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GENE-40714; No.

of pages: 12; 4C:


Gene xxx (2015) xxxxxx

Contents lists available at ScienceDirect

Gene
journal homepage: www.elsevier.com/locate/gene

Research paper

Genome-wide identication, phylogeny, and expression analysis


of the SWEET gene family in tomato
Chao-Yang Feng, Jia-Xuan Han, Xiao-Xue Han, Jing Jiang
The Key Laboratory of Protected Horticulture, Ministry of Education, College of Horticulture, Shenyang Agricultural University, No.120 Dongling Road, Shenyang 110866, PR China

a r t i c l e

i n f o

Article history:
Received 31 March 2015
Received in revised form 14 July 2015
Accepted 15 July 2015
Available online xxxx
Keywords:
SWEETs
Tomato
Phylogeny
Sugar transporter
Gene expression
Gene family

a b s t r a c t
The SWEET (Sugars Will Eventually Be Exported Transporters) gene family encodes membrane-embedded sugar
transporters containing seven transmembrane helices harboring two MtN3 and saliva domain. SWEETs play
important roles in diverse biological processes, including plant growth, development, and response to environmental stimuli. Here, we conducted an exhaustive search of the tomato genome, leading to the identication
of 29 SWEET genes. We analyzed the structures, conserved domains, and phylogenetic relationships of these
protein-coding genes in detail. We also analyzed the transcript levels of SWEET genes in various tissues, organs,
and developmental stages to obtain information about their functions. Furthermore, we investigated the expression patterns of the SWEET genes in response to exogenous sugar and adverse environmental stress (high and
low temperatures). Some family members exhibited tissue-specic expression, whereas others were more
ubiquitously expressed. Numerous stress-responsive candidate genes were obtained. The results of this study
provide insights into the characteristics of the SWEET genes in tomato and may serve as a basis for further
functional studies of such genes.
2015 Elsevier B.V. All rights reserved.

1. Introduction
Carbohydrates are one of the most important energy-storing molecules, which provide building blocks for cell walls and function in signaling to maintain osmotic homeostasis under certain abiotic stress
conditions and for various other purposes. Carbohydrate transport and
partitioning from phototrophic to heterotrophic cells or tissues in
higher plants depends on the activity of diverse transporters (Bttner
and Sauer, 2000; Turgeon and Wolf, 2009). These processes play key
roles in the regulation of plant development and plant responses to
biotic and abiotic stress (Lemoine et al., 2013).
In plants, sugar transport is mediated by proton-coupled sugar/H+
symporters, sugar/H+ antiporters, and uniporters, which belong to the
major facilitator superfamily (MFS family). These transporters are
characterized by 12 transmembrane domains (TMDs) (Doidy et al.,
2012; Reuscher et al., 2014). The plant SWEET (Sugars Will Eventually
be Exported Transporters) family of sugar transporters is a recently
identied seven-TMD protein family (PFAM code PF0383) (Chen

Abbreviations: CDD, Conserved Domain Database; EST, Expressed Sequence Tag; GDB,
Genome Database; MFS family, the major facilitator superfamily; NCBI, National Center for
Biotechnology Information; NJ, neighbor-joining; SGN, sol genomics network; SWEET,
Sugars Will Eventually be Exported Transporters; TCDB, Transporter Classication
Database; TMDs, transmembrane domains.
Corresponding author.
E-mail address: jj_syau@hotmail.com (J. Jiang).

et al., 2010, 2012; Xuan et al., 2013). SWEET genes encode


membrane-embedded proteins consisting of seven transmembrane
helices harboring two MtN3/saliva domains, also referred to as the
PQ-loop-repeat, which comprise a pair of repeats, each spanning two
transmembrane helices connected by a loop (based on the description
in the CDD [Conserved Domain Database]). SWEETs have been identied in various organisms (human, Caenorhabditiselegans, and plants)
based on their capacity to transport glucose across a membrane along
a concentration gradient. The rst plant SWEET transporter to be characterized was AtSWEET1, which acts as a glucose uniporter in various
systems (Chen et al., 2010). AtSWEET11 and AtSWEET12 were recently
identied as key players in sucrose efux from phloem parenchyma
cells (Chen et al., 2012). SWEET17 and SWEET16 are major factors controlling fructose content in the tonoplasts of Arabidopsis thaliana leaves
and roots (Chardon et al., 2013; Klemens et al., 2013; Guo et al., 2014).
VvSWEET4 is a glucose transporter localized to the plasma membrane
in grapevine that is upregulated by inducers of reactive oxygen species
and virulence factors from necrotizing pathogens (Chong et al., 2014).
More recently, SWEET9, a nectary-specic transporter in eudicots, was
shown to function as an essential efux transporter in nectar production (Lin et al., 2014).
According to the TCDB (Transporter Classication Database), typical
MtN3/saliva/SWEET-type proteins can be divided into three clades
(Yuan and Wang, 2013). Phylogenetic analysis has revealed that
MtN3/saliva/SWEET-type proteins in cellular organisms may be derived
from a protein with three transmembrane helices harboring only
one MtN3/saliva domain in prokaryotes, and proteins with seven

http://dx.doi.org/10.1016/j.gene.2015.07.055
0378-1119/ 2015 Elsevier B.V. All rights reserved.

Please cite this article as: Feng, C.-Y., et al., Genome-wide identication, phylogeny, and expression analysis of the SWEET gene family in tomato,
Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.07.055

C.-Y. Feng et al. / Gene xxx (2015) xxxxxx

transmembrane helices harboring two MtN3/saliva domains may have


evolved from this protein in eukaryotes. Although the structures of
these unique proteins harboring MtN3/saliva domains have been authenticated, their exact biological functions are unknown. However, recent studies have shown that SWEET genes are involved in multiple
biological processes; signicant progress has been made toward the
identication and characterization of SWEET genes in Arabidopsis and
rice (Chen et al., 2010; Yuan et al., 2014). While the role of these transporters in sucrose loading into the phloem has been identied, less is
known about how export toward ripening fruits is facilitated. The uptake of hexoses from the fruit apoplast to the storage parenchyma
cells is thought to be achieved by the hexose transporters LeHT1 and
LeHT3 (Ruan et al., 1997; Dibley et al., 2005; McCurdy et al., 2010). A
number of metabolomics studies on the ripening of tomato fruit have
revealed dynamic patterns of accumulation (Roessner-Tunali et al.,
2003; Carrari et al., 2006; Carrari and Fernie, 2006; Schauer et al.,
2006; Carrari et al., 2007; Luengwilai et al., 2010). However, the degree
to which de novo synthesis and import of organic acids contributes
to their nal content in ripe fruit is currently unknown. Moreover,
little is known about this gene family in tomato, which harbors
berry fruits.
Tomato (Solanum lycopersicum) is a major crop plant that is considered to be an economically important vegetable worldwide due to its
high yield and good quality. Whole genome sequencing of tomato
(Heinz 1706) by the Tomato Genome Consortium has enabled
genome-wide identication and functional analysis of the gene families
related to the morphological diversity and agronomic traits of tomato
fruit. Furthermore, tomato fruit is an important resource for developing
potential strategies to genetically improve Solanum-related crops. In the
current study, given the signicance of SWEET genes in diverse biological and physiological processes, including sugar transport, we performed whole genome-wide analysis to identify SWEET proteins in

