from tomato (Solanum lycopersicum). 5’-RACE (Clone-

tech SMART� RACE cDNA Amplification kit) was used to ob-

tain full-length sequence information for LeHT1 and LeHT3

using total RNA isolated from fruit harvested 10–20 d after an-

thesis (DAA) using the RNeasy Plant Mini-Kit (Qiagen). Full-

length clones of LeHT1 and LeHT3 were amplified by PCR from

fruit total RNA using Pfu DNA polymerase (Promega) and then

cloned into pGEM-T Easy (Promega) after A-tailing. Final se-

quence for both LeHT genes was verified by sequencing three

clones in pGEM-T Easy from three independent amplification

reactions. Genbank Accession numbers for LeHT1 and LeHT3

are GQ911580 and GQ911581, respectively.

Phylogenetic Analysis

Phylogenetic analysis, comparing predicted LeHT protein

sequences with selected higher plant hexose transporters,

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was performed by Phylogeny.fr (www.phylogeny.fr; Dereeper

et al., 2008) using ClustalW for multiple alignments, Gblocks

for alignment curation, PhyML for construction of the phylo-

genetic tree using Maximum Likelihood, and TreeDyn for

display.

Functional Analysis in Yeast

Full-length cDNA clones of LeHT1 and LeHT3 were ligated in-

to the yeast expression vector, pDR195 (Rentsch et al., 1995),

and transformed by electroporation into yeast strain

EBY.VW4000 (Wieczorke et al., 1999). Transformation also

was carried out with the vector without an insert. Correct in-

corporation of identical full-length cDNAs after yeast trans-

formation was verified by sequencing plasmid DNA

recovered from yeast. Colonies containing verified hexose

transporter genes were prepared as glycerol stocks for fur-

ther assays. For complementation assays, single colonies of

each hexose transporter clone, as well as the empty vector,

were streaked onto 2% maltose plates (ura–), and single col-

onies re-streaked onto plates (ura–) containing 2% maltose,

glucose, or fructose. Plates were incubated at 30�C for up to

10 d for observation. For hexose uptake assays, single colo-

nies of yeast strain EBY.VW4000, containing a single trans-

porter gene or empty vector, were grown at 30�C in

minimal medium containing 2% maltose to an OD600 of

0.8. Cells were harvested by centrifugation, washed twice,

and re-suspended in 25 mM HEPES–MES, pH 5, unless speci-

fied otherwise. For pH-dependence assays, solutions at spec-

ified pH values were buffered with 25 mM HEPES–MES. A

culture containing 1 3 108 yeast cells was used. Cells were

energized by adding ethanol (100 mM final concentration)

to bathing media containing yeast cells 1 min before

their exposure to [14C]glucose or [14C]fructose in specified

carrier sugar concentrations. For inhibitor assays, final con-

centrations of 50 lM carbonyl cyanide (CCCP), 100 lM 2,4-

dinitrophenol (DNP), or 1 mM phlorizin were added to

energized yeast cells 30 s before addition of a specified

[14C]hexose. For sugar specificity assays, a 10-fold excess of

competing sugar species was used. After incubation for spec-

ified times at 25�C with shaking, cells were collected onto

microfiber filters (0.7 lm pore, GF/C; Whatman) under vac-

uum. Filters were washed twice rapidly with 4 mL of ice-cold

water and thereafter radio-assayed by liquid scintillation

counting. The empty vector transport rates were subtracted

from all transport data except those involving time courses of

hexose uptake.

Analysis of LeHT Gene Expression and Cellular Targeting

of LeHT–GFP Fusion Proteins in Yeast

Total RNA was extracted from transformed yeast using the

RNeasy Mini Kit (Qiagen) and reverse transcribed to cDNA us-

ing a QuantiTect� Reverse Transcription Kit (Qiagen) as per the

manufacturer’s instructions. Semi-quantitative PCR was per-

formed to detect LeHT1, LeHT2, and LeHT3 expression relative

to the RPS3 (ribosomal protein S3) gene in the relevant yeast

strain. For cellular targeting studies, LeHT2 and LeHT3 coding

sequences (minus stop codons) were amplified from their re-

spective pGEM-T plasmids using sequence-specific primers car-

rying SalI and PstI restriction sites on each forward and reverse

primer, respectively. The coding sequence of mGFP6 was am-

plified from pMDC85 (Curtis and Grossniklaus, 2003) using for-

ward and reverse primers carrying PstI and EcoRI restriction

sites, respectively. The relevant hexose transporter amplicons

and mGFP6, each double digested with appropriate restriction

sites, were ligated and inserted into the SalI–EcoRI site of pEN-

TR1A dual selection vector (Invitrogen). The generated plas-

mids were then recombined with pDR196/GW destination

vector (Loque et al., 2007) using LR Clonase Plus (Invitrogen).

