The Sugar Transporter Inventory of Tomato: Genome-Wide
Identification and Expression Analysis
Stefan Reuscher1,4, Masahito Akiyama1,4, Tomohide Yasuda1, Haruko Makino1, Koh Aoki2,
Daisuke Shibata3 and Katsuhiro Shiratake1,*
Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, 464-8601 Japan
Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Gakuen-cho, Sakai, 599-8531 Japan
3
Kazusa DNA Research Institute, Kazusa-kamatari, Kisarazu, 292-0818 Japan
4
These authors contributed equally to this work.
*Corresponding author: E-mail, [email protected]; Fax: +81-52789-4026.
(Received January 8, 2014; Accepted March 31, 2014)
2
The mobility of sugars between source and sink tissues in
plants depends on sugar transport proteins. Studying
the corresponding genes allows the manipulation of the
sink strength of developing fruits, thereby improving
fruit quality for human consumption. Tomato (Solanum
lycopersicum) is both a major horticultural crop and
a model for the development of fleshy fruits. In this
article we provide a comprehensive inventory of
tomato sugar transporters, including the SUCROSE
TRANSPORTER family, the SUGAR TRANSPORTER
PROTEIN family, the SUGAR FACILITATOR PROTEIN
family, the POLYOL/MONOSACCHARIDE TRANSPORTER
family, the INOSITOL TRANSPORTER family, the
PLASTIDIC GLUCOSE TRANSLOCATOR family, the
TONOPLAST MONOSACCHARIDE TRANSPORTER family
and the VACUOLAR GLUCOSE TRANSPORTER family.
Expressed sequence tag (EST) sequencing and phylogenetic
analyses established a nomenclature for all analyzed tomato
sugar transporters. In total we identified 52 genes in tomato
putatively encoding sugar transporters. The expression of 29
sugar transporter genes in vegetative tissues and during fruit
development was analyzed. Several sugar transporter genes
were expressed in a tissue- or developmental stage-specific
manner. This information will be helpful to better understand source to sink movement of photoassimilates in
tomato. Identification of fruit-specific sugar transporters
might be a first step to find novel genes contributing to
tomato fruit sugar accumulation.
Abbrevations: BLAST, base local alignment search tool; DAP,
days after pollination; ERD6, early response to dehydration 6;
EMS, ethylmethane sulfonate; EST, expressed sequence tag;
HT, hexose transporter; INT, inositol transporter; MST,
monosaccharide transporter; pGlcT, plastidic glucose translocator; PM plasma membrane; PMT, polyol monosaccharide
The nucleotide sequences reported in this paper have been
submitted to the DNA Database of Japan (DDBJ) with the
accession number: AB845639–AB845668.
Introduction
In plants, carbohydrates are used as a universal energy currency,
e.g. in the form of sucrose, as building blocks for cell walls, for
signaling, to maintain osmotic homeostasis under certain abiotic
stress conditions and for various other purposes. Carbohydrates
are also one of the most important sources of calories and taste
in cultivated crops and are thus critical for both the nutritional
and the commercial value of crops. Apart from sucrose, monosaccharides such as glucose, fructose, galactose, mannose or
ribose play crucial roles as sources of energy and as metabolites.
Also two classes of carbohydrates related to sugars have complementary roles in plants. Sugar alcohols (also called polyols)
such as sorbitol, mannitol or inositol and organic acids such as
malate or citrate have a variety of functions, often characteristic
of a distinct clade of plants.
Movement of sugars on the whole-plant level is controlled by
loading and unloading of transport tissues, thereby creating sink
and source organs (Bresinsky and Körner 2011). Typical sources
are photosynthetically active tissues, but storage organs, such as
tubers, bulbs or the starchy endosperm of graminaceous plants,
can also act as source tissues. In contrast to this, sink tissues rely
on the supply of photoassimilates from other parts of the plants.
These tissues typically include the roots and leaves during early
stages of development, but developing buds, flowers and fruits
Plant Cell Physiol. 55(6): 1123–1141 (2014) doi:10.1093/pcp/pcu052, available FREE online at www.pcp.oxfordjournals.org
! The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Plant Cell Physiol. 55(6): 1123–1141 (2014) doi:10.1093/pcp/pcu052 ! The Author 2014.
Editor-in-Chief’s choice
Keywords: EST Fruit development Gene expression analysis Sugar transporter Tomato.
transporter; RT-PCR, real-time PCR; SFP, sugar facilitator protein; SOT, sorbitol transporter; ST, sugar transporter; STP,
sugar transporter protein; SUT, sucrose transporter; TMD,
transmembrane domain; TMT, tonoplast monosaccharide
transporter; VGT, vacuolar glucose transporter; VM, vacuolar
membrane.
Regular Paper
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S. Reuscher et al.
are also considered sink tissues. In the majority of plant species
sucrose is the preferred long-distance transport form of carbohydrates in the phloem. Both loading and unloading from
phloem vessels or from companion cells require active transmembrane transport of sugars (Turgeon and Wolf 2009). On
the intracellular level, sugars are transported between different
compartments. Most notably, the vacuole serves as a storage
organ for diverse carbohydrates (Shiratake and Martinoia
2007). The transport of sugars across the plasma membrane
(PM) or the vacuolar membrane (VM) is mediated by transport
proteins either utilizing active transport mechanisms or enabling
passive diffusion at an accelerated rate. In the case of source to
sink transport, active loading and unloading processes are essential to create a sugar concentration gradient which drives the
movement of water and solutes in the phloem.
While the sucrose transporter family (SUCs or SUTs) is a
rather small protein family with three members in tomato and
nine members in Arabidopsis, the proteins that transport monosaccharides are a lot more diverse. Up to 53 members in
Arabidopsis were reported (Doidy et al. 2012). They are separated
into seven families, which can be found across the plant kingdom, including mosses. This suggests that the presence of these
seven families represents an evolutionarily ancient condition
(Johnson et al. 2006). The currently recognized subfamilies are,
in the sequence of descending number of members in tomato:
the SUGAR TRANSPORTER PROTEIN family (STPs or MSTs for
monosaccharide transporters or HT for hexose transporters), the
SUGAR FACILITATOR PROTEIN family (SFPs) which is also
called the EARLY RESPONSE TO DEHYDRATION 6-like (ERD6like) family, the POLYOL/MONOSACCHARIDE TRANSPORTER
family (PMTs or PLTs), the INOSITOL TRANSPORTER FAMILY
(INTs or ITRs), the TONOPLAST MONOSACCHARIDE
TRANSPORTER family (TMTs), the PLASTIDIC GLUCOSE
TRANSLOCATOR family (pGlcTs) and the VACUOLAR
GLUCOSE TRANSPORTER family (VGTs). Members of these
families have been identified in a large variety of plant species,
including grape and rice, leading to an ambiguous nomenclature
(Johnson and Thomas 2007, Afoufa-Bastien et al. 2010). If feasible,
in this study we will follow the nomenclature used for
Arabidopsis family members. While for some families (namely
the SUCs and the STPs) comprehensive data (transport activity,
expression data, subcellular localization and transgenic
approaches) exist, many members from the smaller families are
scarcely characterized, especially in non-model species. Each
family will be described in greater detail in the Results and
Discussion, taking into account the newly gained information
from tomato. All analyzed protein families belong to the major
facilitator super family of proteins (MFS family) which is characterized by 12 transmembrane domains (TMDs). The novel
SWEET family of sugar transporters was identified in 2010 and
belongs to a different superfamily which is characterized by seven
TMDs (Chen et al. 2010, Chen et al. 2012, Xuan et al. 2013).