tomato. We analyzed the gene structures, conserved domains, and


phylogenetic relationships of the SWEET genes in detail. In addition,
we performed comprehensive analysis of tissue- and organ-specic
expression of SWEET genes in tomato and examined their expression
proles in response to abiotic stresses (high salinity and temperature) and exogenous sugar (sucrose, fructose, and glucose). Our
results provide a framework for future functional analyses of
SWEET genes and lay a foundation for the utilization of genes to
breed tomato plants with increased plant production, fruit quality,
and stress resistance.
2. Materials and methods
2.1. Identifying members of the SWEET gene family in tomato
A BLASTP search was performed to identify members of the SWEET
gene family in tomato in the tomato plant GDB database (http://www.
plantgdb.org/) and the Solanaceae Genomics Network database
(http://www.sgn.cornell.edu). To identify tomato SWEET genes, the
protein sequences of 21 rice SWEETs and 17 Arabidopsis SWEETs were
used as queries. A name search for MtN3 also helped identify SWEET
genes in the NCBI database. In total, 29 SWEET sequences were obtained.
These genes were designated S. lycopersicum SWEET (SlSWEET)
genes. The sequences were further used as query sequences for
BLASTN searches against SGN Tomato Whole Genome Scaffolds data
(2.10) (http://www.sgn.cornell.edu/tools/blast/; International Tomato
Genome Sequencing Consortium). Genes were assigned numbers
SlSWEET117 based on their positions (from top to bottom) on tomato
chromosomes 112 (Table 1). The SlSWEET genes were further used
as query sequences to search for members of SWEET gene families
from three other Solanaceae members, namely, potato, Nicotiana
tomentosiformis, and Nicotiana sylvestris using the SGN database

Table 1
Information about tomato SWEET genes.
Gene
name

Gene accession
No.

locus

Exon

Best hit EST


No.

AAa

TMDb

LOCc

MtN3/saliva (PQ-loop
repeat) domain position

Location

Arabidopsis
homologous

E-value

NEC1
SWEET1a
SWEET1b
SWEET1c
SWEET1d
SWEET1e
SWEET1f
SWEET2a
SWEET2b
SWEET3
SWEET5a
SWEET5b
SWEET6a
SWEET7a
SWEET7b
SWEET10a
SWEET10b
SWEET10c
SWEET11a
SWEET11b
SWEET11c
SWEET11d
SWEET12a
SWEET12b
SWEET12c
SWEET12d
SWEET14
SWEET16
SWEET17

Solyc09g074530
Solyc04g064610
Solyc04g064620
Solyc04g064630
Solyc04g064640
Solyc06g060590
Solyc06g060580
Solyc02g071520
Solyc07g062120
Solyc03g007360
Solyc03g114200
Solyc06g071400
Solyc02g086920
Solyc08g082770
Solyc12g055870
Solyc03g097580
Solyc03g097600
Solyc03g097610
Solyc03g097870
Solyc03g097570
Solyc06g072620
Solyc06g072640
Solyc03g097590
Solyc03g097620
Solyc05g024260
Solyc06g072630
Solyc03g097560
Solyc01g099880
Solyc01g099870

LOC101259076
LOC101258946
LOC101259239
LOC101259534
LOC101244279
LOC101255412
LOC101248588
LOC101263567
LOC101249940
LOC101252361
LOC101246817
LOC101266340
LOC101251355
LOC101246848
LOC101263002
LOC101260802
LOC101263195
LOC101265012
LOC101261104
LOC101265905
LOC101265550
LOC101247336
LOC101263500
LOC101262399
LOC101255592
LOC101247627
LOC101265318
LOC101258901
LOC101244429

6
6
6
6
8
6
6
6
6
7
6
5
6
5
5
6
6
6
6
6
6
6
6
5
6
6
6
6
6

FC17DC08
sgn_E313270
FA29CH08
FC10CE02
sgn_E545113
sgn_E341275
Not found
LEFL1019BH05
sgn_E289147
Not found
Not found
sgn_E329522
Not found
LEFL2035M23
Not found
FA14CH11
sgn_E377288
Not found
LEFL1023BG11
Not found
LEFL1098AA07
Not found
sgn_E377412
Not found
sgn_E283622
sgn_E210835
LEFL2019P15
LEFL1055CA05
Not found

286
246
245
250
308
256
252
235
235
257
236
238
233
251
240
302
296
282
300
297
287
282
298
276
295
289
295
293
261

7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7

NU
PM
PM
CL
PM
PM
CL
CL
CL
CL
CL
CS
CL
CL
CL
CL
CL
CL
CL
CL
CL
PM
CL
CL
CL
CL
CL
VM
VM

1196,133218
997,134216
1097,134216
13100,137220
75161,198276
1098,135217
1098,135217
19101,139223
18104,138223
997,132217
993,131217
1094,132218
1098,134219
1098,134219
1095,134219
1097,131217
1096,131217
996,130216
1097,131212
1097,131217
1097,131217
1496,130216
895,129215
1399,134218
1299,133218
1097,131214
794,128214
692,128211
1692, 127208

66359865..66361500
55774237..55776296
55793773..55796459
55801078..55804888
55809536..55813393
38581076..38585285
38577739..38579624
40917928..40921133
64926033..64929612
1910344..1912236
64195079..64203323
43928594..43930300
49505088..49506990
65460893..65463783
61871942..61874589
59900408..59910097
59936826..59938478
59945793..59947917
60189696..60192431
59900408..59910097
44785975..44787871
44798555..44799956
59925733..59927594
59,952,039..59954012
30963398..30965989
44791284..44793146
59893279..59895284
90010299..90013205
90005489..90007706

AT5G13170
AT1G21460
AT1G21460
AT1G21460
AT1G21460
AT1G21460
AT1G21460
AT3G14770
AT3G14770
AT5G53190
AT5G62850
AT5G62850
AT5G62850
AT4G10850
AT4G10850
AT5G50790
AT5G50790
AT5G50790
AT3G48740
AT3G48740
AT3G48740
AT3G48740
AT5G23660
AT5G23660
AT5G23660
AT5G23660
AT4G25010
AT3G16690
AT4G15920

1e-66
1e-85
6e-93
1e-62
3e-73
2e-65
1e-69
1e-66
1e-73
1e-82
9e-87
1e-91
1e-49
3e-66
6e-62
2e-71
3e-73
2e-75
9e-70
1e-72
8e-73
3e-42
5e-78
7e-73
3e-68
1e-61
5e-68
4e-55
5e-53

The amino acid sequence length was either conrmed 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.
b

Please cite this article as: Feng, C.-Y., et al., Genome-wide identication, phylogeny, and expression analysis of the SWEET gene family in tomato,
Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.07.055

