South African Journal of Botany 125 (2019) 176–187

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South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

The SWEET family genes in strawberry: Identification and expression

profiling during fruit development
H.-T. Liu a,b, W.-Y. Lyu b,c, S.-H. Tian b, X.-H. Zou b, L.-Q. Zhang b, Q.-H. Gao a,b,⁎, D.-A. Ni a, K. Duan b,⁎
a
Ecological Technique and Engineering College, Shanghai Institute of Technology, Shanghai 201418, China
b
Shanghai Key Laboratory of Protected Horticultural Technology, Forestry and Fruit Tree Research Institute, Shanghai Academy of Agricultural Sciences (SAAS), Shanghai 201403, China
c
College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai 201306, China

a r t i c l e i n f o a b s t r a c t

Article history: The transport of sugar is essential for plant growth and development, tightly correlated with crop productivity
Received 6 May 2019 and quality. SWEET (Sugar Will Eventually be Exported Transporters) is a family of sugar effluxer first reported
Received in revised form 26 June 2019 a decade ago, whose significance has been reported in a dozen of plant species. Strawberry is an important
Accepted 2 July 2019
crop developing striking fruits. Up to date, the involvement of SWEETs in strawberry is completely elusive. Cur-
Available online xxxx
rent work focuses on the genome-wide identification of SWEET transporter coding genes in diploid strawberry
Edited by E Balázs (Fragaria vesca) and the expression analysis of octoploid strawberry (F. × ananassa) SWEET homoeologs in differ-
ent vegetative and reproductive organs. A total of 20 candidate SWEET loci were identified in Fragaria vesca V4
Keywords: genome via a local blast using the MtN3_slv motif (PF03083) against the protein database of FvH4.0.a2 version.
SWEET Phylogenetic analysis indicated this family falling into four clades. These genes are distributed in all seven chro-
Sugar transport mosomes except for Fvb1, and most members contain six exons divided by five introns, coding a protein with 235
Fruit development to 310 aa and around 7 transmembrane helices. The spatio-temporal expression profiles of SWEET genes in dif-
Expression analysis ferent parts of fruit (cortex, pith and achenes) of cv. Benihoppe were explored and a set of candidate SWEET
Strawberry
genes invoved in sugar transport in strawberry fruits were identified, such as FvSWEET1, -2a, -7, -9a, -9c, -10,
and -11. Our results provide a good foundation for further functional investigation of SWEETs in strawberry
fruit quality formation and other developmental processes as well as responses to pathogens and abiotic stresses.
The information of SWEETs in strawberry also has a reference value for other Rosaceae plants.
© 2019 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction
Soluble sugars are the basic requirements for multicellular organ-
isms, serving as the sources of carbon skeletons, osmolytes, signals,
transient energy storage, and transport molecules (Chen et al., 2015a).
In plants, the mesophyll cells in the mature green leaves are specialized
in providing sugar through photosynthesis and fixation of carbon diox-
ide in chloroplasts, thus the functional leaves are defined as source or-
gans. Other plant organs called sink organs, such as roots, tubers,
flowers, seeds, fruits, and young leaves, depend completely on external
supply of sugars (Delrot et al., 2000). There are several types of sugars,
including sucrose (the most prevalent form), monosaccharides such as
hexose (glucose, fructose, etc.), and sugar polyols (mannitol, sorbitol,
and inositol, etc.). These different sugars could be partitioned into sub-
and intercellular, as well as long distance transport processes
(Zimmermann and Ziegler, 1975). Sugar transport in distinct plant
⁎ Corresponding authors at: Shanghai Key Laboratory of Protected Horticultural
Technology, Forestry and Fruit Tree Research Institute, Shanghai Academy of
Agricultural Sciences (SAAS), Shanghai 201403, China.
E-mail addresses: qhgao20338@sina.com (Q.-H. Gao), kduan936@126.com (K. Duan).
organs and between source and sink organs is not only important for
the growth and development of plants, but also critical for pathogen
susceptibility and responses to the environmental factors (Klemens
et al., 2013; Chong et al., 2014; Chen et al., 2015a; Eom et al., 2015;
Bezrutczyk et al., 2018). Several transport steps in sugar transport pro-
cesses are rate-limiting for the production of valuable compounds in ag-
riculturally important sink organs (Ludewig and Flügge, 2013).
Accordingly, sugar transport is closely associated with seed filling,
crop productivity, and fruit quality (Chen et al., 2015b; Wang et al.,
2015; Yang et al., 2018; Zhang et al., 2018). There are three principle su-
perfamilies of sugar transporters, the Major Facilitator Superfamily
(MFS), the sodium-dependent glucose transporters, and the Sugar
Will Eventually be Exported Transporters (SWEETs) (Xuan et al., 2013).
SWEET protein was first identified in ascidian (Hamada et al., 2005).
Its homologs encode proteins exist in all kingdoms of life including
higher eukaryote, protozoa, metazoan, fungi, bacteria, and archaea,
serving distinct physiological roles (Hamada et al., 2005; Saier et al.,
2006; Feng and Frommer, 2015; Eom et al., 2015). In plant, SWEET
was first defined as a glucose uniporter belonging to a novel transporter
family (PFAM PF03083) in Arabidopsis (Chen et al., 2010). Functional
study on the double mutant atsweet11sweet12 revealed a two-step