Because of these differences and their relative novelty, SWEET
transporters will not be included in this study, although there are
32 SWEET transporters encoded in the tomato genome.
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Sugar accumulation during tomato fruit
development
Developing fruits are strong sinks for sugars, and adequate
supply from photosynthetic tissues is critical for accumulation
of sugars during fruit development (Ho et al. 1982). Sugar
supply for fruit metabolism and storage occurs via the
phloem. After unloading from the phloem, sugars are distributed within the fruit using both apoplastic and symplastic pathways (Ruan and Patrick 1995, Patrick and Offler 1996). Ripe
fruits of commercial tomato variants contain mostly glucose
and fructose at an equimolar concentration but hardly any
sucrose (Klee and Giovannoni 2011). This is due to the combined activity of cell wall-bound and vacuolar invertases which
also contribute significantly to fruit sink strength, although
their exact contribution is not fully understood (Yelle et al.
1991, Klann et al. 1996, Jin et al. 2009, Zanor et al. 2009).
While the transporters for the loading of sucrose into the
phloem have been identified, less is known about how photoassimilates from mesophyll cells reach the phloem or how
export towards developing fruits is facilitated (Riesmeier et al.
1992, Riesmeier et al. 1994). Recently, transporters from the
SWEET family were found to export sucrose to the leaf apoplast
for subsequent loading into companion cells by SUTs (Chen
et al. 2012). Uptake of hexoses from the fruit apoplast to the
storage parenchyma cells is thought to be achieved by the
hexose transporters LeHT1 and LeHT3 based on transport
activity of fruit discs, expression patterns and RNA interference
(RNAi)-mediated knock-down (Ruan et al. 1997, Dibley et al.
2005, McCurdy et al. 2010). Although organic acids are important for fruit metabolism and also contribute to the taste of
tomato fruits, relatively little is known about the factors that
determine their concentration in ripe fruits. A number of
metabolomics studies on the ripening of tomato fruit showed
dynamic patterns of accumulation (Roessner-Tunali et al. 2003,
Carrari and Fernie 2006, Carrari et al. 2006, Schauer et al. 2006,
Carrari et al. 2007, Luengwilai et al. 2010). However, it is
currently unknown how much de novo synthesis and import
of organic acids contribute to the final content in ripe fruits.
A comprehensive transporter inventory.
The genes characterized in the aforementioned studies on
tomato fruit development and sugar accumulation were discovered by reverse genetic methods or through homologous
cDNA cloning. With the complete genome sequence of tomato
available, it became possible to use forward genetic methods to
gain a more complete picture of the genes involved in sugar
accumulation during tomato fruit ripening (Tomato Genome
Consortium 2012). A first step towards this goal is to create a
comprehensive inventory of all putative sucrose and monosaccharide transporters encoded in the tomato genome. Expressed
sequence tag (EST) databases found at, for example, the
Sol Genomics Network (http://www.solgenomics.net/) or
TOMATOMICS (http://www.bioinf.mind.meiji.ac.jp/tomato
mics/) (Aoki et al. 2010) can then be used to confirm the
Plant Cell Physiol. 55(6): 1123–1141 (2014) doi:10.1093/pcp/pcu052 ! The Author 2014.
Identification of tomato sugar transporters
gene models predicted in the reference genome set and to
obtain full-length cDNAs for cloning. Also transcriptome data
(at TOMATOMICS) and metabolomic data of Solanaceae species (KaPPA-View4 SOL at http://www.kpv.kazusa.or.jp/kpv4sol/) are available and can be integrated into the gene inventory
data. A dwarf variety of tomato, called ‘Micro-Tom’ is used as a
model system for tomato genetics and physiology because of its
small size and shortened life cycle compared with commercial
cultivars (Meissner et al. 1997). Ethylmethane sulfonate
(EMS) mutant lines and g-ray irradiation-induced mutant
lines of ‘Micro-Tom’ have been generated and are available
from TOMATOMA (http://www.tomatoma.nbrp.jp/index.jsp)
(Saito et al. 2011).
In this study we comprehensively analyzed the sugar transporter (ST) gene families in tomato. We identified 52 genes
putatively encoding sugar transporter proteins in the tomato
reference genome (Tomato Genome Consortium 2012). Out of
these, 35 showed EST evidence. Using phylogenetic methods,
we assigned all putative ST genes to known families. Within
each family, the amino acid sequences of putative STs from
tomato were compared with those of characterized members
of this family from other plant species. Since it is accepted that
the expression of a gene points to a function at the place and
time of expression, we performed an expression analysis for
selected STs that had EST evidence. By doing this, we hoped
to find genes with interesting expression patterns, with a special
focus on fruit development.
Results and Discussion
Identification of putative sugar transporters
BLAST searches of the translated tomato genome, using
the amino acid sequences of characterized STs as queries,
identified 52 loci putatively encoding full-length sugar
transporters from diverse subfamilies (Table 1). From 35 of
these loci at least one EST was available in public databases.
Loci without EST evidence might only show expression under
certain conditions or in specific tissues, or might represent
pseudogenes. All loci encoding sequences shorter than 100
amino acids were discarded, since they are very unlikely to
encode functional transport proteins. Out of the 35 ESTs,
four clones (annotated as STP9, SFP3, SFP5 and INT2) contained non-sugar transporter cDNAs, contrary to their database
annotation.
Sequencing of EST clones confirmed the predicted exon–
intron structure in most cases. In the case of differences between predicted and experimentally found gene models, the
gene model determined by EST sequencing was used for further
analysis. In the case of SlSTP16 the EST showed a 2 bp deletion
(leading to a premature termination codon) compared with the
reference genome. We assumed this deletion to be an artifact of
EST cloning and used a corrected sequence for further analysis.
Additionally, this deletion might be specific to Micro-Tom and
is thus not found in the Heinz reference genome.
A phylogenetic analysis using all putative tomato ST amino
acid sequences assigned the identified STs to eight subfamilies
(SUC/SUT, STP, SFP/ERD6-like, PMT/PLT, INT/ITR, pGlcT, TMT
and VGT) which is in accordance with the current literature
(Fig. 1). The gene models of the STs analyzed in this study
showed between no intron (STP family) and 17 introns (SFP
family) (Fig. 2). The number of exons and their length were
found to be conserved within the subfamilies, whereas the
length of the introns was more variable. Hereafter, tomato ST
subfamilies and their members will be discussed in comparison
with the current literature.
SUTs/SUCs
In tomato, three amino acid sequences were identified as belonging to the SUCROSE TRANSPORTER family (Fig. 3). All
three putative tomato SUTs showed EST evidence. The proteins
consist of 512 (SlSUT1), 605 (SlSUT2) and 501 amino acids
(SlSUT4), respectively, forming 11 or 12 predicted TMDs.
