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Plant Cell Reports
ISSN 0721-7714
Volume 34
Number 6
Plant Cell Rep (2015) 34:1037-1047
DOI 10.1007/s00299-015-1763-3
Molecular characterization of a
transcriptionally active Ty1/copia-like
retrotransposon in Gossypium
Yuefen Cao, Yurong Jiang, Mingquan
Ding, Shae He, Hua Zhang, Lifeng Lin &
Junkang Rong
1 23
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ORIGINAL PAPER
Molecular characterization of a transcriptionally active
Ty1/copia-like retrotransposon in Gossypium
Yuefen Cao •Yurong Jiang •Mingquan Ding •
Shae He •Hua Zhang •Lifeng Lin •
Junkang Rong
Received: 12 August 2014 / Revised: 3 February 2015 / Accepted: 10 February 2015 / Published online: 19 February 2015
ÓSpringer-Verlag Berlin Heidelberg 2015
Abstract
Key message A transcriptionally active Ty1/copia-like
retrotransposon was identified in the genome of Gos-
sypium barbadense. The different heat activation of this
element was observed in two tetraploid cotton species.
Abstract Most retrotransposons from plants are tran-
scriptionally silent, or activated under certain conditions.
Only a small portion of elements are transcriptionally ac-
tive under regular condition. A long terminal repeat (LTR)
retrotransposon was isolated from the cultivated Sea Island
cotton (H7124) genome during the investigation of the
function of a homeodomain leucine zipper gene (HD1)in
trichome growth. Insertion of this element in HD1 gene of
At sub-genome was related to the trichomeless stem in
Gossypium barbadense. The element, named as GBRE-1,
had all features of a typical Ty1/copia retrotransposon and
possessed high similarity to the members of ONSEN
retrotransposon family. It was 4997 bp long, comprising a
single 4110 bp open reading frame, which encoded 1369
amino acids including the conserved domains of gag and
pol. The expression of GBRE-1was detected under regular
condition in G. barbadense and G. hirsutum, and its ex-
pression level was increased under heat-stress condition in
G. hirsutum. Besides, its expression pattern was similar to
that of the ONSEN retrotransposon. Abundant cis-regula-
tory motifs related to stress-response and transcriptional
regulation were found in the LTR sequence. These results
suggested that GBRE-1was a transcriptionally active
retrotransposon in Gossypium. To our knowledge, this is
the first report of the isolation of a complete Ty1/copia-
type retrotransposon with present-day transcriptional ac-
tivity in cotton.
Keywords Ty1/copia retrotransposon Gossypium
Reverse transcriptase Heat treatment Homeodomain
leucine zipper gene
Introduction
Long terminal repeat (LTR) retrotransposons, which con-
stitute a major component of repetitive regions in plant
genome, widely exist in Eukaryotic genomes (Havecker
et al. 2004). They profoundly affect host genome size and
contribute to genomic evolution by their numerous copy
accumulation and insertion (Hawkins et al. 2006; Wendel
and Wessler 2000). LTR retrotransposons usually encode
the proteins required for their transposition, including a
capsid protein (gag), protease, integrase, reverse tran-
scriptase, and RNase H. By the order of these proteins,
Communicated by M. Prasad.
Y. Cao and Y. Jiang contributed equally to this work.
Electronic supplementary material The online version of this
article (doi:10.1007/s00299-015-1763-3) contains supplementary
material, which is available to authorized users.
Y. Cao Y. Jiang M. Ding S. He H. Zhang J. Rong (&)
The Key Laboratory for Quality Improvement of Agricultural
Products of Zhejiang Province, School of Agriculture and Food
Science, Zhejiang A & F University, Linan,
Hangzhou 311300, Zhejiang, China
e-mail: rjklab_primer@126.com
L. Lin
Nanosphere, Inc, 4088 Commercial Drive, Northbrook,
IL 60062, USA
J. Rong
State Key Laboratory of Cotton Biology, Institute of Cotton
Research, Chinese Academy of Agricultural Sciences,
Anyang 455000, Henan, China
123
Plant Cell Rep (2015) 34:1037–1047
DOI 10.1007/s00299-015-1763-3
Author's personal copy
LTR retrotransposons can be divided into two groups, the
Tyl/copia and Ty3/gypsy (Xiong and Eickbush 1990).