https://doi.org/10.1016/j.sajb.2019.07.002
0254-6299/© 2019 SAAB. Published by Elsevier B.V. All rights reserved.
 H.-T. Liu et al. / South African Journal of Botany 125 (2019) 176–187
mechanism of SWEET-mediated export of sucrose from parenchyma
cells (Chen et al., 2012). As a vacuole-located transporter for both hex-
ose and sucrose, AtSWEET16 overexpression resulted in increased freez-
ing tolerance and improved germination as well as nitrogen use
efficiency in Arabidopsis (Klemens et al., 2013). SWEET17 is the first vac-
uolar fructose transporter identified in plant, responsible for exporting
fructose out of the vacuole in Arabidopsis leaves and roots (Chardon
et al., 2013; Guo et al., 2014). SWEET9 was identified as a nectary-
specific sugar transporter functioning in nectar secretion to reward pol-
linators (Lin et al., 2014). In addition to Arabidopsis, the SWEET gene
family has been reported in many other plants, including tomato
(Feng et al., 2015), cucumber (Hu et al., 2017), grape (Chong et al.,
2014), apple (Wei et al., 2014), orange (Zheng et al., 2014), pear (Li
et al., 2017), soybean (Patil et al., 2015), Oilseed rape (Jian et al.,
2016), sorghum (Mizuno et al., 2016), wheat (Gao et al., 2018), rice
and maize (Sosso et al., 2015). The expression patterns suggest that
SWEET family is involved in the growth and development of all plant or-
gans. There exists a set of SWEET genes closely related with the develop-
ment of flowers, seeds, and fruits in every species.
Strawberry is economically important worldwide, as its fruits are
very nourishing and appreciated not only for the beautiful appearance
but for the unique flavor. The nearest relatives of cultivated strawberry
is woodland strawberry Fragaria vesca, which has a small and high qual-
ity genome sequenced, serving as a valuable reference genomic system
for the Rosaceae plants (Folta and Davis, 2006; Shulaev et al., 2011;
Edger et al., 2018； Li et al., 2019). Fruit growth and development is
closely correlated with the sugar content. The soluble sugar is critical
for the flavor and nutritional quality of all fleshy fruits. In strawberry
fruits, sugar is largely composed of sucrose and two hexoses (fructose
and glucose), and sucrose is mainly in the upper side of cortical and
pith tissues while the two hexoses are equally distributed throughout
the fruits (Enomoto et al., 2018). As a signal, sucrose works to facilitate
strawberry fruit ripening, and the ABA-Stress-Ripening (ASR) transcrip-
tion factor is a key hub gene coordinating the regulation of fruit ripening
by plant hormones and sucrose (Jia et al., 2013; Jia et al., 2016). There is
a proverb familiar to strawberry producers, that no potassium fertilizer,
no sweetness. Consistently, the mRNA levels of two-pore K + (TPK)
channel gene (FaTPK1), are positively correlated with the content of sol-
uble sugar in strawberry fruits, suggesting its important roles in fruit
ripening and quality formation (Wang et al., 2018). The two genes in-
volved in sucrose synthesis and the ripening-related sucrose accumula-
tion, FaSPS3 (Sucrose-6-Phosphate Synthase 3, gene31122) and FaSUS1
(Sucrose Synthase 1, gene12940), are positively regulated by a gibberel-
lin (GA)-related MYB transcription factor FaGAMYB and a FERONIA-like
receptor kinase FaMRLK47 (Vallarino et al., 2015; Jia et al., 2017). A
transcriptional repressor, FaMYB44.2, negatively regulates soluble
sugar accumulation in strawberry. In the white fruit, FaMYB10, the
positive regulator of anthocyanin accumulation, suppresses FaMYB44.2
expression via weakening the interaction between FaMYB44.2 and its
co-repressor FabHLH3, and cooperate with FabHLH3 to activate the ex-
pression of FaSPS (Wei et al., 2018).
These studies have preliminarily revealed the molecular regulatory
network underlying sugar accumulation in strawberry fruits. However,
the involvement of SWEET sugar transporters in strawberry sugar accu-
mulation and quality formation is largely elusive. The objectives of our
study are to define the structure, distribution, phylogeny of SWEET fam-
ily genes in F. vesca and to evaluate the expression profiles and diversi-
fication of SWEET genes in the source and sink organs of cultivated
strawberry. The candidate SWEET loci in F. vesca genome were identified
and their phylogeny and structure were characterized. Furthermore, the
spatio-temporal expression profiles in different organs and fruit parts
(cortex, pith and achenes) were studied in cv. Benihoppe. This work
sets a good basis for future functional investigation on the involvement
of SWEETs in strawberry fruit quality formation and in other develop-
mental or responding processes.
177
2. Materials and methods
2.1. Plant materials
The octoploid strawberry (Fragaria × ananassa cv. Benihoppe) used
in this work was grown in a multi-span greenhouse at Zhuanghang
field trial station of Shanghai Academy of Agricultural Sciences. The col-
onies with six to eight leaves of a similar size were planted in early Sep-
tember. The strawberry fruits at green (15–20 d post anthesis, DPA),
white, half-red (transition stage) and ripe stages, were sampled and dis-
sected into three parts (achene, cortex, and pith). At certain develop-
mental stage, a total of 15 to 30 fruits were collected from 15 plants
and divided into three biological replicates. Simultaneously, the full-
bloom flowers, functional leaf blade (from the third full-expanded com-
pound leaves), whole root system (about 1/4 being light brown, 3/4
being white) cleaned, and the mature proximal parts of stolons were
sampled from 18 fruiting plants for three replicates. Each sample was
from at least five different plants. The sampled materials were immedi-
ately frozen in liquid nitrogen, transferred to lab and maintained at −74
°C until use.
2.2. Identification of SWEET family genes in Fragaria vesca
To identify SWEET family genes in strawberry, the HMMER profile
(Finn et al., 2011) of the MtN3_slv motif (PF03083) was used to search
against the Strawberry Genome v4.0. a2 protein database via a local
blast at Bioedit program (Li et al., 2019). These putative SWEET family
genes (with an E-value b1E-5) were further confirmed through PFAM
search at http://pfam.xfam.org/ (Finn et al., 2016). Finally, a total of 20
SWEET protein sequences were obtained for further analysis. Analysis
of chromosome location was performed against Strawberry Genome
v4.0.a1 chromosomes in Genome Database of Rosaceae (GDR). For
each FvSWEET member, in silico prediction of biochemical characteris-
tics was further performed: trans-membrane helix prediction using
TMHMM Server v. 2.0 at http://www.cbs.dtu.dk/services/TMHMM/,
subcellular localization prediction using PSORT SERVER at https://
psort.hgc.jp/, molecular weight (MW) and theoretical isoelectric point
(pI) prediction using the Prot Param tool at http://web.expasy.org/
protparam/.
2.3. Gene organization, protein module, and phylogenetic analysis
For gene organization analysis, the cDNA sequence of each individ-
ual strawberry SWEET gene obtained from FvH4.0.a2 (Li et al., 2019)
was compared with its genomic DNA sequence from GDR database
(www.rosaceae.org/, Edger et al., 2018). The exon-intron structure
was generated in the Gene Structure Display Server (GSDS, http://
gsds.cbi.pku.edu.cn/) (Hu et al., 2015). To identify the conserved motifs
in FvSWEET proteins, the MEME program at http://meme-suite.org/
(Bailey et al., 2006) was employed using the following parameters: op-
timum width, 15–60; number of repetitions, any; maximum number of
motifs, 7.
For phylogenetic analysis, SWEET orthologs in Arabidopsis and to-
mato (Solanum lycopersicum) were obtained at TAIR (TAIR release 10,
http://www.arabidopsis.org/) and Sol Genomics Network (https://
www.solgenomics.net/locus/1420/view/) (Feng et al., 2015). The pro-
tein sequences of 20 FvSWEETs, 27 SlSWEETs, and 17 AtSWEETs were
aligned through ClustalW program using the format parameters at
MEGA 7.0. A phylogenetic tree was then constructed using the
neighbor-joining (NJ) method with 1000 bootstrap replicates (Kumar
et al., 2016). The resulting NJ tree was visualized using the interactive
Tree Of Life (iTOL) online tool (https://itol.embl.de/version_history.
cgi/) (Letunic and Bork, 2016).
178 H.-T. Liu et al. / South African Journal of Botany 125 (2019) 176–187