While SlSUT1 and SlSUT4 showed comparable exon–intron
structures (four and five exons, respectively), SlSUT2 contained
14 exons. Also, SlSUT2 featured an additional cytoplasmic loop
typical for SUT2-type proteins, which is not found in SUT1 or
SUT4 subfamily members. Our results are consistent with the
earlier identification of three tomato sucrose transporters
(SlSUT1, SlSUT2 from Barker et al. 2000; and SlSUT4 from
Weise et al. 2000). Importantly, we did not identify additional
members of the sucrose transporter family. In a phylogenetic
analysis, the tomato sucrose transporters clustered together
with members from the SUT1 (SlSUT1), SUT2 (SlSUT2) and
SUT4 (SlSUT4) clades from other organisms (Kühn and Grof
2010). Members of the SUT1 clade from dicots were shown to
be localized to the PM in several species, and SlSUT1 was also
reported to localize to the PM (Kühn et al. 1997). Immunogold
labeling localized SlSUT2 to the PM of sieve elements (Barker
et al. 2000). Transient expression in protoplasts showed that
SlSUT4 fusion proteins localize to the vacuolar membrane or
additionally to the endoplasmic reticulum, depending on the
position of the green fluorescent protein (GFP) reporter gene
(Schneider et al. 2012).
SUT family members are well established as sucrose/H+
symporters (Sauer 2007, Shiratake 2007, Kühn and Grof
2010). Proteins from Arabidopsis and tomato were localized
to sieve elements, where they facilitated the loading of sucrose into the phloem. SlSUT1, a high-affinity transporter, is
thought to be the main importer of apoplastic sucrose into
the phloem, and knock-down of the SlSUT1 gene resulted in
accumulation of photosynthesis products in leaves (Hackel
et al. 2006). Members of the SUT2 clade, including SlSUT2,
feature additional cytosolic domains and share sequence
similarities with yeast sugar sensors (Barker et al. 2000). In
tomato, SlSUT2 is probably involved in pollen tube growth
(Hackel et al. 2006). Additional roles for SUT/SUC-type transporters were reported for those found exclusively in
Arabidopsis. For example, SUC2 was found to play a role
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Table 1 Comprehensive nomenclature and feature list of 52 sugar transporters identified in tomato
Gene name
Locus
Best hitEST no.
AAa
TMDb
LOCc
Remarks
SUT
SlSUT1
SlSUT2
SlSUT4
Solyc11g017010.1
Solyc05g007190.2
Solyc04g076960.2
LEFL1045AF06
LEFL2035O24
LEFL1070DB04
512
605
501
11
11
12
PM
PM
VM/ER
LOC (Kühn et al. 1997)
LOC (Barker et al. 2000)
LOC (Schneider et al. 2012)
STP
SlSTP1
Solyc02g079220.2
SGN-E398079
524
12
PM
SlSTP2
SlSTP3
SlSTP4
SlSTP5
SlSTP6
SlSTP7
SlSTP8
SlSTP9
SlSTP10
SlSTP11
SlSTP12
SlSTP13
SlSTP14
SlSTP15
SlSTP16
SlSTP17
SlSTP18
Solyc09g075820.2
Solyc07g006970.2
Solyc04g074070.1
Solyc01g010530.1
Solyc08g080300.1
Solyc03g005140.1
Solyc00g009030.1
Solyc01g008240.2
Solyc03g006650.1
Solyc06g054270.2
Solyc05g018230.2
Solyc03g005150.2
Solyc03g078600.1
Solyc03g093400.2
Solyc03g093410.2
Solyc03g094170.1
Solyc12g008320.1
LEFL1033AD12
LEFL2015N15
Not found
Not found
Not found
LEFL3132D24
Not found
SGN-E360972d
Not found
LEFL1093CG01
LEFL1033AG05
Not found
Not found
LEFL1086CC04e
LEFL1049CD03e
Not found
LEFL3049D18
524
514
510
490
529
488
397
506
519
490
517
488
438
500
515
242
516
12
11
11
11
12
11
7
11
12
11
12
11
8
12
12
4
10
PM
PM
VM
PM
VM
PM
PM
CS
CS
PM
PM
PM
PM
PM
PM
CL
PM
EST not full length (1–377); LeHT1
(Gear et al. 2000)
LOC: PM in yeast, LeHT2 (Gear et al. 2000)
LOC: PM in yeast, LeHT3 (Gear et al. 2000)
SlSFP1
SlSFP2
SlSFP3
SlSFP4
SlSFP5
SlSFP6
SlSFP7
Solyc01g098490.2
Solyc02g005180.2
Solyc01g080680.2
Solyc04g080460.2
Solyc01g098500.2
Solyc12g089180.1
Solyc09g074230.2
Not found
LEFL3012N18e
SGN-E211041d
LEFL1070CE02
LEFL1019BF02
LEFL1001AD10
SGN-E282759
461
396
469
487
462
491
481
12
10
11
12
10
12
12
PM
PM
PM
PM
PM
PM
PM
SlSFP8
SlSFP9
SlSFP10
Solyc01g098560.2
Solyc02g062750.2
Solyc02g085170.2
Not found
Not found
SGN-E547709
466
518
484
12
12
12
PM
PM
PM
PMT
SlPMT1
SlPMT2
SlPMT3
SlPMT4
SlPMT5
SlPMT6
SlPMT7
SlPMT8
Solyc02g078600.2
Solyc07g024030.2
Solyc12g010690.1
Solyc01g109460.2
Solyc02g062890.1
Solyc02g062860.2
Solyc02g062870.2
Solyc02g081710.1
Not found
Not found
LEFL2033E02
LEFL1006BG12
LEFL3155L03
Not found
Not found
SGN-E359943
514
478
520
542
510
498
498
488
10
12
12
10
11
12
12
12
PM
PM
PM
PM
PM
VM
VM
VM
INT
SlINT1
SlINT2
SlINT3
SlINT4
Solyc06g073420.2
Solyc08g048290.2
Solyc12g099070.1
Solyc11g012450.1
SGN-E324058
SGN-E312790d
Not found
LEFL1003DF04
497
527
581
578
10
11
12
12
PM
PM
PM
PM
pGlcT
SlpGlcT1
SlpGlcT2
SlpGlcT3
SlpGlcT4
Solyc02g086160.2
Solyc06g066600.2
Solyc07g020790.2
Solyc07g049310.2
LEFL2053A18
LEFL2053F03e
LEFL2036E03
LEFL2028B16
545
492
484
541
12
10
10
10
MM
PM
CL
CL
TMT
SlTMT1
SlTMT2
SlTMT3
Solyc03g032040.2
Solyc04g082700.2
Solyc02g082410.2
LEFL1012BC07
LEFL1032BA11
Not found
726
739
708
10
11
11
PM
PM
PM
VGT
SlVGT1
SlVGT2
Solyc03g078000.2
Solyc03g096950.2
LEFL1094BD10
LEFL2044B22
546
504
9
12
CL
PM
SFP
a
EST not full length (1–36)
Short C-terminus
Short N-terminus
Short N- and C-terminus
EST not full length (1–12), LeST3
(Garcia-Rodriguez et al. 2005)
Micro-Tom EST differs from reference genome
Failed to grow EST clone
The amino acid sequence length was either confirmed by EST sequencing or predicted using SL2.40 gene models.
