Genome-Wide Function, Evolutionary Characterization and
Expression Analysis of Sugar Transporter Family Genes
in Pear (Pyrus bretschneideri Rehd)
1
Centre of Pear Engineering Technology Research, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University,
Nanjing 210095, China
2
Roy J. Carver Biotechnology Center, University of Illinois Urbana-Champaign, Urbana, IL, USA
*Corresponding author: E-mail, [email protected]; Fax, +86-25-84396485.
(Received September 22, 2014; Accepted June 10, 2015)
The sugar transporter (ST) plays an important role in plant
growth, development and fruit quality. In this study, a total
of 75 ST genes were identified in the pear (Pyrus bretschneideri Rehd) genome based on systematic analysis.
Furthermore, all ST genes identified were grouped into
eight subfamilies according to conserved domains and
phylogenetic analysis. Analysis of cis-regulatory element sequences of all ST genes identified the MYBCOREATCYCB1
promoter in sucrose transporter (SUT) and monosaccharide
transporter (MST) genes of pear, while in grape it is exclusively found in SUT subfamily members, indicating divergent
transcriptional regulation in different species. Gene duplication event analysis indicated that whole-genome duplication
(WGD) and segmental duplication play key roles in ST gene
amplification, followed by tandem duplication. Estimation
of positive selection at codon sites of ST paralog pairs indicated that all plastidic glucose translocator (pGlcT) subfamily members have evolved under positive selection. In
addition, the evolutionary history of ST gene duplications
indicated that the ST genes have experienced significant expansion in the whole ST gene family after the second WGD,
especially after apple and pear divergence. According to the
global RNA sequencing results of pear fruit development,
gene expression profiling showed the expression of 53 STs.
Combined with quantitative real-time PCR (qRT-PCR) analysis, two polyol/monosaccharide transporter (PLT) and
three tonoplast monosaccharide transporter (tMT) members were identified as candidate genes, which may play
important roles in sugar accumulation during pear fruit development and ripening. Identification of highly expressed
STs in fruit is important for finding novel genes contributing
to enhanced levels of sugar content in pear fruit.
Keywords: Genome duplication events Pear (Pyrus bretschneideri Rehd) Positive selection Sugar transporter gene
family.
Abbreviations: AIC, Akaike information criterion; BAC, bacterial artificial chromosome; BLAST, base local alignment
search tool; CDS, coding sequence; DAFB, days after flower
blooming; dN/dS, non-synonymous/synonymous substitution
rate ratios; INT, inositol transporter; MEME, Multiple EM for
Motif Elicitation; ML, maximum likelihood; MST, monosaccharide transporter; Myr, Million years; NJ, Neighbor–Joining;
ORF, open reading frame; pGlcT, plastidic glucose translocator; PLT, polyol/monosaccharide transporter; qRT-PCR, quantitative real-time PCR; RNA-seq, RNA sequencing; SFP, sugar
facilitator transporter; ST, sugar transporter; STP, sugar transporter protein; SUT, sucrose transporter; tMT, tonoplast
monosaccharide transporter; VGT, vacuolar glucose transporter; WGD, whole-genome duplication.
Introduction
In higher land plants, sugars (including sucrose, monosaccharide and polyols) play important roles in plant growth, development and fruit flavor. In addition, sucrose represents the major
photosynthetically assimilated carbon transported via the
phloem between the source and sink (Van Bel 2003). After
release from the phloem, sucrose is directly transported into
sink cells, or is hydrolyzed by an extracellular invertase to fructose and glucose (Sherson et al. 2003). To date, it has been
established that not only the unloading and the loading of
the conducting complex, but also the allocation of sugars
into sink cells and sources are controlled by sugar transporters
(STs) that mediate the transport of polyols (Noiraud et al.
2001a, Juchaux-Cachau et al. 2007), monosaccharides
(Buttner 2007) or sucrose (Kühn 2003, Kühn and Grof 2010).
In previous studies, several ST genes have been isolated from
flora, fauna and other species, such as two hexose transporters
in Juglans regia (Decourteix et al. 2008), seven hexose transporters in Vitis (Fillion et al. 1999, Vignault et al. 2005, Hayes
et al. 2007) and a few polyol transporters in Malus domestica
(Watari et al. 2004), Prunus cerasus (Gao et al. 2003) and Olea
europea (Conde et al. 2007). Among all of the ST gene families,
the SUT gene family is a rather small protein family with six
genes in pear and nine genes in Arabidopsis; however, the MST
family is more diverse. Complete Arabidopsis genome sequencing revealed 53 MST members grouped into seven subfamilies
(Buttner 2007). Furthermore, 52 putative ST genes in tomato
Plant Cell Physiol. 56(9): 1721–1737 (2015) doi:10.1093/pcp/pcv090, Advance Access publication on 16 June 2015,
available online at www.pcp.oxfordjournals.org
! The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
Regular Paper
Jia-ming Li1, Dan-man Zheng2, Lei-ting Li1, Xin Qiao1, Shu-wei Wei1, Bin Bai1, Shao-ling Zhang1 and
Jun Wu1,*
J.-m. Li et al. | Sugar transporter gene family in pear
(Reuscher et al. 2014), 63 putative ST genes in grape (AfoufaBastien et al. 2010), and five SUT genes (Aoki et al. 2003) and 65
MST genes in rice (Johnson and Thomas 2007) were identified
in previous studies, indicating that STs can be found across the
plant kingdom. This result shows that these seven subfamilies
are ancient in higher plants (Johnson et al. 2006). In addition,
the novel SWEET family that can transport sugars was recently
identified. Due to its seven transmembrane domains, the
SWEET family has been reported to belong to a different superfamily (Chen et al. 2010). However, because of its relative novelty and these differences, the SWEET transporter family will
not be included in this study.
In previous research, some transporter gene expression data
in advanced plant and Arabidopsis microarray data (the BAR
database: http://bbc.botany.utoronto.ca) analyses have indicated that the expression of STs could be regulated by environmental and developmental factors, such as in yeast (Rolland
et al. 2002), as well as VvHT1 (Atanassova et al. 2003, Conde
et al. 2006). These results also indicate that the expression of
STs might be regulated distinctly at the transcriptional level,
but usually involves converging signaling pathways, depending
on either metabolic and hormonal signals or developmental
and environmental effects. However, in silico analysis of promoters of different genes involved in sugar storage, transport,
carbon metabolism and mobilization clearly demonstrate the
lack of common sugar-specific cis-regulatory elements (Sheen
et al. 1999, Delrot et al. 2000, Rolland et al. 2006). This agrees
with the fact that different types of transcription factors (AP2,
MYB, bZIP, WRKY, B3 and EIN3) are involved in sugar-linked
regulation of gene expression and are required for sugar signaling in plants (Rolland et al. 2006).
Due to the fast development of sequencing techniques,
more and more genomes in plants have been sequenced in
the past few years, and repeated episodes of small-scale and
large-scale gene duplication events have been shown to play
important roles during the evolution of gene families. Largescale gene duplication includes segmental duplications and
whole-genome duplications (WGDs) (Van de Peer and Meyer
2005). In pear, evidence has indicated that two WGDs occurred
during pear genome evolution, with an ancient WGD event
approximately 140 million years (Myr) ago (Fawcett et al.
2009), and a recent WGD event 30–45 Myr ago (Velasco
et al. 2010). Small-scale gene duplication events, such as
tandem duplications, also play important roles during gene
family expansion (Taylor and Raes 2005). The sum of other
small-scale duplications and tandem duplications are estimated
to contribute duplicates on a scale comparable with large segmental duplications in rice (Yu et al. 2005). The evidence has
indicated that tandem and segmental duplications are important during gene family expansion (Sharoni et al. 2011, Zhao
et al. 2014).
Pear is one of the most important commercial fruits, and is
cultivated in all temperate zone countries of both hemispheres.
Sugar content is an important factor affecting fruit quality to a
large extent. Therefore, the increase in sugar content is directly
related to improvement of fruit quality. Unfortunately, most
previous studies focused on sugar content evaluation in
1722
different pear varieties (Hudina and Śtampar 2000, Ito et al.