2.4. Retrieval of expression values for woodland strawberry FvSWEET genes displayed in Tables S1. The qPCR analysis was performed as previ-
from RNA-seq databases ously described (Liu et al., 2014).

Fragaria vescagenome-wide RNA-seq data submitted by the Uni-

versity of Maryland were used to dissect the expression profiles of
SWEET genes in the floral organs and fruits at different developmen-
tal stages from pre-fertilization to “big green” and in five different
fruit tissues (ovary wall, seed without embryo-ghost, embryo, cor-
tex, and pith) (Hollender et al., 2012; Kang et al., 2013; Hollender
et al., 2014). The fragments-per-kilobase-per-million fragments
mapped (FPKM) values of each gene were obtained and normalized
(Z-score standardization) for Hierarchical clustering in the HemI
package (Deng et al., 2014). Selected F. vesca tissues or organs were
as follows: six organs of seed 2 from fruit at stage 2 (2–4 d post an-
thesis), seedling, leaf, flower1–4 (from floral meristems ranked by
leaf primordia (stage 1) to the emergence of petal primordia spaced
between sepals), perianth 5–6 (flower buds during the initiation of
stamen and carpel primordia to the emergence of carpel primordia),
and receptacle 6–7 (the receptacle dome of flower buds at stage
6–7); 12 gametophytes of different developmental stages (female –
4 samples of 4 stages, male – 8 samples of 6 stages); 14 seed-
related tissues of 5 stages; 14 receptacle-related tissues of 7 stages.
The status of floral organs of stage 1–12 was defined by Hollender
et al. (2012). Five stages of early fruit development are defined by
Kang et al. (2013) as: stage1, full-bloom flower; stage 2, 2–4 d post
anthesis (DPA), stage3, 6–7 DPA; stage 4, 8–10 DPA; stage 5, 10–
13 DPA.
2.5. RNA isolation and RT-PCR
Total RNA was isolated using Plant RNA Kit (OMEGA Bio-tek, Inc.
cat#R6827-01, USA) and reverse-transcribed using the
PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, China).
First-strand cDNAs were used for PCR or real-time quantitative PCR
(qPCR) on Light Cycler 480 (Roche, German). TB Green™ Premix Ex
Taq™ (Till RNaseH Plus) qPCR kit (Takara, China) was used following
the instructions. Reference strawberry gene CHP1 (Conserved hypo-
thetical protein, FvH4_7g15060, MAP Kinase 4) were used as the in-
ternal control for relative expression analysis (Clancy et al., 2013).
Sequence information for RT-PCR primers used in this study was
3. Results
3.1. Identification of FvSWEET genes and phylogenetic analysis
A local BLASTp search against the Rosaceae Genome Database
FvH4.0.a2 using the HMMER profile of the MtN3/saliva domain
(PF03083) was performed, and a total of 20 open reading frames
(ORFs) encoding putative SWEET proteins were identified in the F.
vesca genome (Table 1). To understand the evolutionary relationships
between FvSWEETs and SWEETs from other plants, a phylogenetic
tree was constructed using the amino acid sequences of 20 FvSWEETs,
17 AtSWEETs from Arabidopsis thaliana, and 27 SlSWEETs from Solanum
lycopersicum (Fig. 1). This analysis indicated that strawberry SWEETs
could be grouped into four clades meeting with the previous classifica-
tion (Chen et al., 2010). Strawberry SWEETs were named according to
the nomenclature of Arabidopsis orthologs in the phylogenetic tree.
Each AtSWEET gene corresponds to one to three FvSWEET genes. There
are two loci of strawberry SWEETs homologous to AtSWEET2, -6, -8,
and -15. Three strawberry SWEETs were the homologs of AtSWEET9.
But in woodland strawberry, orthologs of AtSWEET12, -13, and -14
were not identified.
The chromosomal location of FvSWEETs was determined through
BLASTN against strawberry genome v4.0.a1 chromosomes in GDR.
This family is dispersed on all chromosomes except for chromosome 1
(Fvb1) (Fig. 2). Chromosome 3 contains two FvSWEET loci. Fvb2 and
Fvb7 consistently have three SWEET genes. The rest three chromosomes
(Fvb4, 5 and 6) each harbors four loci. In addition, there are two pairs of
tandem-duplicated strawberry SWEET genes, i.e. FvSWEET9b and -9c dis-
tributed on Fvb2, and FvSWEET10 and -11 on Fvb5. Analyzing the trans-
membrane helices (TMH) revealed that the deduced FvSWEET proteins
contain 5–7 TMHs, and a large proportion of this family (n = 17) pos-
sess seven TMHs (Table 1). The relative molecular masses of FvSWEETs
vary from 25.9 kDa (FvSWEET16) to 34.7 kDa (FvSWEET11). The pI
values range from 5.15 (FvSWEET15a) to 9.67 (FvSWEET2a). PSORT
prediction indicated the subcellular localization of most FvSWEETs in
the plasma membrane. FvSWEET9c was also predicted to localize in
chloroplast.