The number of transmembrane domains was predicted by TMHMM Server v2.0.
c
The subcellular localizations were predicted by WoLFPSORT or determined experimentally as indicated under ‘Remarks’. PM plasma membrane, VM vacuolar
membrane, CL chloroplast, MM mitochondrion NU nucleus, ER endoplasmatic reticulum, CS cytosol
d
The ordered EST clone contained another, non-sugar transporter cDNA.
e
The sequenced ESTs contained a 2 bp deletion (STP16) or unspliced intronic sequence (STP15, SFP2, pGlcT2) (assumed to be a cloning artifacts) leading to a
frameshift. Further analyses were performed using the corrected gene models.
b
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Identification of tomato sugar transporters
Fig. 1 Phylogenetic analysis of tomato sugar transporters. Shown is a phylogenetic tree generated by the Neighbor–Joining method derived from
a CLUSTAL alignment of amino acid sequences from tomato. Numbers at internal nodes show the results of bootstrapping analysis (n = 1000).
Colored boxes indicate different sugar transporter subfamilies.
in the retrieval of sucrose along the phloem pathway
(Srivastava et al. 2008, Srivastava et al. 2009, Gould et al.
2012). Another Arabidopsis-specific transporter (AtSUC5)
was recently reported as a biotin transporter, raising the
question of substrate specificity of the SUT/SUC family
(Pommerrenig et al. 2013).
The manipulation of SUTs to improve fruit sugar content
seems to be promising. Although ripe tomatoes contain very
little sucrose thanks to the action of invertases, the developing
tomato fruit is a sink for sucrose (Roitsch and González 2004).
During fruit development, sucrose is imported into the fruit
and subsequently cleaved to glucose and fructose. These in
Plant Cell Physiol. 55(6): 1123–1141 (2014) doi:10.1093/pcp/pcu052 ! The Author 2014.
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Fig. 2 Exon–intron structure of 52 sugar transporter genes identified in tomato. Shown is a graphic representation of the gene models of all 52
sugar transporters identified in this study. Untranslated regions are shown as hatched boxes, exons are shown as black boxes and introns are
shown as black lines. Gene models are based on sequenced ESTs, or, in the case of lack of EST evidence, in silico predictions (ITAG release 2.3
SL2.40) are used.
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Identification of tomato sugar transporters
Fig. 3 Phylogenetic analysis of the SUCROSE TRANSPORTER family. Shown is a phylogenetic tree generated by the Neighbor–Joining method
derived from a CLUSTAL alignment of amino acid sequences from SUT/SUC subfamily members from tomato (this study, bold type label, red
lines) and selected family members from Arabidopsis thaliana AtSUC1 (At1g71880), AtSUC2 (At1g22710), AtSUC3 (At2g02860), AtSUC4
(At1g09960), AtSUC5 (At1g71890), AtSUC6 (At5g43610), AtSUC7 (At1g66570), AtSUC8 (At2g14670), AtSUC9 (At5g06170); Oryza sativa
OsSUT1 (Os03g0170900), OsSUT2 (Os12g0641400), OsSUT3 (Os10g0404500), OsSUT4 (Os12g44380), OsSUT5 (Os02g0576600); Vitis vinifera
VvSUC11/VvSUT1 (HQ323256), VvSUC12 (HQ323257), VvSUC27 (HQ323258), VvSUT2 (HQ323259); Solanum tuberosum StSUT1 (CAA48915),
StSUT4 (AAG25923.2); Zea mays ZmSUT1 (BAA83501); Nicotiana tabacum NtSUT4 (BAI60050); Pisum sativum PsSUF1 (DQ221698), PsSUF4
(DQ221697); Phaseolus vulgaris PvSUF1 (DQ221700); Spinacia oleraceae SoSUT1 (Q03411); Hordeum vulgare HvSUT2 (Q9M423); Lotus japonicas
LjSUT4 (CAD61275); Populus tremula alba PtaSUT4 (HM749900). Numbers at internal nodes show the results of bootstrapping analysis
(n = 1000).
turn contribute to fruit sweetness or can be used as energy for
fruit development. Increasing the sucrose import of developing
tomato fruits might therefore increase the sugar content of the
ripe fruit. However, tomato sugar content depends on many
factors, including source strength, fruit import, invertase activity and intrafruit transport, so increasing sucrose import to the
fruit alone might not be sufficient.
STPs
The SUGAR TRANSPORTER PROTEIN family (STP), also called
the MONOSACCHARIDE TRANSPORTER family (MST) in rice
or the HEXOSE TRANSPORTER family (HT) in grape, is the
largest subfamily of STs in tomato, with 18 members (Fig. 4).
Nine of these were supported by EST evidence. The average
sequence length was about 500 amino acids, encompassing
11 or 12 predicted TMDs. The gene models of most of the
subfamily members featured three or four exons. The proteins
were predicted to localize to the PM, the VM or the chloroplast.
Some proteins were predicted as cytosolic, although >10 TMDs
were present.
All previously characterized STPs are hexose/H+ symporters,
and SlSTP2 and SlSTP3 localized to the PM when expressed in
yeast (Doidy et al. 2012). Their preferred substrate seems to be
glucose, but many STPs show a broader substrate specificity,
including galactose and mannose. Fructose transport activity
was shown for some STPs, but seems to be less common in this
family. While most information is available for the Arabidopsis
STPs, some tomato STPs were characterized (Büttner 2010).
Originally tomato STPs were designated LeHT1–LeHT3 which
does not conform to current nomenclature, so we suggest to
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Fig. 4 Phylogenetic analysis of the SUGAR TRANSPORTER PROTEIN family. Shown is a phylogenetic tree generated by the Neighbor–Joining
method derived from a CLUSTAL alignment of amino acid sequences from STP subfamily members from tomato (this study, bold type label, red
lines) and characterized family members from Arabidopsis thaliana AtSTP1 (At1g11260), AtSTP2 (At1g07340), AtSTP3 (At5g61520), AtSTP4
(At3g19930), AtSTP5 (At1g34580), AtSTP6 (At3g05960), AtSTP7 (At4g02050), AtSTP9 (At1g50310), AtSTP11 (At5g23270), AtSTP13 (At5g26340),
AtSTP14 (At1g77210); Oryza sativa OsMST1 (BAB19862.1), OsMST2 (Os03g39710), OsMST3 (Os07g01560), OsMST4 (AAQ24871.1), OsMST5
(Os08g08070), OsMST6 (AAQ24872.1), OsMST8 (Os01g38670); Vitis vinifera VvHT1 (HQ323260), VvHT2 (HQ323261), VvHT3/VvHT7
(HQ323262), VvHT4 (HQ323263), VvHT5 (HQ323264); Juglans regia JrHT1 (DQ026508), JrHT2 (DQ026509,); Olea europaea OeMST2
(DQ087177) and Ananas comosus AcMST1 (EF460876). Numbers at internal nodes show the results of bootstrapping analysis (n = 1000).
refer to them as SlSTP1–SlSTP3. SlSTP1 and SlSTP2 are functional, energy-dependent glucose transporters when expressed
in yeast (Gear et al. 2000). Four novel tomato STPs (SlSTP6,
SlSTP8, SlSTP10 and SlSTP14) could be grouped together with
SlSTP1, AtSTP1 and VvHT1 in a separate clade. Strikingly, no
EST was available for the novel tomato genes. AtSTP1 was the
first plant STP shown to transport glucose when expressed in
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yeast (Sauer et al. 1990). In grape, VvHT1 was proposed to
transport hexoses during early fruit development based on
gene expression analysis and in situ hybridization experiments
(Fillion et al. 1999, Vignault et al. 2005). SlSTP2 was the only
tomato STP in a separate subgroup together with one STP each
from Arabidopsis, rice and grape, suggesting a conserved origin.