C.-Y. Feng et al. / Gene xxx (2015) xxxxxx

(http://www.sgn.cornell.edu) and NCBI (http://www.ncbi.nlm.nih.gov/)


(Supplemental Table 1).
2.2. Gene structure, motif recognition, multisequence alignment, and
phylogenetic analyses
A schematic diagram of the gene structure of SWEET genes was
constructed using the Gene Structure Display Server (http://gsds.cbi.
pku.edu.cn/) (Hu et al., 2015). To identify the conserved motifs of the
SWEET protein sequences, the online MEME tool (http://meme.sdsc.
edu/meme/meme.html) was used. The deduced amino acid sequences
of the tomato SWEET gene family were submitted to TMHMM Serverv.2.0
(http://www.cbs.dtu.dk/services/TMHMM) in FASTA format with default
settings to predict the presence of protein domains.
All sequences that satised the requirements were analyzed using
the Pfam database (http://pfam.janelia.org/) (Punta et al., 2012), the
SMART database (http://smart.embl-heidelberg.de/) (Schultz et al.,
1998), and the Conserved Domain NCBI database (http://www.ncbi.
nlm.nih.gov/Structure/cdd/wrpsb.cgi/) (Marchler-Bauer et al., 2011) to
eliminate genes that did not contain the known conserved domains
and motifs of the MtN3/saliva/SWEET family members. Genomic information concerning the chromosome locations of the SWEET genes and
the amino acid sequences of SWEET proteins was obtained from the
SGN database (Bailey et al., 2006).
ClustalW (http://align.genome.jp) was used to perform a homologous sequence alignment of the amino acid sequences of tomato
SWEET family proteins using default settings. Then, conservative or
similar amino acid sequences were highlighted with BoxShade (http://
www.ch.embnet.org/software/BOX_form.html). Based on the results
of sequence alignment, an unrooted phylogenetic tree of the tomato
SWEET gene family was constructed using Mega 4.0 (http://www.
megasoftware.net) (Tamura et al., 2007). The neighbor-joining (NJ)
method was applied to construct a phylogenetic tree (Saitou and Nei,
1987), in which Poisson correction, pairwise deletion, and bootstrapping
(1000 replicates; random seeds) served as default values to evaluate the

reliability of the tree. The SGN database (http://www.sgn.cornell.edu)


and NCBI (http://www.ncbi.nlm.nih.gov/) were used to map the positions
of the SWEET genes in the physical maps of the 12 S. lycopersicum chromosomes. The identied CDSs were used to nd EST clones containing
SWEET cDNAs from the TOMATOMICS database (http://www.bioinf.
mind.meiji.ac.jp/tomatomics/) (Aoki et al., 2010) or from the Sol
Genomics Network EST databases (http://www.solgenomics.net/).
2.3. Expression proles of SWEET genes in tomato
The expression proles were obtained by analyzing RNA-seq data
based on locus/gene names. The RNA-seq data were downloaded from
the Tomato Functional Genomics Database (http://ted.bti.cornell.edu/
cgi-bin/TFGD/digital/home.cgi), including sequence data from various
tissues in tomato cultivar Heinz 1706 (S. lycopersicum) and wild
species LA1598(Solanum pimpinellifolium).
Tomato plants (Micro-Tom) were grown in a greenhouse at 26 C
with a 12 h/12 h (light/dark) photoperiod. Plants were treated according to the surface colors of their fruit using ripening stages set by the
USDA. Tomato plants were grown for 6 days at 26 C/15 C with a
12 h/12 h (light/dark) photoperiod at the mature green stage or maturation periods under 1/2 Yamazaki nutrient solution (Yamazaki nutrient
solution, consisting of Ca(NO3)24H2O 1.5 mM, KNO3 4 mM, NaNO3
3.5 mM, NH4H2PO4 0.67 mM, MgSO47H2O 2 mM, H3BO3 0.05 mM,
MnSO44H2O 0.009 mM, ZnSO47H2O 0.7 M, CuSO45H2O 0.32 M,
(NH4)2MoO4 0.1 M, FeSO47H2O 0.05 mM, Na2-EDTA 0.04 mM) and
subjected to various treatments.
The nutrient solution was supplemented with 80 mM NaCl for salt
treatment and 3% (w/v) sucrose, 2% (w/v) fructose, and 2% (w/v) glucose for sugar treatment. The plants were grown at 37 C/15 C with a
12 h/12 h (light/dark) photoperiod for high temperature treatment
and 25 C/6 C with a 12 h/12 h (light/dark) photoperiod for low
temperature treatment. In general, for RNA isolation, plant tissues
were collected after 2 days of treatment, immediately frozen in liquid
nitrogen, and stored at 80C until use.

Table 2
Expression patterns of SlSWEET genes in Heinz 1706 (Solanum lycopersicum).
Gene name

SlSWEET1e
SlSWEET2b
SlSWEET1d
SlSWEET2a
SlSWEET1c
SlSWEET3
SlSWEET6a
SlSWEET1f
SlSWEET7b
SlSWEET11d
SlSWEET11b
SlSWEET10a
SlSWEET11c
SlSWEET12d
SlSWEET10c
SlSWEET12c
SlSWEET1a
SlSWEET11a
SlSWEET12a
SlSWEET5b
SlNEC1
SlSWEET12b
SlSWEET16
SlSWEET10b
SlSWEET1b
SlSWEET5a
SlSWEET7a
SlSWEET14
SlSWEET17

Normalized expression (RPKM) in cultivar Heinz


Roots

Leaves

Unopened
ower buds

Fully opened
owers

1 cm fruits

2 cm fruits

3 cm fruits

Mature green
fruits

Breaker
fruits

Breaker +
10 fruits

8.44
5.49
3.06
8.58
30.44
8.99
0
1.09
0
0
6.61
5.22
0.93
0.31
1.55
3.83
0.14
0.21
2.03
1.85
0.2
0
0
10.98
1.85
0.06
0
0.09
0.73

11.34
8.14
0.19
9.08
0.18
5.11
0
0.58
0
0
5.56
41.51
2.89
1.7
51.94
25.18
12.58
337.64
108.72
0.25
0.23
0
1.41
19.82
0.25
0
0
0.12
0.35

0.88
4.54
0.99
12.32
0.88
1
4.55
0.57
0.42
0.56
2.65
6.31
0.08
0.17
2.61
43.07
117.16
58.86
47.35
617.85
73
0
13.15
41.32
45.54
23.69
24.65
24.68
6.26

3.55
4.26
0.77
6.16
0.61
1.68
0
0
0
0.71
7.85
8.72
0.14
0.1
6.53
18.97
18.52
60.82
140.53
665.33
6.74
0
59.91
164.18
15.68
0
2.73
17.16
0.9

0
0.54
0.68
9.58
4.81
1.21
0
0
0
0.07
0
0
0
0.12
0
0.22
0.33
0.1
0
1.38
0.51
0
0
0.15
27.34
0
19.84
14.61
0.85

0
0.31
0.53
6.55
3.58
1.12
0
0.1
0
0.08
0
0
0.12
0
0
0.41
0.17
0.45
0
0.34
0.44
0
0.35
1.38
42.04
0
20.03
4.76
0.12