2002, Chen et al. 2007), and only a few sugar-related genes
were cloned and studied (Iida et al. 2004). Thus, identification
of important genes related to sugar transportation and accumulation, and understanding their functional mechanism will
help improve sugar quality in pear. STs play an important role
for pear growth and fruit quality; however, there has been
limited reporting of the identification of STs, although they
have been identified from other ligneous or herbaceous species.
Recently, the pear (Pyrus bretschneideri Rehd) genome was
sequenced and assembled by the strategy of BAC (bacterial
artificial chromosome) by BAC, combined with wholegenome shotgun data, i.e. a total of 194 genome coverage
sequencing. The result showed that the assembled sequence
accounts for 97.1% (512 Mb) of the estimated genome size of
pear and includes 2,103 scaffolds with N50 at 540.8 kb. The high
quality of the assembled sequence and annotation was assessed
and confirmed using Sanger-derived BAC sequences along with
RNA sequencing (RNA-seq) of different tissues and public protein database alignment (Wu et al. 2013). The high quality of
the pear genome is suitable for genome-wide identification and
analysis of gene families. The present study is the first to report
on the genome-wide identification of ST genes in pear, together
with phylogenetic, structural and evolutionary analysis. In addition, RNA-seq databases of pear fruit were used to determine
the expression pattern for all ST genes and select key genes
affecting sugar content. This study will help to reveal the
roles of these ST genes in pear fruit development and sugar
quality conformation, as well as provide gene resources for
future genetic improvement of pear. The results obtained will
also provide a reference in sugar regulation and quality improvement for other related fruit species.
Results
Identification and construction of the
phylogenetic tree of the ST gene family in pear
In the present study, a total of 75 open reading frames (ORFs)
encoding putative ST proteins were identified in the pear (cultivar: ‘Dangshansuli’) genome using the HMMER profile and
BLASTp search for further analysis. On the basic of previous
research in Arabidopsis, we renamed the ST of pear as the
STP subfamily, which stands for the sugar transporter protein,
tMT subfamily stands for the tonoplast monosaccharide transporter subfamily, VGT stands for the vacuolar glucose
transporter subfamily, SFP stands for the sugar facilitator
protein subfamily, INT stands for the inositol transporter
subfamily, pGlcT stands for the plastidic glucose translocator
subfamily, PLT stands for the polyol/monosaccharide transporter subfamily and SUT stands for the sucrose transporter
subfamily (Table 1). Phylogenetic analysis of the 75 identified
nucleotide sequences (Fig. 1) reveals that STs could be classified into eight separate subfamilies with two large subfamilies
(the PLT subfamily and STP subfamily) and six small subfamilies
based on the Arabidopsis ST sequences (the phylogenetic tree
which contained the STs of pear and Arabidopsis is not shown).
Plant Cell Physiol. 56(9): 1721–1737 (2015) doi:10.1093/pcp/pcv090
Table 1 Structural and biochemical information of ST members in pear
Gene ID
Chromosome
(Mbp)
PbSTP1
13 (15.1)
PbSTP2
16 (20.6)
PbSTP3
PbSTP4
Strand
Gene model
Genomic
(bp)
cDNA
(bp)
Protein
(amino acids)
E-value
(% identity)
Pbr003615.1
2,280
1,503
501
4.20E-125
Pbr008093.1
2,091
1,503
501
7.00E-127
16 (20.6)
Pbr008083.1
1,996
1,683
561
5.40E-127
16 (20.6)
Pbr008081.1
2,280
1,488
496
3.20E-127
PbSTP5
16 (20.6)
Pbr008096.1
1,958
1,461
487
3.50E-128
PbSTP6
16 (20.6)
+
Pbr008080.1
1,958
1,122
374
3.50E-128
PbSTP7
16 (20.6)
+
Pbr008082.1
3,451
1,368
456
2.00E-124
PbSTP8
13 (15.1)
+
Pbr003614.1
3,701
1,311
437
4.70E-125
PbSTP9
16 (20.6)
Pbr008092.1
5,589
1,497
499
2.50E-120
PbSTP10
16 (20.6)
+
Pbr008084.1
5,401
1,599
533
8.00E-120
PbSTP11
13 (15.1)
+
Pbr003612.1
5,884
1,734
578
6.80E-124
PbSTP12
2 (22.1)
Pbr007315.1
2,947
1,734
578
5.70E-120
PbSTP13
2 (22.1)
Pbr033836.1
2,370
1,734
578
2.30E-120
PbSTP14
16 (20.6)
Pbr008094.1
3,254
1,728
576
3.40E-121
PbSTP15
15 (43.6)
Pbr034294.1
2,936
1,692
564
6.10E-120
PbSTP16
Scaffold 489.0.1 (0.3)
Pbr029168.1
2,914
1,740
580
8.00E-120
PbSTP17
2 (22.1)
Pbr033859.1
2,370
1,602
534
3.00E-116
PbSTP18
16 (20.6)
+
Pbr008095.1
1,970
1,746
582
1.80E-105
PbSTP19
13 (15.1)
+
Pbr003616.1
3,300
1,314
438
1.70E-120
PbSTP20
15 (43.6)
Pbr015498.1
3,027
1,314
438
1.10E-117
PbtMT1
6 (23.1)
+
Pbr015095.1
4,646
1,611
537
1.00E-47
PbtMT2
5 (28.4)
+
Pbr023965.1
4,150
1,611
537
3.90E-52
PbtMT3
5 (28.4)
Pbr033292.1
4,710
1,614
538
9.50E-52
PbtMT4
10 (26.2)
+
Pbr032130.1
3,232
1,581
527
4.70E-52
PbtMT5
15 (43.6)
+
Pbr037349.1
2,895
1,560
520
1.50E-51
PbtMT6
15 (43.6)
+
Pbr037348.1
2,891
1,605
535
6.20E-47
PbVGT1
1 (10.7)
+
Pbr013451.1
3,919
1,578
526
1.10E-96
PbVGT2
7 (15.3)
+
Pbr039977.1
4,191
1,578
526
1.40E-96
PbVGT3
11 (30.3)
Pbr018950.1
3,574
1,578
526
1.50E-94
PbSFP1
13 (15.1)
Pbr014788.1
3,745
1,578
526
1.20E-89
PbSFP2
15 (43.6)
Pbr020127.1
4,139
1,581
527
1.30E-97
PbSFP3
12 (22.8)
Pbr017113.2
3,303
1,584
528
1.20E-47
PbSFP4
8 (17.1)
+
Pbr006144.1
3,746
1,578
526
8.70E-86
PbSFP5
15 (43.6)
+
Pbr005950.1
3,674
1,584
528
3.60E-72
PbINT1
Scaffold 466.0 (0.4)
Pbr028155.1
6,813
1,686
562
1.50E-123
PbINT2
15 (43.6)
Pbr002731.1
6,266
1,455
485
8.00E-118
PbINT3
5 (28.4)
Pbr000049.1
2,941
1,419
473
4.30E-93
PbINT4
10 (26.2)
Pbr038498.1
2,984
1,419
473
1.10E-91
PbINT5
10 (26.2)
Pbr017619.1
2,977
1,473
491
1.10E-91
PbINT6
5 (28.4)
Pbr000048.1
2,607
1,443
481
1.50E-91
PbpGlcT1
17 (25.3)
Pbr010989.1
3,544
1,437
479
3.10E-84
PbpGlcT2
17 (25.3)
Pbr039652.1
5,591
1,437
479
8.40E-100
PbpGlcT3
Scaffold 786.0 (0.2)
Pbr037980.1
5,659
1,317
439
6.10E-92
PbpGlcT4
9 (22.4)
Pbr032591.3
5,974
1,533
511
1.00E-72
PbpGlcT5
Scaffold 477.0 (0.2)
Pbr028640.1
4,397
1,527
509
2.90E-74
PbpGlcT6
Scaffold 477.0 (0.2)
Pbr028631.1
4,397
1,527
509
2.90E-74
PbPLT1
Scaffold 764.0 (0.2)
+
Pbr037512.1
3,119
1,527
509
4.50E-102
PbPLT2
Scaffold 277.0 (0.2)
+
Pbr018464.1
3,188
1,533
511
8.10E-102
PbPLT3
5 (28.4)
Pbr038549.1
2,648
1,533
511
4.60E-104
+
+
+
+
+
+
+
+
(continued)
1723
J.-m. Li et al. | Sugar transporter gene family in pear
Table 1 Continued
Gene ID
Chromosome
(Mbp)
PbPLT4
PbPLT5
PbPLT6
Scaffold 277.0 (0.5)
PbPLT7
7 (15.3)
PbPLT8
7 (15.3)
PbPLT9
Scaffold 764.0 (0.2)
PbPLT10
5 (28.4)
PbPLT11
Scaffold 637.0 (0.3)
PbPLT12
3 (27.4)
PbPLT13
Strand
Gene model
Genomic
(bp)
cDNA
(bp)
Protein
(amino acids)
E-value
(% identity)
5 (28.4)
Pbr038546.1
4,268
1,524
508