In this subgroup, VvHT5 seems to have a specialized role in the
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Identification of tomato sugar transporters
carbohydrate supply to tissues under biotic stress (Hayes et al.
2010). Similarly, AtSTP13 expression was found to be correlated
with programmed cell death following fungal infection
(Norholm et al. 2006). AtSTP13 also positively influenced
plant growth and nitrogen content when overexpressed
(Schofield et al. 2009).
SlSTP3 clustered together with AtSTP7 and VvHT3, all of which
showed no sugar transport activity when analyzed in a yeast
system (Hayes et al. 2007, Büttner 2010, McCurdy et al. 2010).
In this subgroup, two novel STPs, supported by EST evidence, were
identified in the tomato genome (SlSTP12 and SlSTP18). Further
research is necessary to find a transport substrate and ultimately
the function of STPs from this subgroup. The uncharacterized
proteins SlSTP15– SlSTP17 formed a separate clade, together
with VvHT4 and AtSTP3. VvHT4 is a functional hexose transporter
when expressed in yeast (Hayes et al. 2007). AtSTP3 is a low affinity
transporter of glucose and probably other hexoses (Büttner et al.
2000). Another clade is formed by SlSTP4 together with OsMST1,
AtSTP5 and VvHT2. While for VvHT2 no data are available, both
OsMST1 and AtSTP5 showed no glucose transport activity when
assayed in yeast (Toyofuku et al. 2000, Büttner 2010). SlSTP9 was
the only tomato STP protein in a small subgroup together with
AtSTP4, AtSTP9 and AtSTP11. Of these, AtSTP9 and AtSTP11 were
characterized as functional hexose transporters expressed in
Arabidopsis pollen (Schneidereit et al. 2003, Schneidereit et al.
2005). Other pollen-specific STPs from Arabidopsis (AtSTP2 and
AtSTP6) clustered together with SlSTP5, SlSTP7, SlSTP11 and
SlSTP13 (Truernit et al. 1999, Scholz-Starke et al. 2003). It is thus
possible, as shown for Arabidopsis, that tomato also has a relatively high number of STPs which are specifically expressed in
pollen.
SFPs/ERD6-like
In our phylogenetic analysis, 10 amino acid sequences were
identified as members of the SUGAR FACILITATOR PROTEIN
family (SFP), also called EARLY RESPONSE TO DEHYDRATION
6-like family (ERD6-like) (Fig. 5). For six members, EST evidence
was found. The length of full-length SFPs ranged from 396 to
518 amino acids. Full-length SFP proteins featured between 10
and 12 predicted TMDs, and all of them were predicted to
localize to the PM. The proteins encoded by the loci SlSFP4,
SlSFP6, SlSFP9 and SlSFP10 formed a clade together with the
partially characterized AtERD6-like and BvERD6-like proteins.
Another clade was formed by SlSFP1, SlSFP2 and SlSFP5 together with the 11 grape SFPs (VvSFP1–VvSFP11), none of
which is characterized.
The founding member of the family, AtERD6, was shown to
be induced under dehydration and cold stress, but any evidence
for transport activity is lacking (Kiyosue et al. 1998). Also for
two partially characterized proteins from Arabidopsis, AtSFP1
and AtSFP2, no transport evidence is available (Quirino et al.
2001). More recently, studies of AtESL1, another member of the
ERD6-like subfamily, indicated that ERD6-like members facilitate diffusion of glucose and a range of other hexoses (Yamada
et al. 2010). Additionally it was shown that a tri-leucine motif in
the N-terminal region of AtESL1 is necessary for localization to
the VM. This motif is also found in SlSFP1, SlSFP2 and SlSFP8–
SlSfP10 (data not shown), indicating a possible VM localization,
which contradicts the in silico prediction. Unfortunately, no
tomato SFP protein (and also no grape protein) clustered together with the characterized SFPs from Arabidopsis, indicating
different functions of SFPs in those species.
PMTs
Eight loci putatively encoding POLYOL/MONOSACCHARIDE
TRANSPORTER proteins (PMTs or PLTs) with an average
length of approximately 500 amino acids were identified
(Fig. 6). For four family members, EST evidence from tomato
was found. All PMT genes shared a common structure which
consists of two exons separated by an intron, except SlPMT4,
which had three exons. Full-length SlPMT proteins were predicted to localize to the PM (SlPMT1–SlPMT5) or the VM
(SlPMT6– SlPMT8).
The general substrate of PMTs seem to be sugar alcohols such
as sorbitol or mannitol, which replaces sucrose as the long-distance transport form of photoassimilates in some species, most
notably fruit trees in the Rosaceae family (Noiraud et al. 2001b).
In tomato, no major role for sugar alcohols is known, although a
functional sorbitol dehydrogenase gene was found to be expressed ubiquitously (Ohta et al. 2005). In Arabidopsis, six
PMT-type transporters are found. AtPMT5 was described as a
non-specific polyol, hexose and pentose transporter expressed in
various tissues (Reinders 2004, Klepek et al. 2005). In Arabidopsis,
AtPMT1 and AtPMT2 were also characterized as xylitol and fructose transporters expressed in developing xylem and in pollen
(Klepek et al. 2010). Two proteins from Apium graveolens (celery)
were identified as mannitol/H+ symporters and localized to the
PM of phloem cells (Noiraud et al. 2001a, Juchaux-Cachau et al.
2007). PcSOT1 and PcSOT2 were characterized as sorbitol transporters in sink tissues of Prunus cerasus (sour cherry) (Gao et al.
2003). In the phloem of source leaves of apple, expression of
three sorbitol transporters (MdSOT3–MdSOT5) was detected
(Watari et al. 2004). Finally, in Plantago major (common plantain), two sorbitol transporter are expressed in the phloem
(Ramsperger-Gleixner et al. 2004). No tomato PMT was found
to cluster together with the characterized sorbitol transporters.
The amino acid sequence of SlPMT4 seemed most similar to
those of characterized sorbitol transporters, but this was not
supported well according to bootstrap analysis. Together with
VvPMT5, SlPMT4 formed a distinctive subclade. SlPMT5–
SlPMT8 formed a distinct subgroup among the PMTs. Within
this group SlPMT5, SlPMT6 and SlPMT7 are probably the result of
repeated gene duplication events, as these loci are next to each
other on chromosome 2.