The recombinant yeast expression vectors (pDR196/GW:LeHT2/

3–GFP) were transformed into EBY.VW4000 yeast cells and

grown on glucose or maltose plates using ura– selection. Yeast

cells were viewed using a confocal microscope (FV1000, Olym-

pus, Japan) equipped with a 603, 1.3 Numerical Aperature oil

immersion objective and using a 473-nm laser with 500–530 IR

filter.

RNAi Construct Preparation, Transformation, and Plant

Culture

A 201-bp fragment from the 3’-UTR of LeHT3 cDNA (bases

1503–1712) and a 401-bp coding region of LeHT3 (bases

992–1393) were amplified by PCR and introduced in sense

and antisense orientations into the ihpRNA vector pHANNI-

BAL (Wesley et al., 2001) to generate two constructs, ihpRNA–

LeHT3 and ihpRNA–LeHT, respectively. Each pHANNIBAL

construct was sub-cloned into the binary vector pART27

(Gleave, 1992) placing expression of each ihpRNA construct

under control of the CaMV 35S promoter to provide strong

RNAi suppression consistently across all stages of fruit

development (Estornell et al., 2009). These ihpRNA constructs

were transformed into tomato (cv. Money Maker) by

Agrobacterium-mediated transformation according to the

procedure described in Davuluri et al. (2005). Fifty and 58 pri-

mary transgenic lines (T0) were obtained from transforma-

tion with ihpRNA–LeHT3 and ihpRNA–LeHT, respectively,

and of those, 21 and 18, respectively, were identified by real-

time PCR to be carrying single copies of the relevant trans-

gene (data not shown). T1 seed from these single copy

suppression lines (ihpRNA–LeHT3 and ihpRNA–LeHT) showed

20–27% reduction in average seed weight compared with

wild-type plants. Germination percentage for this popula-

tion of seed was also substantially reduced compared to

wild-type, and of those that did germinate, genomic PCR

analysis (.30 tested from both pools) revealed that none car-

ried the ihpRNA constructs. Consequently, clonal propaga-

tion of the original T0 plants was used to generate new

individuals from which all subsequent analyses were per-

formed. For control ‘wild-type’, we chose line LeHTo–003 re-

covered as kanamycin resistant from transformation with the

ihpRNA–LeHT3 construct lacking the ihpRNA–LeHT3 trans-

gene as determined by real-time PCR (data not shown).

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Plants were grown from cuttings of lateral shoots in pas-

teurized potting mix held in a glasshouse under partially con-

trolled conditions, with day and night temperatures ranging

between 24–26 and 18–22�C, respectively. Plants were exposed

to a 16-h photoperiod, with natural lighting supplemented by

metal-halide lamps to ensure a light intensity of at least

100 lmol quanta m�2 s�1 photosynthetically active radiation

throughout the photoperiod.

Collection and Sugar Analysis of Fruit Pericarp

Flowers were tagged at anthesis and pruned to three per truss.

Upon reaching 20 DAA and red ripe, fruit were harvested onto

ice and immediately transferred to the laboratory. Following

determination of fruit fresh weight, the outer pericarp of each

fruit was excised surgically and weighed. Tissue samples from

the pericarp equatorial region were surgically removed and di-

vided into two groups—one for total RNA extraction and the

other for collection of bulk sap for sugar analysis. Both tissue

samples immediately were snap-frozen in liquid nitrogen and

stored at –80�C until processed for transporter gene expression

or sugar analysis. The residual pericarp tissue of each fruit rep-

licate was re-weighed, dried to constant weight in a forced

draught oven at 80�C prior to determining their dry weights

from which estimates of percent dry weight and water content

were obtained.

Undiluted bulk tissue sap was collected by thawing each fro-

zen pericarp sample followed by centrifugation at 4000 g at

4�C for 20 min through a filtered syringe barrel. Eleven lL

of each sap sample, plus 5 lL of a 2 mg mL�1 ribitol internal

standard, was dried by vacuum centrifugation. Derivatiza-

tion/trimethylsilylation and quantification of sugars by GC–

MS were performed as described by Roessner et al. (2006) with

the following modifications. The GC–MS system used was

a 5973A MSD (Agilent Technologies), GC was performed on

a 30-m SGE BPX5 column, and both chromatograms and mass

spectra were evaluated using the Enhanced MSD ChemStation

D.01.02 software (Agilent Technologies). Mass spectra of elut-

ing sugars were identified using the commercially available

mass spectra libraries of the National Institute of Standards

and Technology, the Fiehn GC/MS Metabolomics RTL Library,

and the Wiley Registry� of Mass Spectral Data, and all com-

pounds were verified by subsequent analysis of pure stand-

ards. Absolute quantification of sugars was achieved by

equating normalized areas to a set of standard curves for su-

crose, glucose, and fructose.