5.40E-102
7 (15.3)
Pbr018903.1
2,390
1,530
510
3.30E-104
+
Pbr018463.1
3,279
1,584
528
4.00E-107
+
Pbr018910.1
2,497
1,584
528
2.00E-102
Pbr018908.1
2,497
1,584
528
2.00E-102
Pbr037515.1
2,177
1,554
518
1.10E-105
Pbr038547.1
2,352
1,554
518
4.20E-105
Pbr034137.1
2,652
1,602
534
5.10E-104
Pbr022830.1
2,650
1,554
518
2.20E-103
3 (27.4)
Pbr040466.1
2,609
1,554
518
1.80E-107
PbPLT14
7 (15.3)
Pbr018906.1
2,654
1,653
551
1.10E-102
PbPLT15
8 (17.1)
Pbr019072.1
2,835
1,464
488
1.40E-53
PbPLT16
Scaffold 637.0 (0.3)
+
Pbr034138.1
2,329
1,509
503
1.70E-100
PbPLT17
Scaffold 637.0 (0.3)
+
Pbr034135.1
2,342
1,566
522
2.10E-100
PbPLT18
8 (17.1)
Pbr019074.1
2,293
2,217
739
1.10E-99
PbPLT19
Scaffold 277.0 (0.5)
+
Pbr018465.1
3,429
2,208
736
1.40E-100
PbPLT20
Scaffold 764.0 (0.2)
+
Pbr037511.1
2,509
2,208
736
1.30E-103
PbPLT21
Scaffold 764.0 (0.2)
+
Pbr037514.1
2,844
2,208
736
7.60E-98
PbPLT22
5 (28.4)
Pbr038548.1
4,356
2,259
753
2.70E-99
PbPLT23
8 (17.1)
Pbr019075.1
2,354
2,172
724
4.80E-75
PbSUT1
Scaffold 1139.0 (0.1)
Pbr003266.1
3,314
1,602
534
0
PbSUT2
Scaffold 160.1.13 (0.1)
+
Pbr009635.1
3,167
1,524
508
0
PbSUT3
10 (26.2)
+
Pbr018232.1
3,495
1,524
508
0
PbSUT4
Scaffold 512.0 (0.3)
+
Pbr030158.1
3,286
1,500
500
0
PbSUT5
8 (17.1)
+
Pbr025968.1
7,419
1,551
517
1E-160
PbSUT6
13 (17.1)
+
Pbr039114.1
5,734
1,839
613
1E-140
+
+
Among them, 23 and 20 ST genes were annotated as PLT and
STP (Table 1). Across the maximum likelihood (ML) tree, most
bootstrap values were 80, and eight nodes of each subfamily
clade had a good bootstrap value. In addition, a consensus
Neighbor–Joining (NJ) tree of all 75 ST nucleotide sequences
with 1,000 bootstrap replicates revealed a topology that was
similar to the ML topology (Supplementary Fig. S1).
Combining the NJ topology and ML topology analysis, the
phylogenetic tree of the ST gene family in the present study
is highly reliable.
Conserved motifs and exon–intron organization of
ST genes
Because the sugar transporter domain is essential for catalytic
activity of ST proteins, the Multiple EM for Motif Elicitation
(MEME) motif website search program was used to identify
the conserved motifs from 75 ST proteins in pear. In this
study, three distinct motifs, motif 1, 2 and 3, which all belong
to the ST domain (Fig. 2), were located on the functional domains of all 69 MST proteins, but could not be found in SUT
proteins, suggesting that the three distinct motifs may be necessary for MSTs. Interestingly, when we compared the
1724
conserved motifs between the SUT gene family and MST gene
family, we found that the conserved motifs in the SUT gene
family and conserved motifs in the MST gene family were
quite different (Fig. 1), even though they had the same functional domains (ST domain); this result might due to functional
differences between the SUT gene family (transport sucrose)
and the MST gene family (transport monosaccharide).
Although all MST proteins had three distinct motifs, the
number of motifs in each of the seven subfamilies was different.
As shown in Fig. 1, most genes in the two large MST subfamilies
had 13 motifs, and in the other subfamilies the number of motifs
varied from seven to nine. However, we found that the eight
genes of the PLT subfamily did not have motif 10 (a conserved
domain SH10), suggesting that SH10 was not critical for PLT
member function, despite the conserved sequences and the
fact that it was located on the functional domain of the MST
gene family. Based on the results of the structural analysis, it was
shown that all members with similar structure clustered into the
same subfamily. Interestingly, the two large subfamilies of the
MST family, the STP and PLT subfamily, shared similar structures, except the two motifs SH9 and SH8 that appear in the PLT
subfamily, and SH12 and SH14 that appear in the STP subfamily.
Plant Cell Physiol. 56(9): 1721–1737 (2015) doi:10.1093/pcp/pcv090
Fig. 1 Phylogenetic analysis of pear ST nucleotide sequences. The evolutionary history is inferred using maximum parsimony and maximum
likelihood (ML). An ML tree is created by the PHYML program using 100 bootstrap replicates, and the best fitting substitution models for all data
are determined with the Akaike information criterion (AIC) using ModelTest 3.06. An unrooted ML tree of pear MST nucleotide sequences is
shown. The seven classes are marked by different colors. Different shapes and colors represent different motifs. SH1–15 represents motifs 1–15.
To gain insight into the structure of the ST genes, the exon
and intron boundaries, which are known to play crucial roles in
the evolution of multiple gene families, were analyzed. The
results showed that the exon numbers of 75 ST genes ranged
from two to 18 (Supplementary Fig. S2). Different subfamilies
contained different exon numbers; the fact that PbSFP1 and
PbSFP2 genes have 18 exons, and PbPLT17, PbPLT16, PbPLT20,
PbPLT21 and PbPLT22 have only two exons, indicates that both
exon gain and loss have occurred during the evolution of the ST
gene family, which might lead to functional diversity of closely
related ST genes. However, it was found that within each subfamily, genes usually have a similar number of exons.
Search for cis-elements involved in the
transcriptional regulation of MST genes
In our study, a 2 kb promoter region for each of 62 ST genes was
identified. For the other 13 ST genes, the identified sequence
was shorter than 2 kb, because of the presence of another gene
located <2 kb upstream. Finally, a Plant Cis-acting Regulatory
DNA Elements (PLACE) website analysis was applied in this
research and it identified 254 different cis-elements that have
been classified per ST gene members.
First, a total of 20 common cis-regulatory elements were
identified in the 2 kb promoter region, which were highly conserved among the 62 ST analyzed sequences. For some shorter
promoters, any of the 20 common cis-elements might be missing. Those common cis-regulatory elements are also responsive
to distinct plant hormones, such as cytokinins, and several environmental factors, as well as CO2, light, dehydration stress and
abiotic and biotic stresses (Table 2). At least six of the 20
common cis-acting elements, i.e. GATABOX, EBOXBNNAPA,
IBOXCORE, GTGANTG10, TATABOX5 and GT1CONSENSUS,
are required for transcriptional regulation by light, consistent
with the roles of STs in sugar allocation between sink
1725
J.-m. Li et al. | Sugar transporter gene family in pear
Fig. 2 Sequence logos for three motifs of MST domains using the MEME program. MEME motifs are displayed by stacks of letters at each site.