Smaller families
Four less characterized ST families with a smaller number of
members are recognized in plant species and were also found in
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Fig. 5 Phylogenetic analysis of the SUGAR FACILITATOR PROTEIN/EARLY RESPONSE TO DEHYDRATION 6 protein family. Shown is a
phylogenetic tree generated by the Neighbor–Joining method derived from a CLUSTAL alignment of amino acid sequences from ERD6/SFP
subfamily members from tomato (this study, bold type label, red lines); Arabidopsis thaliana AtERDL6 (At1g75220), AtERD6 (At1g08930), AtSFP1
(At5g27350), AtSFP2 (At5g27360), AtERD6-like1 (At1g08890), AtESL1 (At1g08920), AtESL2 (At1g08900) and 12 additional uncharacterized
Arabidopsis proteins indicated by their AGI-code, and Beta vulgaris (beet) BvERD6l (U43629). Grape proteins sequences were taken from
Afoufa-Bastien et al. (2010): VvSFP1 HQ323290, VvSFP2 HQ323291, VvSFP3 HQ323292, VvSFP4 HQ323293, VvSFP5 HQ323294, VvSFP6
HQ323295, VvSFP7 HQ323296, VvSFP8 HQ323297, VvSFP9 HQ323298, VvSFP10 HQ323299, VvSFP11 HQ323300, VvSFP12 HQ323301, VvSFP13
HQ323302, VvSFP14 HQ323303, VvSFP15 HQ323304, VvSFP16 HQ323305, VvSFP17 HQ323306, VvSFP18 HQ323307, VvSFP19 HQ323308,
VvSFP20 HQ323309, VvSFP21 HQ323310, VvSFP22 HQ323311. Numbers at internal nodes show the results of bootstrapping analysis (n = 1000).
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Identification of tomato sugar transporters
Fig. 6 Phylogenetic analysis of the POLY/MONOSACCHARIDE TRANSPORTER family. Shown is a phylogenetic tree generated by the Neighbor–
Joining method derived from a CLUSTAL alignment of amino acid sequences from PMT/PLT subfamily members from tomato (this study, bold
type label, red lines); Arabidopsis thaliana AtPMT1 (At2g16120), AtPMT2 (At2g16130), AtPMT3 (At2g18480), AtPMT4 (At2g20780), AtPMT5
(At3g18830), AtPMT6 (At4g36670); Vitis vinifera VvPMT1 (HQ323285), VvPMT2 (HQ323286), VvPMT3 (HQ323287), VvPMT4 (HQ323288),
VvPMT5 (HQ323289); Apium graveolens ApMaT1 (AF215837), ApMaT2 (AF480069); Plantago major PmPLT1 (CAD58709), PmPLT2 (CAD58710);
Malus domestica MdSOT1 (AY237400), MdSOT2 (AY237401), MdSOT3 (BAD42343), MdSOT4 (BAD42344), MdSOT5 (BAD4234); Prunus cerasus
PcSOT1 (AF482011), PcSOT2 (AY100638). Numbers at internal nodes show the results of bootstrapping analysis (n = 1000).
the tomato genome (Fig. 7). They are discussed together, although they do not form a distinct clade among the STs. These
families are the H+/Na+ MYO-INOSITOL TRANSPORTERS also
called the INOSITOL TRANSPORTER family (INT or IRT), the
PLASTIDIC GLUCOSE TRANSLOCATOR family (pGlcT), the
TONOPLAST MONOSACCHARIDE TRANSPORTER family
(TMT) and the VACUOLAR GLUCOSE TRANSPORTER family
(VGT). Due to their predicted localization and function, members of the pGlcT, the TMT and the VGT families are promising
targets in the search for novel transport proteins controlling the
movement of sugars between cellular compartments in fruits,
e.g. the accumulation of glucose in the vacuoles of mesocarp
cells.
INTs. In the tomato genome, four loci putatively encoding
INT family members were identified. Of these, SlINT1 and
SlINT4 were supported by EST evidence. The confirmed gene
models of these two SlINTs showed six exons, while SlINT3 was
predicted to have four exons. Members of the INT subfamily
had between 497 and 578 amino acids and featured 10–12
predicted TMDs. All proteins were predicted to localize to
the PM.
INT-type transport proteins are thought to be H+ symporters of inositols. They were first characterized in the halophyte
Mesembryanthemum crystallinum (Chauhan et al. 2000). Later
three INT genes from Arabidopsis were characterized. AtINT1
encodes a myo-inositol transporter localized to the VM
(Schneider et al. 2008), while AtINT2 and AtINT4 were shown
to be localized to the PM (Schneider et al. 2006, Schneider et al.
2007). So far, no physiological role for INT proteins has been
reported.
pGlcTs. Four loci encoding pGlcT-type transporters were
identified in the tomato genome, all of which were supported
by EST evidence. The length of the encoded proteins was 484–
545 amino acids, forming 10 or 12 predicted TMDs.
Confirmation of the predicted gene models by EST sequencing
revealed 12–14 exons. The proteins were predicted to localize
to mitochondria (SlpGlcT1), the PM (SlpGlcT2) or the chloroplast (SlpGlcT3 and SlpGlcT4). Each tomato pGlcT protein
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Fig. 7 Phylogenetic analysis of five tomato sugar transporter protein families. Shown is a phylogenetic tree generated by the Neighbor–Joining
method derived from a CLUSTAL alignment of amino acid sequences from five ST subfamilies from tomato (this study, bold type label);
Arabidopsis thaliana AtINT1 (At2g43330), AtINT2 (At1g30220), AtINT3 (At2g35740), AtINT4 (At4g16480), AtAtTMT1 (At1g20840), AtTMT2
(At4g35300), AtTMT3 (At3g51490), AtSGB1 (At1g79820), AtpGlcT (At5g16150), AtpGlcT-like1 (At1g67300) AtpGlcT-like2 (At1g05030), AtVGT1
(continued)
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Identification of tomato sugar transporters
formed its own respective clade in our phylogenetic analysis
and clustered together with one or two pGlcT proteins from
Arabidopsis, grape and rice.
In plants, plastid-localized glucose transporters were first
identified in spinach with the implication of a role in the
export of starch breakdown products from chloroplasts
during the night (Weber et al. 2000). Similar results were obtained more recently by using Arabidopsis knock-out mutants
of AtpGlcT (Cho et al. 2011). Starch is found in the early stages
of tomato fruit development but is almost completely absent in
ripe fruits (Yin et al. 2010). It can be speculated that the degradation of starch contributes to fruit sugar content. The ability
to export the breakdown products of starch from the plastids
thus makes pGlcT proteins candidates for further research into
fruit sugar accumulation. In olive trees, one pGlcT-type protein
was reported to be expressed during fruit development
(Butowt et al. 2003). Tomato SlpGlcT1 was found to cluster
together with AtpGlcT and OepGlcT in a phylogenetic analysis.
SlpGlcT2, however, was more comparable with AtSGB1
(SUPPRESSOR OF G-PROTEIN BETA1). AtSGPB1 was identified
in a mutant screening as a suppressor of the agb1 (arabidopsis
g-protein ) phenotype, which leads to defects in early development (Wang et al. 2006). Furthermore AtSGB1 was shown to
localize to Golgi vesicles. SlpGlcT4 was found to be most similar
to OsGMST1. Knock-down of OsGMST1 led to reduced tolerance against high NaCl conditions and slightly reduced glucose
and fructose content. In rice, OsGMST1 was shown to localize
to the Golgi apparatus (Cao et al. 2011).