Table 1
The predicted structural and biochemical information of SWEET (Sugars Will Eventually be Exported Transporters) family genes in strawberry (Fragaria vesca).

Gene ID Gene ID (v4.0.a1) Gene ID (v2.0.a2) Chromosome location mRNA Protein TMHa LOCb MWc (kDa) pId

FvSWEET1 FvH4_2g14860 gene00933 13,041,007–13,042,951 1155 243 7 PM 26.8 9.28

FvSWEET2a FvH4_3g06470 gene36140 3,718,352–3,720,903 1267 256 7 PM 29.2 9.67
FvSWEET2b FvH4_3g17630 gene27348 11,218,976–11,223,086 1662 235 7 PM 26.2 9.04
FvSWEET3 FvH4_7g31080 gene13106 22,498,063–22,499,707 1122 251 7 PM 28.3 9.23
FvSWEET4 FvH4_6g35420 gene37800 27,945,823–27,948,018 934 235 6 PM 26.4 9.21
FvSWEET5 FvH4_5g05160 gene32169 3,010,215–3,012,090 1161 237 7 PM 26.6 9.37
FvSWEET6a FvH4_6g50390 gene28377 37,507,889–37,512,657 1114 242 7 PM 26.9 8.57
FvSWEET6b FvH4_6g50580 gene42598 37,626,824–37,628,710 1123 243 7 PM 27.4 9.26
FvSWEET7 FvH4_5g11560 gene25112 6,556,652–6,559,675 1851 253 7 PM 28.1 9.54
FvSWEET8a FvH4_4g13312 gene36658 16,912,096–16,912,975 880 249 7 PM 28.1 9.02
FvSWEET8b FvH4_7g10730 gene38189 10,057,942–10,058,649 708 235 6 PM 26.4 8.27
FvSWEET9a FvH4_6g01150 gene37476 625,207–626,799 1039 257 7 PM 29.0 9.57
FvSWEET9b FvH4_2g24901 gene35839 20,244,213–20,246,889 2018 297 7 PM 33.0 8.96
FvSWEET9c FvH4_2g24900 gene35838 20,240,197–20,242,416 1589 288 7 PM/CL 32.3 8.7
FvSWEET10 FvH4_5g08962 gene06463 5,181,866–5,183,903 1390 292 7 PM 33.0 8.45
FvSWEET11 FvH4_5g08970 gene06462 5,188,507–5,190,910 1559 310 7 PM 34.7 7.65
FvSWEET15a FvH4_4g22110 gene00213 25,050,351–25,051,866 714 237 5 PM 26.5 5.15
FvSWEET15b FvH4_4g23160 gene07246 25,733,918–25,736,393 1624 305 7 PM 34.1 5.8
FvSWEET16 FvH4_7g08940 gene03053 8,720,565–8,722,463 1158 235 7 PM 25.9 8.59
FvSWEET17 FvH4_4g18490 gene06839 22,341,089–22,343,565 1545 241 7 PM 26.6 7.67
a
Number of transmembrane helices, as predicted by the TMHMM Server v2.0.
b
Subcellular localization, as predicted by the PSORT, PM plasma membrane, CL chloroplast.
c
Molecular weight of the amino acid sequence, and kDa for kilo Dalton.
d
Isoelectric point of the FvSWEET.
 H.-T. Liu et al. / South African Journal of Botany 125 (2019) 176–187 179