The total height of the stack is 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. The x-axis represents the width of the motif and the y-axis
represents the bits of each letter.
Table 2 Common putative cis-elements identified in the promoter
sequences of sugar transporters
SITE
Cis-element name
Sequence
Response
S000378
GTGANTG10
GTGA
Pollen
S000454
ARR1AT
NGATT
Cytokinins
S000176
MYBCORE
CNGTTR
Leaf, shoot
S000449
CACTFTPPCA1
CACT
Tetranucleotide
S000039
GATABOX
GATA
Light, leaf, shoot
S000314
RAV1AAT
CAACA
Root, rosette leaves
S000144
EBOXBNNAPA
CANNTG
Light, ABA, seeds
S000198
GT1CONSENSUS
GRWAAW
Light, leaf, shoot
S000494
EECCRCAH1
GANTTNC
CO2-responsive
S000502
MYBCOREATCYCB1
GANNTG
ABA, abiotic stress,
cell cycle
S000447
WRKY71OS
TGAC
Giberellin repressor,
ABA
S000265
DOFCOREZM
AAAG
C-metabolism, leaf
S000493
CURECORECR
GTAC
Copper; oxygen;
hypoxic
S000407
MYCCONSENSUSAT
CANNTG
ABA, leaf, seed, cold
S000199
IBOXCORE
GATAA
Light, leaf, shoot
S000395
INRNTPSADB
YTCANTYY
Light-responsive
S000028
CAATBOX1
CAAT
Seed
S000103
SEF4MOTIFGM7S
RTTTTTR
Seed, storage protein
S000203
TATABOX5
TTATTT
Light
S000415
ACGTATERD1
ACGT
Dehydration stress,
dark
The PLACE website is used for promoter sequence analysis.
SITE, a unique accession number is assigned to a motif sequence. The cis-element
name, the sequence of the promoter and the signaling pathway are presented.
1726
and source organs. A common cis-regulatory element
named MYBCOREATCYCB1 was strongly represented in all
ST genes (Table 2), indicating the importance of the
MYBCOREATCYCB1 sequence in the promoter of pear MST
genes.
Another approach targeted unique cis-element sequences
present in the promoter region of unique transporter genes,
which might indicate expression specificity (Table 3). Finally,
24 unique cis-elements were identified in 17 single promoters.
Interestingly, three unique cis-elements (O2F2BE2S1,
LBOXLERBCS and ABREZMRAB28) out of 24 specific motifs
were present only in PbpGlcT2, and two unique cis-elements
were present in each of PbpGlcT1, PbSTP8, PbSTP19, PbtMT4
and PbINT5. The 11 ST genes have only one unique cis-element.
This result indicated that a limited number of gene-specific ciselements were concentrated in the promoter regions of a few
transporter genes.
Chromosomal localization, gene duplication
events and collinearity analyses
The genomic distribution of ST genes on the pear chromosome
was investigated in this study. Of 75 pear MST genes, 57 were
mapped onto 15 chromosomes, excluding chromosomes 4 and
14, representing unbalanced distribution (Fig. 3). The largest
number of ST genes was mapped onto chromosome 16 with 10
ST genes, and only one MST gene was located on each of
chromosomes 1, 3, 6, 11 and 12. Additionally, 18 out of 75 ST
genes were mapped onto different scaffolds.
During the evolution of a gene family, tandem duplication
and WGD/segmental duplication play important roles in generating new members. Therefore, in order to clarify the
Plant Cell Physiol. 56(9): 1721–1737 (2015) doi:10.1093/pcp/pcv090
Table 3 Unique cis-elements identified only in the promoter sequence of a single MST gene
SITE
Cis-element name
S000489
SORLREP4AT
S000233
HDMOTIFPCPR2
S000496
WRECSAA01
S000085
Sequence
Response
Gene
CTCCTAATT
Light
PbPLT9
CTAATTGTTTA
Pathogen defense
PbSFP4
AAWGTATCSA
Wound
PbSTP9
INTRONUPPER
MAGGTAAGT
Splice junction
PbSFP1
S000154
SPHCOREZMC1
TCCATGCAT
ABA, seed
PbSFP3
S000367
E2FBNTRNR
GCGGCAAA
Cell cycle
PbSTP19
S000163
O2F2BE2S1
GCCACCTCAT
Storage protein, seed
PbpGlcT2
S000397
TE2F2NTPCNA
ATTCCCGC
Cell cycle, tissue
PbINT5
S000022
ARE1
RGTGACNNNGC
Antioxidant
PbSFP5
S000207
GT2OSPHYA
GCGGTAATT
Leaf, leaf, shoot
PbpGlcT1
S000234
AUXRETGA1GMGH3
TGACGTAA
Auxin
PbINT5
S000446
CARGNCAT
CCWWWWWWWWGG
Gibberellin
PbtMT4
S000301
CONSERVED11NTZMATP1
ACGTATTAAAA
Essential for gene expression
PbSTP19
S000361
HBOXPVCHS15
CCTACCNNNNNNNCTNNNNA
Defense
PbtMT4
S000056
HSELIKENTACIDICPR1
CNNGAANNNTTCNNG
Heat shock, pathogen
PbVGT3
S000302
LREBOXIPCCHS1
AACCTAACCT
Light, leaf, shoot
PbPLT14
S000095
RBCSBOX3PS
ATCATTTTCACT
Leaf, shoot
PbSTP8
S000455
PE2FNTRNR1A
ATTCGCGC
Cell cycle
PbpGlcT1
S000183
SP8BFIBSP8AIB
ACTGTGTA
Root
PbSTP8
S000126
LBOXLERBCS
AAATTAACCAA
Light, leaf, shoot
PbpGlcT2
S000133
ABREZMRAB28
CCACGTGG
ABA, seed, shoot
PbpGlcT2
S000343
AGL3ATCONSENSUS
TTWCYAWWWWTRGWAA
Leaf, flower, shoot
PbVGT1
S000368
CACGCAATGMGH3
CACGCAAT
Auxin
PbSTP6
The PLACE website is used for promoter sequence analysis.
SITE, a unique accession number is assigned to a motif sequence. The cis-element name, the sequence of the promoter and the signaling pathway
are presented. Gene indicates the corresponding gene in which the cis-elements are found
potential mechanism of evolution of the MST gene family, both
tandem duplication and segmental duplication events were
investigated in this study. The result of tandem duplication
and WGD/segmental duplication analysis indicated that 24
ST genes could be assigned to WGD/segmental duplication
blocks and nine ST genes were assigned to tandem duplication
(Table 4). In addition, for all cDNA sequences of those genes,
the similarity ranged from 66.91% to 99.68%, and all of the
segmental gene pairs were found to have counterparts on segmental duplication blocks (Fig. 3). Interestingly, there are two
members of the PLT subfamily assigned to the tandem duplication blocks, and none to the segmental duplication blocks in
the pear genome (Table 5). All of six tMT subfamily members
and four out of all SUT subfamily members were assigned to the
segmental duplication blocks. In addition, in order to verify the
reliability of WGD/segmental duplication in our study, both
end sequences of PbtMT2 and PbtMT4 were analyzed as an
example. The result indicated that the genes located on the
ends of PbtMT2 and PbtMT4 are a WGD/segmental duplication
region (Fig. 4).
Estimation of positive selection at codon sites and
history duplications of the ST family
Our results showed that all Ka/Ks paralog pairs of ST genes were
less than one, indicating that ST genes have evolved mainly
under purifying selection (data not shown). Following on
from this, ML estimation of the dN/dS substitution rate ratios
for paralog pairs in which each sequence came from the same
duplication event at nodes in the pear ST nucleotide phylogeny
were calculated using the branch-site models method. The
result showed that six ST paralog pairs present a large
number of codon sites under positive selection (Table 4). We
also estimated duplication ages of ST paralog pairs; a pair of
them (PbpGlcT4–PbpGlcT1) is near to approximately 140 Myr
old, and others ranged in age from approximately 0.83 to approximately 18.11 Myr old. An estimated nucleotide substitution rate of the whole ST family in the pear is 0.05 substitutions
per site per Myr. In addition, the ages of twelve segmental ST
duplication gene events between approximately 2.21 and approximately 543.67 Myr old were estimated; the paralog pairs at
the terminal nodes ranged from approximately 0.83 to approximately 73.61 Myr old in pear, but most of them appear after the
second WGD (Fig. 5). In addition, the divergence times of the
SUT gene family in pear were also explored; as shown in Fig. 5,
estimation of gene duplicate divergence times revealed that the
SUT gene subfamily began to diversify at approximately 8.08
Myr ago, and the same results were also found in the PLT, STP
and SFP subfamily, and according to the result of divergence
time estimation, four of six SUT genes began to diversify after
divergence of pear and apple.