TMTs. Three members of the SlTMT family were detected,
but only SlTMT1 and SlTMT2 had ESTs available. TMTs are
large proteins of >700 amino acids in most species, including
tomato. The gene models consist of five (SlTMT1 and SlTMT2)
or six (SlTMT3) exons. All three proteins are predicted to localize to the PM despite their sequence similarity to
Arabidopsis or rice VM transporters.
So far, TMTs from Arabidopsis and rice have been characterized. In Arabidopsis, AtTMT1 and SlTMT2 were localized to
the VM and characterized as glucose or fructose/H+ antiporters, importing sugars into the vacuole (Wormit et al. 2006,
Wingenter et al. 2010, Schulz et al. 2011). Comparable results
were obtained for the rice OsTMT1 and OsTMT2 proteins (Cho
et al. 2010). While the Arabidopsis TMTs respond to environmental cues, such as cold stress, on the transcript level, the rice
TMTs did not.
VGTs. The smallest family of STs analysed in this study were
the VGTs, with two members identified in tomato. Both members had EST clones available. The sequenced ESTs were found
to encode proteins of 546 (SlVGT1) and 504 (SlVGT2) amino
acids, respectively. They had nine and 12 predicted TMDs, respectively, and were predicted to localize to the chloroplast and
the PM, despite their name. The gene models showed 14 exons.
VGT proteins form a small subfamily with two or three
members in all species analyzed here. Only one VGT protein
from Arabidopsis has been characterized so far (Aluri and
Büttner 2007). In yeast and in Arabidopsis protoplasts,
AtVGT1 localized to the VM and was shown to import glucose
and to a lesser extent also fructose into the vacuole in an ATPdependent manner. Although Atvgt1 mutants were shown to
have a germination defect and a late flowering phenotype, no
specific physiological function was shown.
Expression analysis
Although Micro-Tom ESTs from 35 STs could be identified, only
29 of those ESTs encoded a full-length ST protein. To get insight
into putative functions of ST genes, we analyzed gene expression of these 29 STs in different Micro-Tom tissues by semiquantitative real-time PCR (RT-PCR) (Fig. 8). Since we were
most interested in the functions of STs during fruit development, we included tissues from developing fruits at nine different time points in our analysis. Several STs showed tissue- and
development-specific dynamic expression patterns, which is a
first hint at their putative functions.
In our analysis, we found SlSUT1 and SlSUT4 to be expressed
ubiquitously, with a slight increase in the course of fruit development. In contrast to previous data, we could not detect
SlSUT2 expression in any tissue. Barker et al. (2000) used RNA
gel blotting to detect a weak signal of SlSUT2 in leaves, stems
and roots. Their method seems to be more sensitive but probably also less specific than the PCR-based detection used here.
SlSUT2 was reported to be expressed predominantly in anthers,
which were not sampled separately in this study (Hackel et al.
2006). As in other species, SlSUT1 and SlSUT4 are thought to
import sucrose into specialized phloem cells in source leaves
(Sauer 2007). The fact that they are also expressed in typical
sink tissue such as roots or the developing fruit is a strong
indication that they also play a role in sink tissues, for example
in phloem unloading or import into sink parenchyma cells.
Thus, SlSUT genes should be considered targets for the manipulation of tomato sweetness.
Fig. 7 Continued
(At3g03090), AtVGT2 (At5g17010), AtVGT3 (At5g59250); Oryza sativa OsITR1 (Os04g41460), OsITR2 (Os07g05640), OsITR3 (Os04g43210), OsTMT1
(Os10g39440), OsTMT2 (Os02g13560), OsTMT3 Os03g03680, OsTMT4 (Os11g40540), OsTMT5 (Os02g58530), OsTMT6 (Os11g28610), OspGlcT
(Os01g04190), OsGMST1 (Os09g23110); OspGlcT-like1 (Os02g17500), OspGlcT-like2 (Os09g27900), OsVGT1 (Os03g60820), OsVGT2 (Os10g42830),
Vitis vinifera VvINT1 (HQ323314), VvINT2 (HQ323315), VvINT3 (HQ323316), VvTMT1 (HQ323282), VvTMT2 (HQ323283), VvTMT3 (HQ323284),
VvpGlT (HQ323320), VvpGlcT2(HQ323319), VvpGlcT3 (HQ323318), VvpGlcT4(HQ323317), VvVGT1 (HQ323312), VvVGT2 (HQ323313), Olea europaea
OepGlcT (AY036055), Spinacia oleracea SopGlcT (AF215851), Mesembryanthemum crystallinum McITR1 (AF280431), McITR2 (AF280432). Numbers at
internal nodes show the results of bootstrapping analysis (n = 1000). Colored backgrounds indicate different subfamilies of STs.
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Fig. 8 Expression analysis of selected tomato sugar transporters. Shown is a semi-quantitative RT-PCR analysis of tomato sugar transporters.
RNA was extracted from the indicated tissues, transcribed to cDNA and used as a template for PCR. + indicates reactions using the respective
EST-containing vector as a template. Gene-specific primers (amplicons 200 bp) were used to analyze expression levels by PCR. UBQ indicates a
tomato ubiquitin gene used as a constitutively expressed control gene. DAP, days after pollination. Results are representative of two technical
replicates for each tissue.
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Identification of tomato sugar transporters
SlSTP1 and SlSTP3 were reported to be expressed predominantly in sink tissue, with the highest expression in young fruits
(Gear et al. 2000). In our analysis, SlSTP1 transcript could be
detected in all analyzed tissues, while SlSTP3 was expressed at
detectable levels only in flowers and developing fruits 14 DAP
(days after pollination). SlSTP2 expression was detected in
young leaves, in shoots, in developing fruits 14 DAP and to a
lesser extent also in mature leaves. Expression of SlSTP7 was
restricted to the roots. No expression of SlSTP11 and SlSTP16
could be detected in any tissue despite EST evidence. SlSTP12
was expressed in all analyzed tissues, with the strongest expression in leaves and fruits 14 DAP. Also SlSTP15, which is expressed only in developing fruits, showed the strongest
expression at 14 DAP. Additionally, SlSTP2, SlSTP3 and
SlSTP18 showed a distinct expression at 14 DAP. This expression profile indicates that the expression of SlSTP genes in developing fruits at around 14 DAP plays a critical role for sugar
accumulation in ripe fruits. Expression of SlSTP2 and SlSTP3 was
analyzed previously in the pericarp layer (Dibley et al. 2005).
The developmental expression profile in the pericarp differed
from that found here in the whole fruit, which might be explained by the different sampling and perhaps by the use of
different tomato cultivars. Based on expression patterns,
SlSTP2, SlSTP3 and SlSTP18 might play a role in fruit development around 14 DAP, whereas SlSTP12 and SlSTP15 are important during the later stages of fruit development. Expression
around 14 DAP might be in connection with the proposed
symplastic to apoplastic switch of sugar supply to the developing tomato fruit (Ruan and Patrick 1995). In the earlier stages of
fruit development, sugars are thought to be distributed within
the fruit mainly symplastically through plasmodesmata. At
some point in development, the sucrose which reaches the
fruit is exported to the apoplast and cleaved to glucose and
fructose by cell wall-bound invertases. These monosaccharides
then need to be distributed apoplastically and reimported into
fruit parenchyma cells, which might be the reason for the
increased expression of several SlSTP genes at 14 DAP.