0
0.61
0.81
2.06
2.32
1.09
0
0
0
0
0
0
0.05
0.05
0.05
0.4
0.18
0.29
0
2.61
0.23
0
0.38
0.32
52.11
0
17.85
1.85
0.09

0.05
0.62
0.77
0.51
2.28
0.41
0
0
0
0
0.68
2.26
0
0
0
172.52
0.43
0.49
0.18
1.06
0
0
0.37
0.54
20.24
0
3.06
6.51
0

0.05
0.27
0.96
0.04
0.14
0.09
0
0
0
0
0
0
0
0
0
50.43
0.13
0.32
0.05
0.17
0
0
0.2
0.46
0.38
0
0
3.01
0

0.04
0.09
1.23
0
0.07
0
0
0
0.05
0
0
0
0
0
0
95.86
2.48
0.23
0
0.07
0.11
0
0.1
0.42
0.04
0
0.04
1.1
0

Please cite this article as: Feng, C.-Y., et al., Genome-wide identication, phylogeny, and expression analysis of the SWEET gene family in tomato,
Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.07.055

C.-Y. Feng et al. / Gene xxx (2015) xxxxxx

2.4. Total RNA isolation and validation of SWEET gene expression proles
via quantitative real-time PCR
Total RNAs were extracted using an RNA Simple Total RNA Kit
(Tiangen, Beijing, China) according to the manufacturer's instructions.
Then, the quality of total RNAs was tested using agarose gel electrophoresis. The RT reactions were performed using M-MLV Reverse Transcriptase (Takara, Japan) as directed by the manufacturer. Quantitative realtime PCR was performed with SYBR Green PCR Master Mix (Tiangen,
Beijing, China) for qRT-PCR analysis using an ABI Prism 7500 Sequence
Detection System and Software (Applied Biosystems). Amplication
was initiated with a denaturation step of 10 s at 95 C, followed by
40 cycles of 95 C for 10 s and 60 C for 30 s. All reactions were
performed in triplicate, and negative controls (no template and no
RT) were included for each gene. The specicity of the reactions was
veried by melting curve analysis (Supplemental Table 2).

3. Results
3.1. Identifying tomato SWEET gene family members and their distribution
on chromosomes
The SWEET genes were identied based on BLAST searches against
the SGN and NCBI databases. A total of 29 SlSWEETgenes with conrmed
conserved domains were identied from the S. lycopersicum genome
(Tables 1, 2). The SlSWEET genes were named according to their
homologous genes in A. thaliana. Each AtSWEET gene corresponds to
approximately one to six SlSWEET genes.
The SlSWEET genes are distributed on 10 out of 12 chromosomes (all
except 10 and 11). The ORFs of the SlSWEET genes range from 699 bp to
924 bp in length, encoding polypeptides 233 aa to 308 aa in length. The
predicted subcellular localizations of the SlSWEET genes vary, including
the plasma membrane, vacuolar membrane, chloroplast, mitochondrion,

Fig. 1. Distribution of 29 tomato SWEET genes on chromosomes. indicates backwards transcription, and indicates forward transcription.

Please cite this article as: Feng, C.-Y., et al., Genome-wide identication, phylogeny, and expression analysis of the SWEET gene family in tomato,
Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.07.055

C.-Y. Feng et al. / Gene xxx (2015) xxxxxx

and nucleus. BLAST searches of the translated tomato genome using the
AA sequences of characterized SWEETs in Arabidopsis as queries identied 29 loci putatively encoding full-length SWEETs in tomato
(Table 1). A total of 29 ESTs were identied based on tblastn searches
against the EST MicroTom database (http://www.kazusa.or.jp/jsol/
microtom/), and ESTs with scores 100 or E values 1010 were selected
as the best ESTs of each SWEET gene. At least one EST was available in
public databases for 19 of these loci. Loci without EST evidence might
only be expressed under certain conditions or in specic tissues.
The chromosomal location of each SWEET gene was determined
based on the genomic sequences of tomato. Fig. 1 shows the distribution
of the 29 genes on a total of ten tomato chromosomes. No SlSWEET gene
was found on chromosomes 10 or 11. Most genes are distributed on the
third, fourth, and sixth chromosomes, which harbor ten, four, and six
genes, respectively. Only two genes (each) are present on chromosomes
1 and 2. Only one SlWEET is located on chromosomes 5, 7, 8, 9, and
12. Three pairs of genes (SWEET1e/SWEET1, SWEET1c/SWEET1d, and
SWEET16/SWEET17) on the chromosome are tightly linked. These
homologous genes have bootstrap values of 99.
3.2. Gene structure and conserved domain analysis of SWEET family genes
To analyze the structural characteristics and conserved regions of
the SlSWEET genes, we mapped their gene structures, including exons
and introns, examined their conserved regions and motifs, and aligned
their putative protein sequences. We obtained the gene structures
using the online software Gene Structure Display Server (http://gsds.
cbi.pku.edu.cn/index.php). In the tomato MtN3/saliva/SWEET gene
family, all SlSWEET genes contain introns in their genomic sequences.
SWEET1d contains eight exons, SWEET3 contains seven exons, and
SWEET5b, SWEET7a, SWEET7b, and SWEET12b contain ve exons; the
remaining gene family members all contain six exons (Fig. 2).
According to information in the Pfam database, we determined that
all 29 SWEET genes encode seven transmembrane helices (Fig. 3) harboring two MtN3/saliva domains (Supplemental Fig. 1). There are
approximately 90 aa in these two MtN3/saliva domains, all at the
same positions in the proteins, as shown in Table 1. We deduced the

amino acid sequences of the 29 members of the tomato MtN3/saliva/


SWEET gene family and submitted them to the TMHMM Server v.2.0
(http://www.cbs.dtu.dk/services/TMHMM) in FASTA format with
default values. We predicted that the protein domains encoded by all
29 genes have a seven-transmembrane-domain structure, while the
areas outside the membrane are generally very short (Fig. 3).
Pair-wise comparisons of these SlSWEET protein sequences revealed
that their similarity levels range from a low of 44% (between SlNEC1 and
SlSWEET12c) to a high of 90% (between SlSWEET11a and SlSWEET12a),
and the N-terminal DBDs of SlSWEET proteins are highly conserved
(Fig. 4). Among these, there are 16 SlSWEET genes with bootstrap values
higher than 90, including SWEET11/SWEET12, SWEET11C/SWEET12d,
SWEET16/SWEET17, SWEET5/SWEET5b, SWEET7a/SWEET7b, SWEET2a/
SWEET2b, SWEET1e/SWEET1f, and SWEET1c/SWEET1d.
Sequence alignment revealed that the MtN3/saliva domain is highly
conserved within the inner side of the membrane domains and relatively conserved in the transmembrane area. There is a possible serine
phosphorylation site in each of the two highly conserved segments in
the inner side of the membrane area. Two proteins (SlSWEET5a and
SlSWEET10b) contain threonine in the rst MtN3/saliva domain instead
of serine, while the rst MtN3/saliva domain in SlSWEET3 contains
asparagine. All of these amino acids can be phosphorylated except
asparagine. These results indicate that the inner side of the membrane
area of SWEET family proteins is likely to be their important functional
area or protein structure or their active regulatory region, and its regulation is likely subjected to reversible phosphorylation/dephosphorylation
controlled by some protein kinase/phosphatase. This nding may
represent a breakthrough in the analysis of the function of the tomato
SWEET family.
3.3. Phylogenetic analysis of SWEETs in tomato and other members of the
Solanaceae family
To study the phylogenetic relationships among SWEET genes in
tomato, Arabidopsis, and rice, an unrooted phylogenetic tree was
constructed by aligning 29 SlSWEET protein sequences, 17 AtSWEET
protein sequences, and 21 OsSWEET protein sequences using ClustalX