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J.-m. Li et al. | Sugar transporter gene family in pear
Fig. 3 Chromosomal distribution and gene duplications of the ST gene family. The scale on the circle is in Mega bases. The gene IDs on the
chromosomes indicate the positions of centromeres; the numbers of each chromosome are shown inside the circle of each bar. The WGD or
segmental duplication genes are connected by a black line. In addition, after each gene ID, *0, *1, *2, *3 and *4 indicate singleton duplication,
dispersed duplication, proximal duplication, tandem duplication and WGD or segmental duplication, respectively.
Expression of ST gene family in pear
To investigate the transcript pattern of ST family genes during
fruit development, the expression patterns during six developmental stages of pear fruit, from the early to mature stage, were
analyzed using the RNA-seq database available from our previous research (Wu et al. 2013). Finally, a hierarchical cluster with
the logarithm of average values for the 53 ST family members
was generated. As shown in Fig. 6, ST family genes can be
divided into two major groups based on their expression profiles. Group A contained 15 MST genes, 13 of them exhibiting
1728
preferential expression in some stages or low expression in
other stages, and two of them, PbSTP12 and PbPLT1, with
low expression in all stages, indicating that those genes may
not play important roles in sorbitol accumulation during the
whole of pear fruit development. In addition, 38 MST genes
belong to group B, which showed high expression in different
stages; among them, PbPLT9 and PbPLT22 had the highest expression levels during fruit development. The genes in group B
could be further divided into three subgroups, B1, B2 and B3.
Subgroup B1 included 17 ST genes, which showed high
Plant Cell Physiol. 56(9): 1721–1737 (2015) doi:10.1093/pcp/pcv090
Table 4 Analysis of the MST gene families in pear
Paralog pairs
Positive
No. of
selection? codon
sitesa
Likely age of Duplicate
duplication type
(Myr)
PbpGlcT3–PbpGlcT2 Yes
6/2**
PbpGlcT4–PbpGlcT1 Yes
19/1**/3* 124.65
PbpGlcT5–PbpGlcT6 Yes
24/2**/1* 1.09
18.11
WGD/segmental
PbVGT1–PbVGT2
No
79.63
WGD/segmental
PbSFP2–PbSFP4
No
27.87
WGD/segmental
PbSFP4–PbSFP5
No
26.08
PbPLT9–PbPLT13
No
73.61
PbPLT19– PbPLT21
No
11.27
PbPLT20–PbPLT6
No
13.54
PbPLT1–PbPLT2
No
4.74
PbPLT11–PbPLT18
No
3.09
PbPLT16–PbPLT23
No
8.20
PbPLT7–PbPLT8
Yes
PbtMT5–PbtMT1
No
10/4*
Discussion
0.88
312.31
WGD/segmental
PbtMT2–PbtMT4
No
77.58
WGD/segmental
PbtMT3–PbtMT2
No
9.13
WGD/segmental
PpINT1–PpINT2
No
9.25
PpINT4–PpINT3
No
237.88
PpINT4–PpINT5
No
1.31
PbSTP9–PbSTP10
No
3.62
PbSTP9–PbSTP4
No
543.67
PbSTP2–PbSTP3
No
2.42
PbSTP4–PbSTP18
No
PbSTP5–PbSTP6
Yes
WGD/segmental
WGD/segmental
25.33
4/2*
0.83
PbSTP7–PbSTP14
No
PbSTP15–PbSTP16
No
11.10
PbSTP17–PbSTP15
No
73.88
WGD/segmental
PbSTP12–PbSTP13
No
2.21
WGD/segmental
PbSTP17–PbSTP12
Yes
1.53
1
Finally, the results of qRT-PCR analysis indicated that the expression levels of five genes are closely connected to the change
of sugar content during pear fruit development (Fig. 7), one of
them differing from the RNA-seq data. On the basis of the RNAseq data analysis, the expression pattern of the PbtMT4 gene is
up-regulated during the whole of pear fruit development.
However, according to the results of the qRT-PCR analysis, the
expression pattern of the PbtMT4 gene is up-regulated from
May 1 to July 29, and down-regulated from July 29 to
September 4. With the exception of the PbtMT4 gene, the
other four ST genes show a similar trend to the RNA-seq data,
indicating that our RNA-seq data are reliable.
1.27
PbSUT1–PbSUT4
No
7.56
WGD/segmental
PbSUT2–PbSUT3
No
8.05
WGD/segmental
a
Values without an asterisk show codon sites with posterior probability (PP)
>50%; a single asterisk and double asterisks indicate PP >95% and PP >99%,
respectively.
expression during all developmental stages, but almost all lower
than the expression of subgroup B3. Subgroup B2 consisted of
eight ST genes that displayed higher expression before July 28,
and with low expression during the later stage of fruit development. Subgroup B3 comprised 13 ST genes that displayed
higher expression than the other two subgroups in almost all
developmental stages, suggesting that these 13 ST genes may
play a more important role than other genes in pear fruit
development.
Verification of gene expression by qRT-PCR
On the basis of the RNA-seq database, combining the content of
sucrose, glucose, fructose and sorbitol analysis, we found that
the expression levels of five ST (PbtMT2, PbtMT3, PbtMT4,
PbPLT9 and PbPLT22) genes were closely related to sugar accumulation levels during pear fruit development and ripening, and
may play more important roles than other genes. In order to
verify that these genes were associated with sugar content
during pear fruit development, the expression levels of five
genes was analyzed by quantitative real-time PCR (qRT-PCR).
Identification, and phylogenetic and structural
analysis of the ST gene family in pear
The genome sequence and RNA-seq profiles of pear provide a
large amount of useful data to explore the functional diversity
of the ST gene family from multiple perspectives. In this study,
the search for the ST gene family in the pear translated genome
has identified 75 STs, and, among them, six ST genes belong to
the SUT family, indicating that SUT is a small family within the
ST gene family, similar to results found in other plants, such as,
four putative SUT genes were identified in grape (AfoufaBastien et al. 2010), five in rice (Aoki et al. 2003) and three in
tomato (Reuscher et al. 2014). In addition, 69 were putative
MSTs, showing that the number of MST members in pear is
larger than in Arabidopsis (53 genes) (Buttner 2007), grape (61
genes) (Afoufa-Bastien et al. 2010), rice (65 genes) (Johnson and
Thomas 2007) and tomato (49 genes) (Reuscher et al. 2014). We
also compared the different subfamily members and gene duplication events among pear, Arabidopsis and rice (Table 5).
Interestingly, as in rice, STP and PLT form the largest subfamilies
in pear, perhaps due to the repeated regions encompassed by
STP and PLT genes. As expected, conserved domains and phylogenetic analysis performed with these MST proteins revealed
seven distinct subfamilies (Fig. 1). The same result has been
found in grape, rice, Arabidopsis and tomato (Johnson et al.
2006, Afoufa-Bastien et al. 2010, Reuscher et al. 2014), which
indicated that the classification of pear MST families was reliable and reasonable. As shown in Fig. 1, different subgroups
have similar conserved domains, indicating that under normal
circumstances the same subfamily members had the same
function due to similar conserved domains. In addition, the
results of the determination of exon–intron organization of
ST genes have shown that the numbers of exons in 75 ST
genes ranged from two to 18 (Supplementary Fig. 2), similar
to tomato, in which the exon numbers of all ST genes ranged
from one to 18 (Reuscher et al. 2014).
Cis-elements involved in the transcriptional
regulation of ST genes
Based on sequences of cis-elements, ST gene promoters contained highly repetitive regions and several common motifs.