Fig. 1. Phylogenetic relationship of SWEET proteins from Fragaria vesca, tomato Solanum lycopersicum and Arabidopsis thaliana, marked with red star, yellow circle, and green triangle,
respectively. Neighbor-joining (NJ) phylogenetic tree of the SWEET family protein sequences was constructed using MEGA7.0 software with 1000 bootstrap re-samplings and
visualized using the interactive Tree Of Life (iTOL). SWEET proteins fall into four clades indicated as different colors. The number indicated at each branch/clade stands for bootstrap
supports from 1000 replicates. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2. Distribution of strawberry SWEET genes on chromosomes. The scale ruler indicates the physical distance of the chromosomes. The relative positions and the direction of
transcription of FvSWEETs are marked as triangles. Black scale line represents the length of chromosome.
180 H.-T. Liu et al. / South African Journal of Botany 125 (2019) 176–187
3.2. The exon–intron organizations for FvSWEET genes
According to the similarity of their nucleotide sequences, the 20
FvSWEET genes could be classified into four distinct clades, consistent
with the phylogenetic analysis based on amino acid sequences. Most
FvSWEET genes (n = 15) commonly harbor six exons divided by
five introns (Fig. 3). Two members (FvSWEET6a and FvSWEET7)
possessing five exons were included in clade II. In clade III,
FvSWEET15a contains 4 exons. FvSWEET8a and -8b were most notably
for one exon without intron. Fifteen FvSWEET members containing six
exons were present in all four clades. Among the family, the second
exon and the one next to the last are largely conserved in length, ca.
37 and 120 bp, respectively.
3.3. Domain architectures of putative FvSWEET proteins
Based on an amino acid alignment analysis of all 20 FvSWEET pro-
teins (Supplementary Fig. S1), furtherly the conserved motifs in the 20
FvSWEETs were identified using the MEME program. This analysis re-
vealed a total of seven conserved motifs, and the consensus of each
motif was identified (Fig. 4). The length of the conserved motifs
ranged from 11 to 41 amino acids, being shorter or longer than a
TMH (approximate 23 amino acid residues). All members except for
FvSWEET8b and -15a contain the whole set of seven motifs.
FvSWEET8b lacks the fifth motif (acid blue), and the third (purple)
and fourth (dark blue) motifs are absent in FvSWEET15a. Multiple se-
quence alignment logo of seven motifs in FvSWEETs highlighted the
conserved core structural amino acid residues (Fig. 5). Being the mid-
dle part of a second MtN3_slv domain, Motif 5 is the most conserved
one. Seven amino acid residues in this motif are identical in nearly all
FvSWEET members harboring this motif. In detail, the proline (P, 5th),
Leucine (L, 6th), Valine (V, 12th and 18th), Serine (S, 17th), Methio-
nine (M, 21th), and Phenylalanine (F, 23th) of motif 5 are largely con-
served in this family, which might be important for sugar intercellular
exchange in strawberry. Notably, Motif 2 contains one completely
identical Tyrosine (Y, 5th) site in whole family members. In addition,
there is at least one Glycine residue (G) conserved in motifs 1, 3,
and 4 among all family members.
Fig. 3. Exon-intron organization of 20 strawberry SWEET genes of FvH4.0.a2 version. The structural model was generated from the Gene Structure Display Server (GSDS) website (http://
gsds.cbi.pku.edu.cn/chinese.php/). The neighbor-joining tree of these genes was constructed using 1000 bootstrap replicates by MEGA7.0.
3.4. Expression patterns of SWEET genes in woodland strawberry from RNA
sequencing
Previous work provided RNA-seq data for all woodland strawberry
SWEET genes (Kang et al., 2013; Hollender et al., 2014). The expression
profiles of FvSWEETs were analyzed in a total of 46 samples, which were
sorted into four groups (Fig. 6). RNA-seq data showed that all 20
FvSWEET members were expressed during the development of the
male and female gametes (Fig. 6A). Three genes including FvSWEET4,
-8a, and -8b were not expressed in carpel of stage 7 to 12. FvSWEET8a
and -8b seem to be specifically expressed in pollen, peaking at distinct
stages with varying levels, while FvSWEET4 transcript was limited in
the style of stage 2. In pollen, the expression levels of most FvSWEETs
were lower than that of FvSWEET6a, -1 and -5. Notably, these three
genes displayed expression peaks at anther of stage 9, 10, and 12, re-
spectively. However, FvSWEET5 and -6a are predominantly expressed
in anther and pollen, while FvSWEET1 is expressed in both anther and
carpel. Similarly, FvSWEET2a, -2b, -7, -10, and -15b are significantly
expressed in both male and female organs. In both sexual organs, sev-
eral additional members including FvSWEET2b, -10, and -16 are
expressed at a moderate level, while FvSWEET3, -8a, -9a, -11, and -15a
at a low level.
Transcripts of 15 FvSWEETs are detectable in 14 samples covering the
early to middle development of strawberry fleshy fruit (Fig. 6B). Seven
members including FvSWEET1, -2a, -2b, -7, -15b, -16, and -17 are signif-
icantly expressed in all 14 samples examined, including the cortex, pith
and the whole fleshy fruit. At the early stage of fruit development, ex-
pression of FvSWEET1 and -7 seems to be preferential in the cortex
and pith of stage 1–2. These two genes are also clearly expressed in
the fruits at 15–20 DPA. FvSWEET9c shows a moderated expression in
the fleshy fruits of early and middle stages. The remaining seven
genes are expressed at low levels in the fruits. For example, a weak ex-
pression of FvSWEET9a is present in the cortex of stage 1–2, and of
FvSWEET11 is limited in the pith of stage 3 and 5, while FvSWEET10 is
weakly expressed in the cortx and pith of stage 2. Notably, a very
weak expression of FvSWEET4 and -8a is specific to the cortex of stage
2 and the pith of stage 2, respectively.
All FvSWEETs except for FvSWEET8a and -8b were detected in 14
samples referring to seed (achene) development (Fig. 6C). Of them,
 H.-T. Liu et al. / South African Journal of Botany 125 (2019) 176–187
Fig. 4. Motif compositions of FvSWEET proteins identified by the MEME program. The gray lines represent the non-conserved sequences. Different colored boxes indicate distinct motifs
identified by MEME. The lengths of the motifs in each protein are proportional. E-value, the overall match of the motif models.
FvSWEET3, -4, -5, -6a, -6b, -9a, and -15a are weakly expressed in cer-
tain tissues. A very weak expression of FvSWEET5 and -9a is limited in
the wall and the seed of certain stages. FvSWEET6a is expressed at a
low level in the ghost and embryo, while FvSWEET15a expression
presents in the wall, ghost, and embryo similarly at a low level. On
the contrary, FvSWEET1, -2a, 2b, -7, 9b, 10, 11 and -15b are ubiqui-
tously expressed during seed development at notable levels.
FvSWEET1 is highly expressed in the embryo of stage 4 and the
ghost of stages 5, respectively. FvSWEET7 stably exhibits a higher ex-
pression in the ghost and wall of seed than in the embryo. Further-
more, FvSWEET11 and FvSWEET15b display an expression peak in the
ghost of stage 4. FvSWEET9b and -10 are highly expressed in the
ghost of stage 4–5.
The expression patterns of this family in seven samples nearly cover-
ing the whole life cycle of strawberry were displayed in Fig. 6D. The re-
sults showed that 15 of 20 FvSWEETs were transcribed. At least five
genes including FvSWEET1, -2a, -2b, -16, and -17 were ubiquitously
expressed, while the remaining 10 members were specifically
expressed in certain tissues. FvSWEET1 is ubiquitously expressed but ex-
hibits higher expression in the leaf, seedling, shoot meristem and floral
meristem. FvSWEET2a is expressed in all tissues but peaks in leaf. There
are three genes including FvSWEET2b, -7, and -17 highly expressed in
the roots, and FvSWEET2b is also highly expressed in the roots under
phosphate limitation (root P).
RNA-seq data are in agreement with a role of FvSWEETs in sugar
transport in whole life cycle. Expression of more than half family mem-
bers is closely relative to the development of sexual gametes and seeds,
while only five members including FvSWEET1, -2a, -2b, -16, and -17 are
obviously expressed in leaf, a vegetative organ.
3.5. Expression analysis of SWEET genes in different organs of octoploid
strawberry
For the SWEET family genes in strawberry, we were most interested
in those involved in sugar intercellular exchange in octoploid straw-
berry, especially during fruit development and ripening. To gain insights
into the functions of SWEETs in cultivated strawberry, semi-quantitative
PCR and qRT-PCR were first performed in this work. The transcript accu-
mulation of 20 homoeolgous SWEETs was examined in a total of 16 sam-
ples from cv. Benihoppe (Fig. 7, Fig. S2). The expression of all SWEETs
181
except for SWEET6a, -8a and -8b were detected. Results from semi-
quantitative PCR were largely consistent with the expression profiles
determined by qRT-PCR.
Of the family, six genes (SWEET1, 2a, -2b, -7, -9a, -15b, and -16) were
ubiquitously expressed in all 16 tissues, although at varied levels.
SWEET3, and -9c were obviously expressed in all 16 tissues exception
for certain vegetative organ achene, or root (Fig. 7). SWEET genes varied
significantly concerning expression level. In most tissues, SWEET7 was
expressed at the highest level of this family, which was comparable to
that of the reference FaCHP1. However, SWEET5, and -6b were expressed
at levels significantly lower than the reference gene.
A number of SWEETs were significantly expressed in flowers and
during fruit development, with a lower level of expression or non-
expression in vegetative organs. Indeed, there were 16 SWEET
genes expressed in the full-bloom flowers (except for SWEET6a,
-8a, 8b, -11), 14 genes in the achenes, 12 genes in the fleshy
parts of the fruits. The expression of SWEET5 was highly tissue-
specific, being confined in the flower. Expression of SWEET11 was
specifically present in the achenes. SWEET10, -15a, and -15b were
preferentially expressed in the achenes and flowers, with low or
non-expression in the vegetative organs. Interestingly, SWEET3
was expressed with a pattern complementary to that of SWEET11,
which was non-expressed in achenes but expressed in all other tis-
sues, mainly expressed in full-bloom flowers and the fleshy part of
green fruits. Overall, none of the 15 SWEETs detected in fruits
displayed a stable pattern during fruit development. In the cortex
and pith parts, expression of SWEET1, -9b, and -9c was up-
regulated during fruit ripening. Expression of SWEET2b and -3
was down-regulated following fruit development, with a higher
expression in green fruits compared to ripe fruits. Expression of
SWEET2a, -6b and -16 peaked at the white fruit stage, and SWEET9a
was highly expressed at the transition stage. In the achenes, five
genes SWEET2a, -2b, -7, -10, and -15b displayed a clear decreasing
tendency as during fruit development. The other three genes
SWEET6b, -16, and -1 were expressed at a peak level in the achenes
from the transition, the transition, and the red/ripe stage,
respectively.
Notably, SWEET17 was the unique one mainly expressed in the root,
a vegetative organ. This gene was also moderately expressed in the sto-
lon, weakly in the leaf, flower and the pith of ripe fruit. The ubiquitously
182 H.-T. Liu et al. / South African Journal of Botany 125 (2019) 176–187