Total RNA Extraction from Fruit and Real-Time PCR of LeHT

Gene Expression

Total RNA was extracted from frozen pericarp segments using

an RNeasy Plant Mini Kit (Qiagen) and reverse transcribed to

cDNA using a QuantiTect� Reverse Transcription Kit (Qiagen)

as per the manufacturer’s instructions. Real-time quantitative

PCR was performed using GoTaq� qPCR Master Mix (Promega)

on a Corbett RG-6000 cycler as per the manufacturer’s instruc-

tions, with a hot start at 95�C for 2 min and 40 cycles of a two-

step (95 and 60�C) PCR program. Quantitative analysis was per-

formed as described by Pfaffl (2001) using EF1-a as a house-

keeping gene.

Estimating Rates of Photoassimilate Production and

Phloem Transport

The analysis was conducted on a truss containing fruit at

30 DAA with measures of leaf photosynthesis and phloem

transport undertaken during the mid-component of the pho-

toperiod. Photoassimilate production was evaluated by esti-

mating photosynthetic rates and areas of the two leaves

inserted immediately either side of each test truss as described

below.

Steady-state net rates of photosynthesis of two opposing

leaflets of each leaf were measured using a Li-Cor 6400 porta-

ble infrared gas analyzer (Li-Cor Biosciences, Lincoln, NE, USA)

configured in the open position at light saturation (600 lmol

quanta m�2 s�1 of photosynthetic active radiation), a gas

stream carbon dioxide concentration held at 380 ppm, and

a leaf temperature of 28�C. Thereafter, leaf discs, 8 mm in di-

ameter, were excised with a cork borer from these leaf sites,

taking care to avoid major veins. The tissue discs from each leaf

were bulked, immediately snap-frozen in liquid nitrogen, and

stored at –80�C until processed for total RNA extraction and

determination of LeHT isoform expression by real-time PCR

(see previous section). At the conclusion of collecting phloem

sap (see below), the test leaves were excised and held between

sheets of water saturated paper on ice. On transfer to the

laboratory, the area of each leaf was determined using

a calibrated Delta-T leaf area meter (Delta-T Devices Ltd, Cam-

bridge, UK).

Following determination of photosynthetic rates and col-

lecting leaf discs, one fruit from each test truss was excised

by severing its pedicel immediately above knuckle using

a sharp razor whilst continuously irrigating the pedicel with

a 20-mM EDTA solution buffered at pH 7.0 with 100 mM

Tris-HCl. Thereafter, the cut surface of each pedicel was im-

mersed in a 0.8% agarose gel prepared using the EDTA

solution described above and contained in a 0.5-mL microcen-

trifuge tube. Once positioned correctly, each tube was sealed

onto the pedicel stump with a strip of Parafilm to hold it in

place and prevent evaporative loss. By chelating calcium, EDTA

acts to prevent sieve pore occlusion by callose such that

phloem sap is freely exuded from the cut ends of severed sieve

tubes (Valle et al., 1998). Collecting phloem exudates was ter-

minated after 6 h by removing the agarose tubes, immediately

snap-freezing these in liquid nitrogen, and thereafter storing

at –80�C. The solution phase was collected from the agarose

matrix by centrifugation and processed for sugar analysis as

described for the bulk saps derived from fruit pericarps (see

above).

SUPPLEMENTARY DATA

Supplementary Data are available at Molecular Plant Online.

1060 | McCurdy et al. d Characterization of Tomato Hexose Transporters

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FUNDING

The research described was supported by an ARC SPIRT–APA (Indus-

try) Grant (C00002632) in partnership with Seminis Vegetable Seeds

Inc. and a University of Newcastle Pilot Project award. Stephen Dib-

ley is grateful for a Research Higher Degree scholarship jointly

funded by the SPIRT-APA (Industry) Grant.

ACKNOWLEDGMENTS

We thank Brett Chambers for excellent technical assistance with the

transport studies, Peter Waterhouse for supplying the pHANNIBAL

construct and advice in its use, Joe Palys (Seminis Vegetable Seeds

Inc.) for performing the transformation work, and Kevin Stokes for

provision of healthy experimental plant material. No conflict of

interest declared.

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