Two members of the SFP family, SlSFP2 and SlSFP5, were expressed during the later stages of fruit development. SlSFP4 expression was weak and restricted to the leaves. Expression of
SlSFP6 was restricted to vegetative tissues and the early stages
of fruit development. SlSFP10 was expressed in the roots and in
flowers. Based on these data SlSFP2 and SlSFP5 might contribute
to fruit development. However, since the substrate specificity of
SFP-type transporters is not very well characterized, it is not
possible to propose a more specific function. Of the PMT
family, only SlPMT4 showed strong dynamic expression during
the late stages of fruit development. SlPMT3 transcript was not
detectable in any tissue. SlPMT5 showed only weak expression in
mature leaves and roots. Expression of SlpGlcT1 was detected in
all tissues, except fruits 3 DAP. SlpGlcT2 and SlpGLcT3 were expressed at several stages during fruit development, with SlpGlcT3
showing strong expression at 7 DAP. Expression of SlpGlcT4 was
not detected in any tissue. The two SlTMT genes and SlINT4 were
expressed in most analyzed tissues. SlTMT2 showed strong,
consistent expression in vegetative tissues, while during fruit development expression was weaker, except 14 DAP and during the
breaker stage. While SlVGT1 was expressed in all analyzed tissues,
SlVGT2 was expressed only in leaves and during the later stages of
fruit development, with the strongest expression during the
orange stage of fruit development. Because of its increasing expression levels in the course of fruit development, SlVGT2 is
worth further investigation.
Conclusion
In this study 52 putative sugar transporter genes were detected
in the tomato genome. An expression analysis of selected genes
revealed tissue- and developmental stage-specific gene expression of some members. In the next step, specific roles for these
genes should be found. This will probably result in a more
complete picture regarding sugar mobility during development
of fleshy fruits (Ludewig and Flugge 2013). For example, it is
unclear what the individual contribution of each SUT to the
different steps in phloem transport is. It is also worth investigating the function of the monosaccharide transporters found
to be expressed in a dynamic, fruit-specific manner. After cleavage of sucrose to glucose and fructose in the developing fruit,
the hexoses have to be transported within the fruit to the
storage parenchyma for storage, presumably into the vacuoles.
These steps require monosaccharide transporters, which have
not been identified yet.
Materials and Methods
Identification of Solanum lycopersicum sugar
transporter genes
Tomato loci putatively encoding sugar transporter (ST) proteins
were retrieved from the tomato reference genome (ITAG release
2.3 SL2.40) (Tomato Genome Consortium 2012). The TBLASTX
tool on http://www.solgenomics.net was used to identify loci
encoding putative STs. Amino acid sequences of known members from each recognized ST subfamily from tomato or from
Arabidopsis were used as queries. The retrieved gene models and
amino acid sequences were then used for phylogenetic analysis
and in silico predictions of gene features. The identified coding
sequences (CDSs) were used to find EST clones containing ST
cDNAs from the TOMATOMICS database (Aoki et al. 2010) or
from the Sol Genomics Network. After consolidation of the data,
the most similar EST clone for each putative ST locus was obtained and sequenced to verify the current gene model. All EST
sequences were submitted to the DNA Data Bank of Japan
(http://www.ddbj.nig.ac.jp/) (accession nos. DDBJ: AB845639 to
DDBJ: AB845668).
Multiple sequence alignments and phylogenetic
analysis
Final classification of ST genes into subfamilies and subgroups
was done according to phylogenetic analysis. Multiple sequence
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alignments, using amino acid sequences from predicted proteins (ITAG release 2.3 SL2.40) or from sequenced ESTs, were
made using the CLUSTAL alignment function in the CLC Main
Workbench software (CLC Bio). Phylogenetic trees were build
using the Neighbor–Joining algorithm in the same software and
visualized using Treeview (Page 2002) and Dendroscope (Huson
and Scornavacca 2012).
In silico prediction of subcellular localization and
transmembrane helical domains
Prediction of subcellular localization of putative STs was performed using the WoLFPSORT algorithm (http://wolfpsort.seq.
cbrc.jp) (Horton et al. 2007). Prediction of transmembrane
helical domains was performed using TMHMM Server v.2.0
(http://www.cbs.dtu.dk/services/TMHMM/) (Krogh et al.
2001).
Plant material and growth conditions
Solanum lycopersicum plants for gene expression analysis
were of the dwarf cultivar ‘Micro-Tom’. Plants were grown on
soil in a growth chamber (Biotron LPH-350S, NK Systems)
with a light regime of 16 h light/8 h darkness at 25 C and
60% relative humidity. Plants were watered twice a week with
tap water. Fertilizer (Otsuka Chemicals) was applied once per
week.
bromide. Primer sequences and PCR conditions are described in
Supplementary Table S1.
Supplementary data
Supplementary data are available at PCP online.
Funding
This work was supported by the Bio-oriented Technology
Research Advancement Institution (BRAIN) [Program
for Promotion of Basic and Applied Researches for
Innovations in Bio-oriented Industry]; the Japan Society for
the Promotion of Science (JSPS) [Grant-in-Aids for Scientific
Research].
Acknowledgments
We thank Dr. Shogo Matsumoto and Dr. Shungo Otagaki for
helpful discussions, and the National Bioresource Project
(NBRP)-Tomato in Japan and the Sol Genomic Network
(SGN) for providing cDNA clones. We also thank Dr. Takashi
Akihiro from the University of Shimane for comments on our
manuscript.
RNA isolation and cDNA synthesis
Plant tissues from young leaves, mature leaves, roots, shoots
and flowers, and from developing fruits 3, 7, 14, 21 and 28 d
after fertilization and during the breaker, orange and red stages
of fruit development were harvested into liquid nitrogen.
Vegetative tissues were harvested from approximately
6-week-old plants. Samples of young leaves included developing, not fully expanded leaves, and samples of mature leaves
included fully expanded, non-senescent leaves. RNA from developing fruits 14 and 21 d after fertilization was isolated using
the RNA Suisui-R kit (Rizo). RNA from all other tissues was
isolated using TRIzol reagent (Life Technologies) following the
manufacturer’s protocol. Quality and quantity of the RNA were
assessed using a spectrophotometer. RNA was stored at –80 C.
cDNA was prepared using the PrimeScript RT reagent Kit with
gDNA Eraser (Clontech) according to the manufacturer’s specification. For each 20 ml reaction, 500 ng of total RNA was used.
RT-PCR expression analysis
Semi-quantitative RT-PCR was performed using 0.1 ml of cDNA
as a template and EmeraldAmp GT PCR Mastermix (Clontech).
For each primer pair, the PCR program was empirically
adjusted. All primers were tested for specificity by trying to
obtain a PCR product using vector DNA containing ESTs
from other subfamily members as a template (data not
shown). As a control, the constitutively expressed SlUBQ
(Solyc01g056940) gene was used. PCR products were analyzed
using 1% agarose gels stained for nucleic acids with ethidium
1138
Disclosures
The authors have no conflicts of interest to declare.
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