Among them, motifs such as DOFCOREZM (DNA-binding
1729
J.-m. Li et al. | Sugar transporter gene family in pear
with one finger) may play an important role not only in terms
of response specificity via a combinatory control, but also in the
regulation of gene expression in terms of activity levels for the
ST. A similar result has been identified for AtSUC2 (Schneidereit
et al. 2008), the expression of which in the companion cell is
regulated by the close co-operation of binding sites for a putative HD-Zip transcription factor and a DOFCOREZM protein. In
previous studies, several transporter gene promoters showed an
important concentration of sugar-responsive elements, indicating their transcriptional regulation via sugars. The first to be
clearly demonstrated was the transcriptional regulation of
VvHT1 by glucose (Atanassova et al. 2003, Conde et al. 2006),
confirmed by the fact that the VvHT1 promoter has the largest
number of sugar-responsive motifs. In the present study, 20
common cis-regulatory elements were conserved in the promoter regions of ST gene family members (Table 2), the same
number as has been identified in the ST gene family of grape
(Afoufa-Bastien et al. 2010). Finally, the MYBCOREATCYCB1
promoter, which is required for transcriptional regulation of
cyclin B1 during G1/S to G2/M transition in the cell cycle
(Tréhin et al. 1997), was identified in all ST gene family members. However, it is different from a previous finding that the
MYBCOREATCYCB1 sequence was exclusively found in SUC/
SUT promoters in grape (Afoufa-Bastien et al. 2010). The
MYBCOREATCYCB1 promoter could be found in SUT promoters for sucrose-dependent induction of Cyclin D3 gene expression (Riou-Khamlichi et al. 2000); this result indicated a
possible concomitant regulation of some SUT genes in the
cell cycle. In addition, it was interesting to find that the
MYBCOREATCYCB1 promoter could be found in MST gene
family members, which would provide a new research direction
for cis-elements in the MST gene family of pear. In addition, this
also indicated that different species might have a different transcriptional regulation mechanism in the MST gene family.
The ST gene family arose mainly through WGD/
segmental duplication, accompanied by tandem
duplications
It had been reported that a primary driving force of new functions in the evolution of genomes and genetic systems is gene
duplication (Moore and Purugganan 2003), which is one of the
major evolutionary mechanisms leading to functional speciation and diversification (Lynch 2000). As previously reported,
the pear genome had undergone two rounds of WGD events,
which have a great impact on the amplification of members of a
gene family. In the present study, we found 75 ST genes in the
pear genome that could be classified into eight subfamilies
(Fig. 1) and distributed on 15 chromosomes or some scaffolds
(Fig. 3). In addition, the results of the pear tandem duplication
and segmental duplication analysis showed that 24 ST genes
could be assigned to WGD/segmental duplication blocks and
nine ST genes were assigned to tandem duplication. This result
indicated that some ST subfamilies have increased rapidly
during the course of evolution, and segmental duplication is
Table 5 Comparative analysis of ST gene families in pear, Arabidopsis and rice
Subfamily
Number of genes
Pear
Duplicates
Tandem
STP
20
5
Arabidopsis
Segmental
6
Duplicates
Rice
Tandem
14
2
Duplicates
Tandem
29
14
VGT
3
0
2
3
0
2
0
PLT
23
2
0
6
2
15
10
INT
6
2
2
4
0
3
0
tMT
6
0
6
3
0
6
0
SFP
5
0
2
19
13
6
4
pGlcT
6
0
2
4
0
4
0
SUT
6
0
4
9
1
5
0
Total
75
9
24
62
18
70
28
Fig. 4 Collinearity relationships of ST genes in pear; 100 kb on each side flanking the genes PbtMT2 and PbtMT4. Segmental duplication pairs are
connected with bands. Each black line represents a chromosome segment, and the chromosome number is to the right of the line. ST genes are
shown in red, other genes are shown in green, arrowheads represents genes and their transcriptional orientation, and the suffix of the gene name
is next to the line.
1730
Plant Cell Physiol. 56(9): 1721–1737 (2015) doi:10.1093/pcp/pcv090
Fig. 5 Calibrated phylogenetic tree with gene duplicate divergence time estimates for the ST family. The 75 gene sequences of ST from pear and
the phylogenetic tree topology constructed by PHYML. Dates are estimated using the ML method. Calibration points of different subfamilies are
shown on the external nodes. Red square symbols at the external nodes indicate segmental duplication gene pairs, and black square symbols at
the external nodes indicate paralog gene pairs.
the main mechanism for expansion of this ST gene family,
accompanied by tandem duplications. The same phenomenon
has also been found in the WRKY transcription factor family of
soybean genes (Yin et al. 2013), where a majority of WRKY
genes arose through segmental duplication, accompanied by
tandem duplications, but different from Arabidopsis and rice,
which experienced more tandem duplicated genes than WGD/
segmental duplicated genes in the MST gene family (Johnson
and Thomas 2007). For example, PLT and STP subfamilies are
greatly expanded, with tandem duplications in rice accounting
for 10 and 14 of those subfamily members, respectively
(Table 5). In addition, between the vascular and the nonvascular lineages, STP and PLT subfamilies vary significantly in
size, suggesting that the expansion of STP and PLT subfamilies
could be related to the evolution of vascular plants, and indicating the increased importance of the sugar transporters in
vascular plants (Johnson et al. 2006).
Positive selection and history of duplication of ST
family genes
For functional proteins, many amino acids are not free to
vary under functional constraints and strong structural traits.
Thus, in order to detect positive selection of only a few amino
acid residues, one should determine the variation in selective
pressure among sites (Yang et al. 2000). In previous studies, the
extent of positive selection on many protein families has been
shown via phylogeny-based analyses of codon substitution
(Smith and Eyre-Walker 2002, Weinberger et al. 2010), and
1731
J.-m. Li et al. | Sugar transporter gene family in pear
Fig. 6 Expression patterns for pear ST genes from the RNA sequence. Heat map showing expression patterns of pear MST family genes in six
stages. The fruit samples of the ‘Dangshansuli’ cultivar at April 22 (15 days after full blooming, DAFB), May 13 (36 DAFB), June 27 (81 DAFB), July
28 (110 DAFB), August 30 (145 DAFB) and September 21 (167 DAFB) were collected in 2011, which include the key stages of pear fruit
development from early fruit setting to the mature stage. The expression of all ST genes identified in this research is measured by RNA-seq
analysis using six stages of pear fruit development. The genes are located on the right and the different stages are indicated at the top of each
column. The color scale represents reads per kilobase per million normalized log2-transformed counts, where light red indicates a high level, light
green indicates a low level and black indicates a medium level.
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Plant Cell Physiol. 56(9): 1721–1737 (2015) doi:10.1093/pcp/pcv090
Fig. 7 Expression profiles of five ST genes during pear fruit development by qRT-PCR analysis. Five fruit stages of ‘Dangshansuli’ were sampled
depending on the status of pear development in 2013, May 1 (31 DAFB), May 27 (57 DAFB), June 23 (84 DAFB), July 29 (120 DAFB) and
September 4 (157 DAFB), for qPT-PCR analysis. The relative mRNA levels of individual ST genes are normalized with respect to the housekeeping
gene, tubulin, at different stages. The value on the x-axis represents the five stages of pear fruit development. The y-axis represents the relative
mRNA (fold). The results were expressed using May 1 as a reference for each gene (relative mRNA level 1). Values represent the experiment
among three independent biological repetitions.
has determined that positive selection at some codons is an
important driver of protein evolution (Yang and Bielawski
2000). In this study, some of the members of the ST family
have positive selection sites (Table 4). The PbpGlcT2–
PbpGlcT3, PbpGlcT4–PbpGlcT1 and PbpGlcT5–PbpGlcT6 gene
pairs had more positive selection sites than other gene pairs.
Interestingly, all six of these genes belong to the pGlcT subfamily, which indicates that the pGlcT subfamily has evolved under
positive selection to survive during evolution. In addition, positive selection appears in most duplicate pairs younger than
approximately 20 Myr old in pear, except the PbpGlcT4–
PbpGlcT1 (124.65 Myr old) gene pair, which also indicates
that positive selection appears in most duplicate pairs after
divergence of pear and apple. However, positive selection in
Arabidopsis is seen in any duplicate pairs older than approximately 34 Myr in the MST gene family (Johnson and Thomas
2007).