Most retrotransposons are transcriptionally inactive
(Kumar and Bennetzen 1999), or activated at the particular
developmental stages, or under a certain biotic and abiotic
stresses, including phytopathogens or wounding (Grand-
bastien 1998; Kimura et al. 2001). Several retrotransposons
such as FaRE1,CIRE1,Tto 1,Tnt 1,Tnp2,Tos 17, and
OARE-1have been isolated, and their transcriptional ac-
tivities are identified under the stressful conditions or in
particular tissues (Grandbastien et al. 1989; Grandbastien
1998; Hirochika 1993; Hirochika et al. 1996; Kimura et al.
2001; Rico-Cabanas and Martı
´nez-Izquierdo 2007). Re-
cently, a heat-stressed activated LTR retrotransposon
named ONSEN was discovered in Arabidopsis thaliana by
Ito et al. (2011). This retrotransposon preferred to integrate
into genes or open chromatin structures in Arabidopsis
species (Ito et al. 2013). Heat activation of ONSEN was
detected in other five Brassicaceae species, while another
four species showed ONSEN transcripts even under non-
heat-stressed condition (Ito et al. 2013). It suggests that the
activation of some retrotransposons might be due to the
stressful environmental factors, the inserted region in host
genome and the expression of neighboring genes, and
homeostatic active domain in retrotransposon promoter
(Araujo et al. 2001; Hollister and Gaut 2009; Ito et al.
2013). Based on previous evidences, retrotransposon is
believed to play a significant role in plant biology.
The genus Gossypium is composed of 50 species in-
cluding 45 diploid members and 5 tetraploid members that
distribute in many tropical and subtropical semi-arid re-
gions of the world (Cronn et al. 2002; Wendel and Cronn
2003). Diploid members of the genus are divided into eight
groups based on chromosome pairing behavior and fertility
in interspecific hybrids (Hendrix and Stewart 2005). All
diploid members have 13 chromosomes with genome sizes
ranging from 885 Mb per haploid nucleus in the American
D-genome species to 2572 Mb per haploid nucleus in the
Australian K-genome species (Hendrix and Stewart 2005).
In cotton genome, there exist numerous transposable ele-
ments. Differential lineage-specific amplification of trans-
posable elements is responsible for genome size variation
in Gossypium (Hawkins et al. 2006). Hawkins et al. (2006)
found that 40–65 % of each genome was composed of
transposable elements in three diploid members of Gossy-
pium, and that copia,gypsy, and non-LTR LINE retro-
transposons were major components of the Gossypium
genome (Hawkins et al. 2006,2008). Bioinformatics ana-
lysis suggested that non-LTR retrotransposons may be
more actively transcribed than LTR retrotransposons in
Gossypium (Hu et al. 2010).
Although transposable elements are the main compo-
nents of the cotton genome and widely distribute around
entire genome, individual LTR retrotransposon has not yet
been characterized at the DNA and RNA level. Moreover,
few studies were conducted to identify the effect of its
insertion into genes on a typical phenotype. AtML1 is a
HD-Zip IV family gene that encodes for a transcription
factor and is confirmed to be related to epidermal cell
differentiation and trichome growth by working together
with AtPDF2, another member of HD-Zip IV. Addition-
ally, two cotton homologous genes of AtML1 (GhHD-1and
GbML1) were isolated from G. hirsutum and G. bar-
badense and were proved to be related to growth and de-
velopment of cotton trichome and fiber (Walford et al.
2012; Zhang et al. 2010). In our study on the function of
HD1 on G. barbadense trichome phenotype, we found a
very close association of a retrotransposon insertion in At
subgenome HD1 gene with trichomeless stem phenotype
(data unpublished). In the present work, a full length copy
of an active LTR retrotransposon was isolated in G. bar-
badense and designated as GBRE-1. Its structure, tran-
scriptional activity, and evolution history in diploid cottons
were reported.
Materials and methods
Plant materials
G. barbadense modern variety H7124 and primitive culti-
vated form GB1596 were used to isolate the entire retro-
transposon. H7124 is a modern cultivar planted in China.