Fig. 5. Sequence logos for seven motifs of FvSWEET proteins identified by the MEME program (http://meme-suite.org/). Motifs are displayed by stacks of letters at each site. The total
height of the amino acid stack indicates the “information content” of that site in the motif in bits. The height of each letter in a stack is the probability of the letter at that site
multiplied by the total information content of the stack. X-axis, the width of the motif; Y-axis, the bits of each letter.
 H.-T. Liu et al. / South African Journal of Botany 125 (2019) 176–187 183

Fig. 6. The expression profiles of FvSWEET genes from RNA-sequencing in woodland strawberry. Gene expressed in each tissue with an average reads per kilobase per million (RPKM)
higher than 0.3 were combined for analysis. The heatmap of FvSWEET genes expression was hierarchically clustered using the HemI package. The color scale represents RPKM
normalized log2-transformed counts, where red indicates a high level, blue a low level, green a medium level, and gray for non-expressed (RPKM ≤ 0.3). (A) The expression of
FvSWEETs in 13 samples corresponding to the different stages of male and female gametes as previously defined (Hollender et al., 2012). (B) Expression in 14 berry-related samples
post fertilization from early fruit setting to the transition stage as previously stipulated (Kang et al., 2013). Stage 1, open flower; 2, 2–4 day post anthesis (DPA); 3, 6–7 DPA; 4, 8–10
DPA; 5, 10–13 DPA. (C) Expression in 12 seed/achene related samples. Stage 1–5 was consistent with that in (B). (D) Expression profiles of FvSWEET genes in seven samples including
root, leaf, seedling, and meristems. Root_P for root under phosphate limitation. (For interpretation of the references to color in this figure legend, the reader is referred to the web
version of this article.)