Fig. 2. Exon-intron structures of 29 SWEET genes identied in tomato. Shown is a graphic representation of the gene models of all 29 SWEETs identied in this study. Exons are represented
by green boxes and introns are represented by black lines, upstream/downstreams are represented by blue boxes. Gene models are based on sequenced ESTs; when EST data were lacking,
in silico predictions (ITAG release 2.3 SL2.40) were used. 0 indicates an intron located between two codons, 1 indicates an intron inserted into the rst base of a codon, and 2 indicates an
intron inserted into the second base of a codon. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)

Please cite this article as: Feng, C.-Y., et al., Genome-wide identication, phylogeny, and expression analysis of the SWEET gene family in tomato,
Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.07.055

C.-Y. Feng et al. / Gene xxx (2015) xxxxxx

Fig. 3. Unrooted neighbor-joining phylogenetic tree and predicted domain structures of the 29 tomato SWEET proteins. Blue line indicates cytosol localization, pink line indicates apoplast
localization, and red line indicates transmembrane domain. Bootstrap values are indicated on the branches. (For interpretation of the references to color in this gure legend, the reader is
referred to the web version of this article.)

and MEGA4.0. These SWEET sequences fall into two broad classes. Class
I is divided into three subclasses, namely Ia, b, and c. Subclass Ia contains
nine SlSWEETs (SlSWEET1a1f, 2a, 2b, and 3), three AtSWEETs
(AtSWEET1, 2, and 3), and six OsSWEET (OsSWEET1a, 1b, 2a, 2b, 3a,
and 3b). Subclass Ib contains ve SlSWEETs (SlSWEET5a, 5b, 6a, 7a,
and 7b), ve AtSWEETs (AtSWEET48), and nine OsSWEET (OsSWEET4,
5, 6a, 6b, and 7ae). Subclass Ic contains two SlSWEETs (SlSWEET16
and 17), two AtSWEETs (AtSWEET16 and 17), and one OsSWEET
(OsSWEET16). Class II contains 13 SlSWEETs (SlNEC1, SlSWEET10ac,
SlSWEET11ad, SlSWEET12ad, and SlSWEET14), seven AtSWEETs
(AtSWEET915), and ve OsSWEETs (OsSWEET1115). A total of 22 sister pairs, including four SlSWEET-AtSWEET, six SlSWEET-SlSWEET,
seven OsSWEET-OsSWEET, one OsSWEET-SlSWEET, and four
AtSWEET-AtSWEET pairs are present in the combined phylogenetic
tree. Class I and II include SlSWEET, OsSWEET, and AtSWEET (Supplemental Fig. 2).
Using tomato SWEET protein sequences as query sequences, BLASTP
searches against sequences from other Solanaceae plant gene index projects and the SGN database resulted in the identication of 35 SWEET
genes in potato (Solanum tuberosum), 31 in tobacco (N. tomentosiformis),
and 36 in tobacco (N. sylvestris), while no SWEET gene was found in petunia or pepper because of incompleteness of the database now. The characteristics of the databases used for these search, as well as the number of
genes, are shown in Supplemental Table 1. The nomenclature used for
all SWEETs from different Solanaceae family members is similar to that
used for SlSWEETs, i.e., Solanum tuberosum SWEET (StSWEET) genes,
N. tomentosiformis SWEET (NtSWEET) genes, and N. sylvestris SWEET
(NsSWEET) genes. The combined phylogenetic tree of 168 SWEET protein
sequences from various Solanaceae members resulted in formation of four
groups, namely IIV. The number of SWEET members in each group

varies. In total, 56 sister pairs were identied from Solanaceae plants,


including 26 SlSWEET-StSWEET, 24 NsSWEET-NtSWEET, four AtSWEETStSWEET, one NsSWEET-NsSWEET, and one StSWEET-StSWEET pair
(Supplemental Fig. 2).

3.4. Expression patterns of SWEET genes in cultivated and wild tomato


Digital expression proling (or RNA-seq) is a powerful, efcient
approach for large-scale gene expression analysis (Wang et al., 2009).
We therefore examined the expression patterns of SWEET genes based
on data available in the public databases (RNAseq database: http://ted.
bti.cornell.edu/). Based on this analysis, four genes, SlSWEET1f,
SlSWEET7b, SlSWEET11d, and SlSWEET12c, are not expressed in Heinz
1706 (S. lycopersicum) or wild tomato LA1589 (S. pimpinellifolium). In
Heinz 1706, the expression of the remaining SlSWEET genes displayed
spatial variation in different tomato organs. Some of these genes were
constitutively expressed in every organ investigated, and their expression levels were high, especially in young immature fruit, and they
declined during fruit ripening; such genes include SlSWEET1C and
SlSWEET2a from group A, as well as SlSWEET1b, SlSWEET7a and
SlSWEET2a, whereas SlSWEET12c shared a similar expression pattern,
with high expression in mature fruit (Table 2). In wild tomato
LA1589, SlSWEET12c and SlSWEET14 were expressed in every organ examined and their expression levels were relatively high in ripening fruit
(33 DPA). Some of the genes, such as SlSWEET10a and SlNEC1, were highly
expressed, especially at 20 DPA, and their expression declined during fruit
ripening. SlSWEET5b and SlSWEET16 showed similar expression patterns,
with high expression in anthesis owers (0 DPA) and low expression
levels at other stages (Table 3). These results suggest that the SlSWEET

Please cite this article as: Feng, C.-Y., et al., Genome-wide identication, phylogeny, and expression analysis of the SWEET gene family in tomato,
Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.07.055

C.-Y. Feng et al. / Gene xxx (2015) xxxxxx

Fig. 4. Unrooted neighbor-joining phylogenetic tree of SWEETs in tomato, rice, and Arabidopsis. Scale bar represents 0.1 substitutions per amino acid position.