In a previous study, gene duplicate divergence time estimates revealed that protogenes of each MST subfamily type
were present in organisms leading to the land plant lineage at
least as far back as the middle Proterozoic (Johnson and
Thomas 2007). The result of gene duplicate divergence time
of ST members in pear estimated in this study showed that the
ST family had comparatively few members at 140 Myr ago, and
the expansion of ST genes into large subfamilies continued after
the second WGD (Fig. 5, 40 Myr ago). This result is similar to
the research in Arabidopsis and rice, where the expansion of
large subfamilies continued through the Cenozoic (65–0 Myr)
(Johnson and Thomas 2007). Additionally, it was reported that
pear and apple diverged from each other at 21.5–5.4 Myr ago
(Wu et al. 2013). In this study, most members of the ST gene
family were distributed at 20–0 Myr in phylogenetic trees according to the divergence time estimation (Fig. 5), indicating
that most ST family members mainly arose after divergence of
pear and apple. The comparison among pear, rice and
Arabidopsis indicated that the different rounds of genomewide duplication events and polyploidy led to the ST gene
family expansion at inconsistent times among different species
(Vision et al. 2000, Bowers et al. 2003). So, we can conclude that
the ST family experienced large expansions resulting from the
WGDs or multiple segmental duplications and continued to
expand through the second WGD, and that most of the ST
family began to expand after divergence of pear and apple.
Expression of the ST gene family during pear fruit
development
For the ST gene family, we were most interested in those which
play important roles during pear fruit development and ripening. In this report, transcript data showed that a total of 53 MST
genes were expressed during pear fruit development. This result
indicated that these expressed genes are functionally active,
with 38 of them being expressed in all six stages during fruit
development and ripening (Fig. 6). For PLT subfamily genes,
nine of them had one motif missing during evolution, but genes
PbPLT20, PbPLT21, PbPLT19 and PbPLT10 are still expressed
during development of pear fruit. These results indicated that
the loss of this N-terminal domain does not affect gene
function.
Sucrose is a major phloem-translocated component and
photosynthetic product in most plants. However, some
plants synthesize carbohydrates other than sucrose in source
leaves and translocate them to sink organs, such as polyols
(often named sugar alcohols). Polyols are highly soluble, low
molecular weight non-reducing compounds, which means they
are suitable as translocating compounds. In many species of
Rosaceae, the major phloem component is sorbitol, such as
in pear, apple, apricot, peach, cherry and prune. In the
phloem of apples, sorbitol comprises about 80% of translocated
carbohydrates, and in mature apricot leaves up to 65–75% of
translocated carbon is from sorbitol (Kühn et al. 1999, Lalonde
et al. 2003). Even though sorbitol is very important in Rosaceae,
the mechanism for phloem loading of sorbitol is still unclear
(Noiraud et al. 2001b). In a previous study, two PmPLT genes
were isolated from Plantago major, which can transport sorbitol
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J.-m. Li et al. | Sugar transporter gene family in pear
(Ramsperger-Gleixner et al. 2004); the results indicated that
PmPLT1 and PmPLT2 proteins were localized specifically in
companion cells of source leaf phloem and showed their importance in phloem loading of sorbitol. Six PLT genes were
detected in Arabidopsis, and have been described as nonspecific hexose, pentose and polyol transporters expressed in
different tissues (Klepek et al. 2005, Reinders et al. 2005). In
addition, two sorbitol transporters (PcSOT1 and PcSOT2)
were identified in sour cherry with high expression levels in
fruit (Gao et al. 2003), and three MdSOT genes were isolated
from apple source leaves (Watari et al. 2004). All these findings
indicate that the PLT subfamily might play an important role in
long-distance transport of assimilative sorbitol from leaves to
fruits in Rosaceae species. In our study, 13 genes of the PLT
subfamily were expressed during fruit development (Fig. 6).
Out of all of them, the expression levels of PbPLT9 and
PbPLT22 were believed to correspond to changes in sorbitol
levels in pear fruit, with up-regulation from the early stage to
the middle stage, and a slow decrease from the middle stage to
near ripening (Fig. 6). In addition, the expression levels of two
genes (PbPLT9 and PbPLT22) have been verified by qRT-PCR
analysis (Fig. 7), on the basis of which we found that the expression levels of two PLT genes have similar trends with RNAseq analysis. Based on our previous research, it was found that
the content of sorbitol in pear fruit increased from the early
stage to the middle stage, and slowly decreased from the middle
stage to near ripening (data not shown). Thus, PbPLT9 and
PbPLT22 should be considered as important candidate genes
for the manipulation of sorbitol transport and accumulation for
pear fruit.
The tMT subfamily has also been characterized in rice and
Arabidopsis, as AtTMT1 and AtTMT2 were characterized as
fructose/H+ or glucose antiporters and localized to the vacuolar membrane (Wormit et al. 2006, Schulz et al. 2011). In this
study, all of the tMT subfamily genes were detected as expressed over the whole course of fruit development. Among
them, the expression levels of three genes (PbtMT2, PbtMT3
and PbtMT4) were strongly believed to correspond to fructose
and glucose levels during fruit development (Fig. 6), and similar
results have been found in qRT-PCR analysis (Fig. 7). Fructose
and glucose in ‘Dangshansuli’ have low levels at early stages,
increasing sharply from the middle stage to near ripening
during fruit development, with glucose levels then slowly
decreasing during pear fruit ripening. In addition, previous studies have shown that PbtMT4 can be detected as differently
expressed proteins and considered as candidate genes that
could improve fruit quality during pear fruit development
and ripening (Li et al. 2015). So, on the basis of RNA-seq, proteome and qRT-PCR analysis, PbtMT2, PbtMT3 and PbtMT4 are
highly reliable candidate genes, which can increase fructose and
glucose content during pear fruit development.
Conclusion
A total of 75 ST genes were identified in a genome-wide survey
of the pear genome, and can be classified into eight subfamilies,
with two large subfamilies (the STP subfamily and PLT subfamily) and six small subfamilies, as supported by the organization
1734
of conserved domains and phylogeny. Gene duplication analysis
indicated that during expansion of the ST gene family, many
subfamilies have evolved, and WGD/segmental duplications
have played a more important role during the expansion of
the VGT, tMT, pGlcT and SFP subfamilies in pear. Large expansions of the ST family continued through the second WGD,
especially after the divergence of pear and apple. In addition,
the estimation of positive selection at codon sites showed some
amino acid sites belonging to pGlcT members under positive
selection. The analysis of promoter sequences indicated that
different species have different transcriptional regulation in the
MST gene family, such as the MYBCOREATCYCB1 sequence
exclusively found in SUC/SUT promoters in grape, but present
in all ST gene families in pear. Finally, expression analysis revealed that most ST genes are expressed during fruit development. Among them, two PLT members and three tMT
members showed consistent trends with sugar accumulation
in fruit. Our results help to clarify the biological function of MST
genes in pear development and have a significant influence on
our knowledge of woody plant STs.
Materials and Methods
Identification of ST protein in pear
A search for all ST genes in the pear genome was performed using HMMER
software (Eddy 1998) for all ST subfamilies. First, an ST domain (PF00083)
downloaded from Pfam (http://pfam.sanger.ac.uk/) was used to search the
pear protein database by HMMER software. Then, 167 putative ST proteins
were identified in the pear genome with an E-value <1E-5. Secondly, all ST
proteins were downloaded from the Arabidopsis database (http://www.arabidopsis.org/). Each Arabidopsis ST protein sequence was used as a query sequence in a Base Local Alignment Search Tool (BLAST) search (Altschul et al.
1997), searching against the 167 putative ST protein database of pear to find its
best match sequence. Finally, a total of 75 STs protein sequences were identified
for further analysis.
Alignment and phylogenetic tree analysis of the
ST gene family
Multiple alignments of nucleotide sequences were performed using the Muscle
program and Gblocks software (Castresana 2000). For phylogenetic tree analysis, the 75 pear ST nucleotide sequences using maximum parsimony and ML
were used. An ML tree was created by the PHYML program (Guindon et al.