expressed SWEET7 also displayed an obvious expression in the root. In
addition, there were four and five SWEET members clearly expressed
in the functional leaves, and stolons, respectively.
Based on the above results, we summarized the expression of SWEET
genes in different organs of octoploid strawberry in a schematic figure
(Fig. 8). It is proposed that after sucrose is synthesized by photosynthe-
sis in the source organs, SWEET1, -2b, 9c and -16 primarily contribute to
the efflux of sucrose from the leaves, SWEET7 and SWEET17 mainly me-
diate the unloading and transport of sugar in the roots, and SWEET7 and
SWEET9c preferentially contribute to the unloading of sugar in the sto-
lons. During flower development, SWEET7, -9a and -9c might play im-
portant roles in sugar unloading to support the development of
gametophyte. When sugar unloaded into the fruits, the major member
SWEET2a, -7, -9b, and -9c might contribute to strawberry sugar
accumulation and quality formation of the berry, while SWEET1, and -
10 might role in seed filling.
4. Discussion
Plant SWEET protein was first reported a decade ago in Arabidopsis
(Chen et al., 2010). The growing availability of genomic sequence data
has facilitated the identification of SWEET homologs in more than 15
plant species in the last decade (Chen et al., 2010; Chong et al., 2014;
Feng et al., 2015; Jian et al., 2016; Li et al., 2017; Mizuno et al., 2016;
Patil et al., 2015; Sosso et al., 2015; Wei et al., 2014; Zheng et al.,
2014). Growing evidence indicate that SWEETs are important trans-
porters for sugar efflux in all kingdoms of life and play diverse physio-
logical roles. Phylogenetic studies integrated with gene structure and
184 H.-T. Liu et al. / South African Journal of Botany 125 (2019) 176–187

Fig. 7. Quantitative RT-PCR analysis of SWEETs in F. × ananassa cv. Benihoppe. The amount of cDNA templates in each sample was normalized by the amplification of FaCHP1 (Clancy et al.,
2013). Fruits of four stages (G, green; W, white; T, half red half white; R, ripe) were dissected into three parts: achenes (A), cortex (C), and pith (P). Root (the whole root system), stolon
(the proximal mature part), leaf (the third fully expanded leaf blades), and full-bloom flower were simultaneously sampled from the fruiting plants. Y-axis indicates gene expression levels
relative to the common FaCHP1 in certain organs. X-axis shows different organs. Error bars represent the standard deviations within the triplicates. (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.)
 H.-T. Liu et al. / South African Journal of Botany 125 (2019) 176–187 185

Fig. 8. Schematic models of preferential expression of strawberry SWEET genes in different organs and during fruit development. This summary is based on the expression data in cv.
Benihoppe revealed in the present work. For a certain gene, it is shown in those organs with an expression value (relative to reference FaCHP1) higher than its average value in all 16
samples. Those genes with a preferential expression in certain organ significantly higher than the rest members are indicated in bold. Fruits of different stages were dissected into
three parts: achenes (A), cortex (C), and pith (P).