Table 3
Expression patterns of SlSWEET genes in wild tomato species LA1598 (S. pimpinellifolium).
Gene name

SlSWEET1e
SlSWEET2b
SlSWEET1d
SlSWEET2a
SlSWEET1c
SlSWEET6a
SlSWEET3
SlSWEET1f
SlSWEET7b
SlSWEET11d
SlSWEET11b
SlSWEET10a
SlSWEET11c
SlSWEET12d
SlSWEET10c
SlSWEET12c
SlSWEET1a
SlSWEET11a
SlSWEET12a
SlSWEET5b
SlNEC1
SlSWEET12b
SlSWEET16
SlSWEET10b
SlSWEET1b
SlSWEET5a
SlSWEET7a
SlSWEET14
SlSWEET17

Normalized expression (RPKM) in wild species LA1598


HYPO

COTYL

MERI

ROOT

YL

ML

YFB

0DPA

10 DPA

20DPA

33DPA

15.21
7.21
2.61
11.85
3.3
0
1.62
0.23
0.01
0.01
14.18
1.32
56.58
5.03
52.62
42.92
4.5
136.16
90.91
0.57
0.18
0
1.01
40.37
4.3
0
0
0
0.57

2.53
6.11
0.54
11.82
0.36
0
3.55
0.03
0.01
0.04
17.16
8.45
2.33
1.62
31.74
72.67
5.51
225.12
61.34
0.59
4.21
0
2.96
15.11
3.25
0
1.4
0.71
0.12

2.91
6.26
1.43
13.75
1.74
0
1.57
0.1
0.03
0.02
21.33
2.39
14.21
3.53
43.94
6.15
7.71
61.25
58.77
0.39
0.62
0
2.44
12.8
1.88
0.02
0.08
0.14
2.27

19.64
6.8
6.43
7.91
91.45
0.07
134.55
0.33
0.01
0
9.23
1.3
21.47
6.14
20.36
15.24
0.02
7.99
22.83
0.37
1.22
3.9
14.35
198.79
0.05
0
0
0.05
0.9

0.13
6.37
1.8
12.38
0.21
0.03
0.19
0.02
0
0.02
1.12
1.25
0.58
0.3
3.77
33.93
10.68
32.17
14.7
0.76
0.26
0
2
1.95
2.29
0
0.12
0.39
7.42

6.65
3.28
0.38
5.58
0.93
0
6.94
0.05
0
0.1
4.61
7.88
7.52
2.21
46.1
159.59
2.35
625.24
97.82
3.18
0.23
0.04
6.43
8.07
0.16
0
0
0.05
0.22

0.04
6.19
1.85
11.22
0.02
0.01
0.07
0.02
0.02
0.01
1.1
1
1.01
0.39
1.77
27.18
17.63
18.4
12.77
0.61
0.51
0.06
5.37
8.56
13.43
0
1.13
3.81
11.25

2.03
6.7
1.42
4.8
1.68
0.09
2.37
0.01
0
0
1.89
3.55
0.28
0.2
2.71
38.8
10.71
114.38
89.42
1217.85
4.76
0.56
145.92
66.91
23.7
0.64
1.71
18
3.36

0.49
1.37
0.82
3.15
3.36
0.2
35.91
0
0.05
0
21.12
184.66
0.53
0.24
2.72
57.89
27.25
33.27
7.08
6.23
66.48
0
1.34
4.43
52.66
0
22.74
12.56
0.09

0.29
2.18
1.76
0.86
4.89
2.28
22.06
0
2.37
0
72.61
1169.49
0.14
0.01
0.26
86.2
7.62
8.02
1.72
0.43
469.53
0.04
0.15
1.68
22.46
0
48.08
15.51
0

0.29
0.55
2.89
0.12
0.83
0.96
0.23
0
3.2
0
7.71
113.62
0.9
0.03
0.39
576.39
9.44
4.38
0.53
0.14
2.12
0
0.03
0.7
4.04
0
0.15
45.26
0

Hypo hypocotyl, cotyl cotyledon, meri vegetative meristem, YL young leaf, ML mature leaf, YFB young ower bud, 0 DPA ower at anthesis, 10 DPA F at 10 DPA, 20 DPA F at 20 DPA, 33 DPA
F at 33 DPA.

Please cite this article as: Feng, C.-Y., et al., Genome-wide identication, phylogeny, and expression analysis of the SWEET gene family in tomato,
Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.07.055

C.-Y. Feng et al. / Gene xxx (2015) xxxxxx

gene family may play important roles in tomato fruit development in


addition to functioning in non-reproductive tissues.
3.5. Expression proles of SWEET genes under abiotic stress in tomato
In the RNA-seq experiment, the differential expression patterns of
SlSWEET11b observed between ripening stages of cultivar Heinz and
wild LA1598 fruits indicate that SlSWEET genes might play an important
role during ripening in tomato. Since only a few SWEET genes are
represented in the Genevestigator database, we examined the expression of all SWEET genes in Micro-Tom fruit by real-time PCR to elucidate
the spatio-temporal expression of each member of the gene family at
the transcriptional level. The heatmap representation of the global
expression patterns reveals the clustering of tomato SWEETs. Based on
Fig. 5A and B, data analyses indicated high variability in transcript abundance of the SWEET genes in tomato. Most SWEET genes exhibited
opposite expression patterns in roots versus leaves, such as SlSWEET1c,
7b, 14, 10a, 12a, 11a, 11b, 11c, and 11d. Notably, SlSWEET10a, 10b, 10c,
11a, 11b, 11c, 11d, 12a, and 12c exhibited similar expression patterns
in response to sugar treatment, salt, and temperature stress and were
therefore grouped together (Fig. 5A). These genes were upregulated
several fold in leaves under these treatments, while they were dramatically downregulated in roots. However, during the development of mature green fruits, these genes produced high transcript levels upon
fructose and sucrose treatment or low temperature stress (Fig. 5B);
these results indicate that these genes have spatio-temporal expression
characters. Genes with high transcript levels at the onset of fruit ripening may play essential roles in determining fruit quality and yield.
We used Cluster software for sequence analysis of gene expression
and displayed the results in a tree diagram using TreeView software.
Based on the patterns of gene expression in tomato fruit, SWEET family

genes were divided into two clusters: Cluster A (19 genes) and Cluster B
gene (10 genes). Cluster A and Cluster B were each divided into two
groups. The genes in Group A1 were not regulated in mature green
fruit in response to treatment; however, these genes were suppressed
in red ripening fruit. Likewise, Group A2 genes were suppressed under
fructose and sucrose treatment in MG fruits, suggesting that they play
a negative role under high sugar conditions. SlSWEET10a, SlSWEET10b,
SlSWEET10c, SlSWEET11a, SlSWEET11b, SlSWEET11c, and SlSWEET12a
were generally not regulated by high fructose and sucrose treatment
but were upregulated by low temperature stress, in MG fruits. These
genes were therefore clustered into one group, B1. In Group B2,
SlSWEET12c was suppressed under high sugar conditions and both salt
and temperature stress in mature green fruits; however, this gene was
continually induced after the above treatments in red ripening fruits.
Remarkably, even the homologous SWEET genes exhibited varied
response patterns, suggesting the stress-responsive functional divergence of the homologous genes. Expression proling represents an
initial step in the functional analysis of SWEET genes in tomato; further
studies are needed to elucidate the potential functions of these genes in
abiotic stress adaptation, which would be highly valuable.
3.6. Stress-relevant cis-elements in the promoters of SlSWEET genes
To identify the cis-acting elements in the upstreams of SWEET genes.
We gained the sequences of promoters of 29 SlSWEET genes from
GeneBank and identied the cis-acting elements in the upstreams of
these genes. Then the CAAT-box and TATA-box were identied in the
2000-bp upstream genomic sequences of these SlSWEET genes as the
milestones to conrm the TSS (transcription start site). At last, the
1500-bp DNA sequence upstream of the TSS was truncated to identify
cis-acting motifs via PlantCARE (http://bioinformatics.psb.ugent.be/