2010) using 100 bootstrap replicates, and the best fitting substitution models
for all data were determined with the Akaike information criterion (AIC) using
ModelTest 3.06 (Posada and Crandall 1998). The model selected was
GTR + I + G, gamma distribution with four categories, and an estimated
shape parameter of 1.5159. Representations of the calculated trees were constructed using Figtree. In addition, an NJ phylogenetic tree was created by
MEGA 6.0 (Tamura et al. 2013).
Conserved motifs, cis-elements and gene structure
of ST genes
ST protein sequences were analyzed by the MEME program (http://meme.nbcr.
net/meme/cgi-bin/meme.cgi) to confirm the conserved motifs. MEME was
employed using the following parameters: maximum number of motifs, 600;
number of repetitions, any; optimum width, 15–60; and maximum number of
motifs, 15. The results were generated as a txt file. Finally, the iTOL (interactive
tree of life) program (Letunic and Bork 2011) (http://itol.embl.de/other_trees.
shtml) was used to integrate the phylogenetic and structural tree. Promoter
sequences (2,000 bp) of ST family genes were obtained from the
pear Annotation Project database in our previous study (Wu et al. 2013).
Plant Cell Physiol. 56(9): 1721–1737 (2015) doi:10.1093/pcp/pcv090
The cis-elements of promoters were identified by PLACE Web Signal ScanPLACE (http://www.dna.affrc.go.jp/PLACE/signalup.html). The gene structure
display server 2.0 (GSDS, http://gsds.cbi.pku.edu.cn) was used to illustrate exon
and intron organization for individual ST genes by comparison of the cDNAs
with their corresponding genomic DNA sequences from the pear genome database website (http://peargenome.njau.edu.cn).
Chromosomal locations and gene duplications of
all ST genes
The chromosome Map Tool was used to determine the location of ST genes on
pear chromosomes. The duplication pattern for each ST gene was analyzed in
this study. In brief, the 42,812 protein-coding genes from the pear genomic
database were analyzed using an all-vs-all local BLAST search with E-value
<1E-5. The BLAST search outputs were imported into MCScanX software
(http://chibba.pgml.uga.edu/mcscan2/) and 42,812 protein-coding genes
were classified into various types of duplications including tandem, WGD/
segmental, dispersed and proximal under a default criterion. If the pairs of
genes were on the two segmental loci and are collinear gene pairs, we considered the gene pairs as segmental duplication gene pairs.
Estimation of positive selection at codon sites
To explore whether positive Darwinian selection drove the evolution of the ST
gene family, the non-synonymous/synonymous substitution rate ratios (dN/dS)
of all paralog pairs were analyzed using the coding sequence (CDS) of ST gene
paralogs. If the dN/dS ratio was >1, it indicates that the gene pairs were under
positive selection, or, alternatively, a dN/dS ratio of <1 indicates purifying
selection and a dN/dS ratio = 1 indicates neutral evolution. In addition, ML
estimation of the dN/dS for paralog pairs in which each sequence came from
the same duplication event at nodes in the pear MST nucleotide phylogeny
(square symbols in Fig. 5) was calculated using the branch site models method.
The branch site test2 of positive selection was used in this study, as described in
a previous study (Johnson and Thomas 2007), comparing the null model A1
(model = 2, NS sites = 2 and fix omega = 1) with the alternative model A (model = 2, NS sites = 2 and fix_omega = 0) to find codon sites under probable positive selection. The test of positive selection, with significance cut-offs of 5.41
and 2.71 at the 1% and 5% levels, respectively, was used. Codon sites under
probable positive selection and genes with positive selection at the 5% level
were identified using the Bayes Empirical Bayes method (Yang et al. 2005).
Estimation of divergence times
The 75 gene sequences of ST from pear and the phylogenetic tree topology
constructed by the PHYML (Guindon et al. 2010) program were used. To estimate molecular evolutionary rates and divergence times, a Bayesian method
implemented in MCMCtree in PAML (Yang 2007) and the independent rates
model was applied to estimate the prior of rates among internal nodes. Each
subfamily calibration was included through the time prior: the pGlcT subfamily
divergence time was set at 967 (±58) Myr ago, the SFP subfamily divergence
time was set at 866 (±53) Myr ago, and the VGT subfamily, STP subfamily, also
called the hexose transporter family in grape, INT subfamily, PLT subfamily and
tMT subfamily divergence times were set at 478 (±43), 835(±41), 689(±54),
833(±61) and 311(±14) Myr ago, respectively. All seven nodes were constrained
with maximum and minimum ages (Johnson and Thomas 2007).
Genome-wide expression analysis of the ST gene
family
To investigate the expression of ST gene family members, pear fruit samples of
the ‘Dangshansuli’ cultivar on April 22 (15 days after full blooming, DAFB), May
13 (36 DAFB), June 27 (81 DAFB), July 28 (110 DAFB), August 30 (145 DAFB)
and September 21 (167 DAFB) were collected in 2011, which included the key
stages of pear fruit development from early fruit setting to the mature stage.
RNA-seq libraries of six fruit developmental stages were constructed using an
Illumina standard mRNA-Seq Prep Kit (TruSeq RNA and DNA Sample
Preparation Kits version 2). The RNA-seq data can be downloaded from our
center website (http://peargenome.njau.edu.cn/). Expression values of each
gene were log transformed, and the cluster analyses were performed using
cluster software with the hierarchical cluster method of ‘complete linkage’
and Euclidean distances. Finally, the Treeview program was used to display
the results of the cluster analysis.
RNA extraction and first-strand cDNA synthesis
In our research, five fruit stages were sampled depending on the status of pear
development in 2013, May 1 (31 DAFB), May 27 (57 DAFB), June 23 (84 DAFB),
July 29 (120 DAFB) and September 4 (157 DAFB) for qPT-PCR analysis. Total
genomic RNA was extracted from pear fruit according to the CTAB (cetyltrimethyl ammonium bromide) method (Gasic et al. 2004), and then DNase I
(Invitrogen) was used to remove genomic DNA contamination. Finally, about
2 mg of total RNA was used for first-strand cDNA synthesis using a ReverTra
Ace-aFirst Strand cDNA Synthesis Kit (TOYOBO Biotech Co. Ltd.) according to
the manufacturer’s protocol.
Real-time PCR analysis
The primers used for amplifying five ST genes are listed in Supplementary
Table S1. In the present study, the LightCycler 480 SYBR GREEN I Master
(Roche) was used according to the manufacturer’s protocol. Each reaction
mixture contained 10 ml of LightCycler 480 SYBR GREEN I Master, 0.4 ml of
each primer, 1 ml of diluted cDNA and 7.4 ml of nuclease-free water. The qRTPCR was performed on the LightCycler 480 (Roche) and all reactions were run
as duplicates in 96-well plates. Each cDNA was analyzed in triplicate, and then
the average threshold cycle (Ct) was calculated per sample. The qRT-PCR conditions were as follows: pre-incubation at 95 C for 10 min and then 40 cycles of
94 C for 15 s, 60 C for 30 s, 72 C for 30 s, with a final, extension at 72 C for
3 min, and reading the plate for fluorescence data collection at 60 C. A melting
curve was performed from 60 to 95 C in order to check the specificity to the
amplified product. Finally, the average threshold cycle (Ct) was calculated per
sample; Pyrus tubulin (accession No. AB239681) was used as the internal control, and the relative expression levels were calculated with the 2–Ct method
descripted by Livak and Schmittgen (2001).
Supplementary data
Supplementary data are available at PCP online.
Authors’ contributions
J.L. carried out the experiments and data analysis, and produced
a draft of the manuscript. D.Z., L.L. and X.Q. participated in the
collinearity analysis, data analysis and preparation of figures.
B.B. and S.W. contributed to sample collection and data analysis. S.Z. contributed with consultation. J.W. managed and designed the research and experiments.
Funding
This work was supported by the National Science and
Technology Support Funds [2013BAD02B01-2], National
Natural Science Foundation of China [31171928]; the
Ministry of Education Program for New Century Excellent
Talents in University [NCET-13-0864].
Disclosures
The authors have no conflicts of interest declared.
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