domain architecture analysis consistently support the defining of four
SWEET clades in every plant species characterized (Gao et al., 2018).
Previous study identified 10 SWEET homologs in F. vesca (Li et al.,
2017). Hereon, through a genome-wide search based on the updated
FvH4.0.a2 database, a total of 20 FvSWEET homologs were identified in
F. vesca, distributed in all chromosomes except for Fvb1. All FvSWEETs
deduced proteins were predicted to be plasma-membrane localized,
and all except for FvSWEET15a and -15b have a theoretical pI larger
than 7.0. As an important parameter of a protein, pI is determined by
the relative contents of amino acid residues of different pH, and it affects
a protein in its stability and protein physiological role or activity
(Gasteiger et al., 2005). FvSWEET15a and -15b have a pI being 5.15
and 5.80, respectively, while their arabidopsis homolog AtSWEET15
(SAG29) has a pI of 8.19. This observation triggers a speculation that
SWEET15 may function in a way in strawberry different from that in
Arabidopsis. Future functional study is needed to clarify this deduction.
The draft genome of F. vesca, a short-read-based reference genome,
was available eight years ago (Shulaev et al., 2011). Recently, based on
single-molecule real-time sequencing, a near-complete genome of F.
vesca was revealed and ~24.96 Mb novel sequences were added to the
new version of genome (Fragaria vesca Genome v4.0), ~ 300-fold im-
proved than the previous version (Fragaria vesca Genome v1.0) (Edger
et al., 2018). Direct comparison between F. vesca V4 and V1 revealed nu-
merous, large-scale scaffolding errors in previous genome. Using
MAKER-P annotation pipeline, 28,588 gene models were identified in
F. vesca V4, containing 1496 newly predicted gene models (Edger
et al., 2018). Recently, an updated annotation of the wild strawberry
Fragaria vesca V4 genome identified 34,007 protein coding genes in
FvH4.0.a2 version (Li et al., 2019). In this work, we identified 20
SWEET loci using the conserved the MtN3_slv (PF03083) domain via
local blast against FvH4.0.a2 protein database, and 18 out of 20
FvSWEETs with two intact PF03083 domains, and the remained two
FvSWEETs (-8b, -15a) only miss 1 or 2 motifs of the 7 motifs in two do-
mains. By contrast, our previous identification and re-annotation
against the previous database suggested that 8 out of the 19 family
members had no whole MtN3_slv domain, and the exon-intron organi-
zation for 11 of 19 members was inconsistent with FGENESH analysis at
Softberry website (data omitted). Clearly, the updated FvH4.0.a2 ge-
nome database provides an annotation of high quality and will facili-
tates functional genome research in strawberry greatly.
Newly, the origin and evolution of octoploid strawberry genome was
elucidated and reported for cv. Camarosa, which represents a milestone
in exploring the life secrets of modern cultivated strawberry (Edger
et al., 2019). As compared with the submissive subgenomes from F.
nipponica, F. iinumae, and F. viridis, the last diploid progenitor F. vesca
was considered as the dominant subgenome provider, based on the sig-
nificantly greater gene content, gene expression abundance, and biased
exchanges between homoeologous chromosomes. Moreover, metabolic
pathways including those controlling strawberry flavor and aroma are
largely controlled by F. vesca subgenome, whose homoeologs in octo-
ploid strawberry are responsible for more than 95% of the biosynthesis
of fructose associated with sweetness (Edger et al., 2019). In current
work, qPCR analysis of SWEET homoeologs in cv. Benihoppe focused
on fruit development, where the sampled achenes and cortex/pith in
green fruits were comparable to the seeds and receptacle used in
RNA-seq analysis of F. vesca, concerning the developmental stage
186 H.-T. Liu et al. / South African Journal of Botany 125 (2019) 176–187
(Kang et al., 2013). In achenes from green fruits, a set of preferentially
expressed homoeologs including SWEET7, -10, -9b, -1, -2a, and -2b
displayed a pattern biased toward F. vesca -like, being largely consistent
with the transcriptome data of FvSWEETs (Fig. 6C). However, in pith and
cortex of young fruits, two distinct sets of SWEET homoeologs were
identified as the preferential ones in diploid F. vesca and octoploid F. ×
ananassa, although the expression patterns of FvSWEET2a and -7 were
similarly observed in cv.Benihoppe. The pair of FvSWEET9a and its octo-
ploid homoeolog provide a typical example displaying significantly var-
ied expression pattern in woodland and cultivated strawberry. In cv.
Benihoppe, SWEET9a was ubiquitously and highly expressed, while its
homoeolog in F. vesca Yellow Wonder was not detected in leaf and
young fruits, with transcripts limited to young flowers at a very low
level. There are three SWEETs (SWEET6a, -8a, and -8b) not detected in
cv. Benihoppe in this work, awaiting for future characterization in
other organs. Expression of all their homoeologs was detected in F.
vesca, although limited to young floral organs or seeds at certain
developmental stage, largely at very low levels. Generally, there is a
clear proportion of sugar efflux transporter genes SWEETs maintaining
F. vesca-biased expression pattern in octoploid. For specific SWEET
gene, it is very common being expressed in cultivated strawberry with
a pattern different from that of its diploid homoeolog.
Characterized angiosperm genome often harbors 17–59 loci for
SWEET paralogs serving distinct physiological roles. The conservation
in expression and tightly correlated function of some SWEET homologs
in distinct species reinforces that SWEETs are essential for plant devel-
opment and defense responses relevant to agricultural traits such as
crop yield and quality. The vegetative organs expressed FvSWEET17
showed the highest level of expression in roots, consistent with its
ortholog in Arabidopsis, of which the root-highly expressed AtSWEET17
mediates fructose transport (Guo et al., 2014). In Arabidopsis, SWEET9
is essential for nectar secretion (Eom et al., 2015). Herein the expression
of FaSWEET9 peaked in the full-bloom flowers, about 30-fold higher
than in other organs. AtSWEET11, -12 and -15-mediated sucrose efflux
from seed coat to embryo is important for seed filling in Arabidopsis, and
in rice SWEET11 and SWEET15 consistently are two key players in seed
filling (Chen et al., 2015b; Ma et al., 2017; Yang et al., 2018). Similarly,
we observed the achenes-specific expression of FvSWEET11 and an ob-
vious expression of FvSWEET15a and -15b in achenes. In fruit crops, dif-
ferent sets of fruit-expressed SWEET genes had been identified in apple
(Wei et al., 2014), grape (Chong et al., 2014), orange (Zheng et al.,
2014), and pear (Li et al., 2017). Expression of SWEETs in these crops
is dynamically changed during fruit development, and distinct sets of
SWEETs are expressed in the young and mature fruits. In octoploid
strawberry, nine SWEETs were clearly transcribed in the fleshy parts of
fruits, displaying a gradually increasing (FvSWEET1, -4, -9b), decreasing
(FvSWEET2b, -3, -7) or fluctuated (FvSWEET2a, -6b, -9a, -16) patterns,
respectively, following fruit development. A greater number (n = 12)
of SWEETs were achene-expressed in octoploid strawberry, and these
members were down-regulated or irregularly changed during fruit
development. Further functional study is required to verify SWEET-
mediated sugar-efflux during fruit development and clarify their func-
tions in sugar accumulation and flavor formation.
Till now, only the sucrose transporter FaSUT1 was found to be a
major sugar transporter component responsible for sucrose accumula-
tion during strawberry fruit development (Jia et al., 2013). Recently,
over 60 sugar transporter (ST) genes belonging to the sucrose trans-
porter family (SUC) and seven monosaccharide transporter (MST) fam-
ilies were identified and expression-profiled in woodland strawberry
(Jiu et al., 2018). How these transporters in cultivated strawberry
share out sugar transport and cooperate with one another will be a
major subject in exploration for the sweetness and the basis of berry fla-
vor. The current work provides a basic understanding of the SWEET
genes in strawberry and will benefit for the future study to elucidate
the detailed roles of SWEET-mediated sugar efflux in strawberry and
other Rosaceae crops.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.sajb.2019.07.002.
Author contributions
K.D. conceived the project. Q-H. G. and D-A. N. planned, designed,
and supervised the research. H-T. L. performed the experiments and
data analysis. W-Y. L.and S-H. T. contributed to sample collection. L-Q.
Z. and X-H. Z. roled in bioinformatic analysis. H-T. L. and K. D. prepared
the manuscript. All authors contributed to the revising of the manu-
script, had read and approved the final manuscript.
Declaration of Competing Interest
The authors have no conflicts of interest to declare.
Acknowledgments
This work was supported by funds from Shanghai Municipal Agricul-
tural Commission (Key Basic Research Project (2014) No. 7-2-2 and
(2016) No. 6-1-4; Hu Nong Ke Zhong Zi-2017-2-1), and the Science
and Technology Commission of Shanghai Municipality (Key Program,
16391901400). We are grateful to Miss Hai-Xin Diao of Shanghai Insti-
tute of Technology for polishing Fig. 8.
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