Fig. 5. Hierarchical clustering and heat map representation of tissue- or organ-specic SWEET gene expression proles in Micro-Tom. The expression levels of genes are presented using
fold-change values transformed to Log2 format. The data obtained by quantitative RT-PCR correspond to the levels of SWEETs in total RNA samples extracted from Roots (R), Leaves (L),
Mature Green (MG), and Red Ripening (RR) fruit. The data indicate the relative expression levels normalized to that of the internal control Actin2 and correspond to three independent
biological repetitions. Red and green correspond to strong and weak expression of the SWEET genes, respectively. (For interpretation of the references to color in this gure legend, the
reader is referred to the web version of this article.)

Please cite this article as: Feng, C.-Y., et al., Genome-wide identication, phylogeny, and expression analysis of the SWEET gene family in tomato,
Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.07.055

Element

ABRE ARE

ABA

SlNEC1
SlSWEET1a
SlSWEET1b
SlSWEET1c
SlSWEET1d
SlSWEET1e
SlSWEET1f
SlSWEET2a
SlSWEET2b
SlSWEET3
SlSWEET5a
SlSWEET5b
SlSWEET6a
SlSWEET7a
SlSWEET7b
SlSWEET10a
SlSWEET10b
SlSWEET10c
SlSWEET11a
SlSWEET11b
SlSWEET11c
SlSWEET11d
SlSWEET12a
SlSWEET12b
SlSWEET12c
SlSWEET12d
SlSWEET14
SlSWEET16
SlSWEET17

1
1
1

AuxRR-core Box-W1 CGTCA-motif circadian ERE

Anaerobic Auxin
2
2
4

Fungal

2
1
1
1

1
2

MeJA
1
1
2
2
1
1
1

1
1
4
1
1
2
1

2
1
1
2
1

1
1

1
1

1
1

1
2

1
1
2

2
1
1
1

1
2
2

TATC-box

1
2

1
1

2
3

1
1
2
1
2
4
1

2
1
1
2

1
4
2

1
2

1
1

3
3

1
1
1

P-box

1
1
1
1

1
2

1
1
1

1
3
2

3
2
1
3
2

TCA-element TC-rich
repeats

TGACG-motif TGA-element WUN-motif TOTAL

1
1
2
2
1
1
1

1
1

1
1

2
1

3
1

1
1

1
1
1

1
1
1
1
5
3
2
5
2
2
1
1

1
2
2
4
1

2
2

Auxin

Wound

1
1
1

1
1

1
2

1
2

2
2
1
2
2

1
1
1

1
1
1

1
1

1
1
1

1
2
2

MBS

Circadian Ethylene Gibberellin Heat LowDrought Gibberellin Gibberellin Salicylic acid Defense
MeJA
control
temperature
and stress

2
4

1
2
1

GARE-motif HSE LTR

1
2
2

1
1

1
1
1

1
2

1
1
1
1
1

1
1

11
10
20
10
13
9
11
9
11
10
14
23
7
13
8
12
13
3
7
12
7
11
3
11
9
5
12
16
9

C.-Y. Feng et al. / Gene xxx (2015) xxxxxx

Please cite this article as: Feng, C.-Y., et al., Genome-wide identication, phylogeny, and expression analysis of the SWEET gene family in tomato,
Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.07.055

Table 4
Kinds and numbers of known stress-related and hormone-related elements in the upstream regions of SlSWEET genes.

10

C.-Y. Feng et al. / Gene xxx (2015) xxxxxx

webtools/plantcare/html/). As shown in Table 4, 18 known stressrelated elements were identied in the promoters of these SlSWEET
genes. No less than three stress-responsive elements were found in
the 29 SWEET promoters, indicated that SWEETs participate in stress
response.
4. Discussion
The availability of high quality sequence data from the S. lycopersicum
Heinz 1706 genome has greatly advanced the study of tomato. The tomato genome sequence can be mined to identify gene families, analyze
genetic relationships, and determine gene family distribution across the
chromosomes. For example, such studies have led to the identication
of putative Receptor-like Kinases (RLKs) (Sakamoto et al., 2012), putative
SUN, OFP, GABBY, transcriptions factors (Huang et al., 2013), 146 putative
ERF proteins (Pirrello et al., 2012), ERECTA genes (Villagarcia et al., 2012),
and MAPK (Kong et al., 2012), helicase (Xu et al., 2013), WRKY (Huang
et al., 2012), and nucleobaseascorbate transporter (NAT) gene family
members (Cai et al., 2014). With a comprehensive inventory of all sucrose
and monosaccharide transporters encoded in the tomato genome,
Reuscher et al. (2014) identied 52 genes putatively encoding sugar
transporter proteins in the tomato reference genome. All these sugar
transporter protein families belong to the MFS family which is characterized by 12 transmembrane domains (TMDs) and driven by the protonmotive force. In this investigation, we generated 29 genes putatively
encoding SWEET protein, which belonged to a different sugar transporter
superfamily. The tomato SWEET protein sequences exhibited a typical
MtN3/Saliva domain, and almost all of them were phylogenetically
clustered with at least one member of the Arabidopsis SWEET family.
MtN3/saliva/SWEET-type proteins are thought to be seven TMDs of
sugar transporters and independent of the proton gradient, therefore
they were named SWEET proteins (Chen et al., 2010, 2012).
The Solanaceae family is one of the most morphologically diverse
plant families, with more than 3000 described species (Knapp et al.,
2004) distributed worldwide. Bioinformatics studies based on the
tomato sequence, which serves as a reference genome for other
Solanaceae species, have demonstrated that the tomato genome may
serve as a true reference genome for closely related Solanaceae and
may enable studies of orthologous genes and gene families both within
tomato and among more divergent plants (Menda et al., 2013). Systematic analysis of the phylogeny and expression of these genes during
reproductive developmental stages in tomato will be helpful for mining
candidate genes for subsequent detailed characterization. In the present
study, 29 SWEET genes were identied from tomato, while 35, 31, and
36 orthologous proteins were identied from other members of the
Solanaceae family, namely potato, N. tomentosiformis, and N. sylvestris,
respectively. Tomato has served as a model system to study fruit development and ripening, and several SWEETs have been shown to be
involved in various stages of fruit set and development. Expression analysis of SlSWEETs during fruit development in tomato in the current
study suggests that several SlSWEETs are involved in the regulation of
fruit ripening. The comprehensive data generated in this study will be
helpful for conducting functional genomics studies to elucidate their
precise roles during fruit development in tomato as well as the roles
of other members of the Solanaceae family. No SWEET gene was identied from petunia, as the EST resources for this plant are still inadequate.
As the number of available ESTs increases, the number of SWEETs
identied in petunia, pepper, and N. benthamiana may also increase. In
total, we identied 56 sister pairs, including 26 SlSWEET-StSWEET, 24
NsSWEET-NtSWEET, four AtSWEET-StSWEET, one NsSWEET-NsSWEET,
and one StSWEET-StSWEETpair. StSWEETs were found to be more closely
related to their tomato homologs than to NtSWEETs. All 26 StSWEETs
genes were found to be more closely related to SlSWEETs genes than
to SWEET members of other Solanaceae plants, thus providing insights
into the evolutionary relationship between the Solanaceae members
examined in this study. Since it is known that gene numbers and gene

positions in almost all Solanaceae species are conserved, the identication and characterization of SlSWEET genes from tomato may help
lead to the identication of these genes (and their putative functions)
in other Solanaceous species.
Using tomato gene ChIP-Seq information, we found that more than
one SWEET gene is responsive to sugar, salt, and temperature stress,
suggesting that these genes play important regulatory roles in the stress
response and that there may be functional redundancy. Based on the
expression patterns of the family members, SlSWEET genes have specic
temporal and spatial expression patterns. Some SWEET family genes
were expressed during fruit development, suggesting that they participate in regulating the process of tomato fruit ripening, but their specic
functions require further study.
More comprehensive ChIP-seq databases are available for A. thaliana
and rice than for tomato, and the functions of some SWEET genes in
A. thaliana and rice had been more widely veried compared with
those of other species. Based on cluster analysis, tomato, Arabidopsis,
and rice SWEET family genes have a relatively conservative evolutionary
relationship. In Arabidopsis and rice, several SWEET genes are involved
in many developmental processes, such as reproductive development,
senescence, environmental adaptation, and hostpathogen interactions
(Yuan and Wang, 2013). Although the roles of SlSWEET genes in these
processes are not yet known, the possibility that SlSWEET genes, like
AtSWEETs and OsSWEET genes, may be involved in these developmental
processes in tomato cannot be ruled out.
The cis-element LTR which is responsible for low-temperature
responsiveness of many plant genes (Thomashow, 1999), is located in
the promoter regions of SWEET1a, 5a and 5b. The heat stress-responsive
element HSE-motif is located in the 21 SWEET promoters, but not in the
promoters of SWEET1a, 1c, 1e, 2b, 6a, 11a, 12a and 12c (Table 4).
Although many temperature-related elements were also found in the
upstream of the SWEETs, these mRNA were detected as downregulated,
but not upregulated. The reason is that downregulation is a much more
complicated process. This type of regulation cannot be simply answered
through stress-related cis-element analysis.
Analysis of the phylogenetic relationships among Arabidopsis, rice,
and tomato SWEET genes revealed that these SWEET sequences could
be divided into two broad classes. Class I was further divided into
three subclasses. Subclass Ia contains three AtSWEETs (AthSWEET13),
six OsSWEETs (OsSWEET1a3b), and nine SlSWEETs (SlSWEET1a3).
ArabidopsisAtSWEET1, which functions as a bidirectional low-afnity
glucose transporter, mediates glucose uptake across the plasma membrane and efux into the endoplasmic reticulum (Chen et al., 2010).
Rice OsSWEET2a exhibits relatively high expression levels in owers or
panicles at various developmental stages, suggesting that it plays a
role in reproductive development (Wang et al., 2010). Members of
this subclass are mainly expressed in oral organs. Based on their high
sequence similarity and similar expression patterns, we proposed that
SlSWEET1a3 also function in glucose transport in tomato. Subclass Ib
includes ve SWEETs (AthSWEET16, AtSWEET17, OsSWEET16, SlSWEET16,
and SlSWEET17). In Arabidopsis, AtSWEET17 and AtSWEET16 are highly
expressed in the root cortex, and they encode proteins that function
as Fru-specic uniporters on the root tonoplast (Chardon et al., 2013;
Klemens et al., 2013; Guo et al., 2014). Subclass Ic contains ve
AtSWEETs (AthSWEET48) and nine OsSWEETs (OsSWEET47e).
Arabidopsis AtSWEET4, AtSWEET5, AtSWEET7, and AtSWEET8/RPG1
function as bidirectional low-afnity glucose transporters, which mediate glucose uptake across the plasma membrane and efux into the endoplasmic reticulum in the human embryonic kidney cell line HEK293T
and in Xenopus oocytes (Chen et al., 2010; Yuan and Wang, 2013). Members of this subclass are mainly expressed in oral organs. Tomato
SlSWEET5b/Lestd1 is specically expressed in maturing pollen grains
(Salts et al., 2005).
Class II contains 13 SlSWEETs (SlNEC1, SlSWEET10ac, SlSWEET11ad,
SlSWEET12ad, and SlSWEET14), seven AtSWEETs (AtSWEET915), and
ve OsSWEETs (OsSWEET1115). AtSWEET13 functions as a low-afnity

Please cite this article as: Feng, C.-Y., et al., Genome-wide identication, phylogeny, and expression analysis of the SWEET gene family in tomato,
Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.07.055

C.-Y. Feng et al. / Gene xxx (2015) xxxxxx

glucose transporter when expressed in HEK293T cells. AtSWEET11 and


AtSWEET12, both harboring two MtN3/saliva domains, function as lowafnity transporters for the efux of sucrose from phloem parenchyma
cells into the apoplast (Chen et al., 2010, 2012). We proposed that class
II family proteins are also related to glucose and sucrose transport in
tomato.
5. Conclusions
In the present study, we identied 29 SWEET genes in tomato and
investigated characteristics of SlSWEETs including locus/gene name,
chromosomal localization, exon number, number of nucleotides and
amino acids (length), EST, and predicted localization. The separation
of the tomato SWEET proteins into four subclasses was mutually supported by their exon/intron structures, phylogeny, and distribution of
conserved motifs. We also focused on the response patterns of the
SWEET genes to high sugar conditions, high salinity conditions, and
temperature stress treatments, revealing numerous candidate stressresponsive genes in tomato. The putative involvement of SWEET genes
in the stress response opens up new avenues of research into the roles
of these genes in the adaption to various stresses. As the levels of
conservation (at the macro- and micro-synteny levels) among various
Solanaceae members are known, the comparative, phylogenetic, and
expression analyses of SlSWEET gene family members will provide a
platform for identication and comprehensive functional characterization of the SWEET gene family from various other Solanaceae species.
Conict of interest
The authors have declared that no competing interests exist.
Acknowledgments
This study was supported by grants from National Natural Science
Foundation of China (No. 31372054) and State key laboratory of plant
physiology and biochemistry open project (SKLPPBKF1404). Authors
also acknowledge use of the tomato genome sequence, which was generated by the International Tomato Genome Sequencing Consortium
(http://solgenomics.net/tomato).
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.gene.2015.07.055.
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