GB1596 originated in Central American was kindly pro-
vided by Dr. James Frelichowski and kept in US Cotton
Germplasm Collection at Taxes. The TM-1 is a G. hirsu-
tum standard genetic line. 8 diploid D-genome and 2
diploid A-genome species including two diploid ancestors
of tetraploid cottons (Table 3) were also included for in-
vestigation of LTR region together with 2 tetraploid vari-
eties. All materials were planted in our university campus
at Linan, Zhejiang, China.
DNA and RNA extraction
Genomic DNA was extracted from fresh young leaves
using the CTAB method (Murray and Thompson 1980).
Total RNA was extracted with CTAB method (Jiang and
Zhang 2003) and treated with Recombinant DNase I
(TaKaRa, Japan) to remove the residual DNA.
Polymerase chain reaction (PCR) and cloning of PCR
product
A pair of primers (contig24f, 50-TCTTCTGAGGTCTGTC
TCTG-30; down2, 50-AGAC [G/T] ACGTTCATTGCAA
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CAAT-30) inside of HD1 was used to amplify the retro-
transposon. DNA amplification was performed in 20 lL
reaction solution containing 20 ng genomic DNA, each
dNTP at 0.2 mM, each primer at 0.4 lM, 0.2 U of LA Taq
DNA polymerase (TaKaRa, Japan), and LA Taq PCR
buffer with 2.5 mM MgCl
2
. The following program was
used for PCR reaction: 94 °C for 5 min, followed by 32
cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for
5 min, and ending with an elongation step of 72 °C for
5 min. The amplicon was electrophoresed in 1 % regular
agarose gel and purified with AxyPrep
TM
DNA Gel Ex-
traction Kit (Axygen, USA), then sequenced directly. To
amplify a 665 bp sequence from RNase H to LTR (RH-
LTR) in the retrotransposon, another pair of the primers
LTR-L3 (50-TAGGTGTTTCAAATGAAGGTCC-30) and
LTR-L (50-TGTTGGAAACGTGATGGGCC-30) was de-
signed. The PCR mixture was the same as above except for
using GoEasy Taq DNA polymerase (Bioinshin, China)
instead of the LA Taq DNA polymerase. PCR was per-
formed under the following conditions: 94 °C for 5 min,
followed by 30 cycles of 94 °C for 30 s, 57 °C for 30 s,
and 72 °C for 10 s, and ending with an elongation step of
72 °C for 5 min. The products were purified from gel with
AxyPrep
TM
DNA Gel Extraction Kit (Axygen, USA), li-
gated into the pMD
Ò
18-T Vector (TaKaRa, Japan), and
transformed into E. coli DH5acompetent cells. Recombi-
nant plasmids were isolated with AxyPrep
TM
plasmid
Miniprep Kit (Axygen, USA) for sequencing.
Heat treatment and molecular analysis
Cotton seedlings were planted in soil with 18 h pho-
toperiod at 25 °C for 10 and 17 days, and then subjected
to 37 °C for 24 h as heat-stress treatment, with original
growth condition (25 °C) as control. Total RNA was
isolated from cotyledon (10-day seedling) and euphylla
(17-day seedling) of these plants, respectively. For RT-
PCR, reverse transcriptase M-MLV (TaKaRa, Japan) was
used for the 1st strand synthesis with Oligo d (T) 18
Primer and GoEasy Taq DNA polymerase (Bioinshin,
China) for PCR with specific primers RVT-F (50-GTC
TACCGATTGAAGAAATCTTTG-30) and RVT-R (50-GG
ATCAGCTGGCGTGACT-30). EF1a(GenBank no.
AF120093) was amplified as an internal control (Ya-Jun
et al. 2008). PCR was performed under the following
conditions: 94 °C for 3 min, followed by 30 cycles of
94 °C for 30 s, 55 °C for 30 s, and 72 °C for 10 s, and
ending with an elongation step of 72 °C for 5 min. The
PCR was repeated three times (supplemental Fig. 2). The
significance of relative abundance of GBRE-1transcripts
was analyzed by paired-samples Ttest with 95 % confi-
dence interval.
Sequence analysis
DNA sequencing was performed by BGI (Shenzheng,
China). Sequences were annotated with LaserGene pro-
gram (DNASTAR, Madison, WI, USA), including nu-
cleotide statistics analysis, open reading frame (ORF)
prediction, and protein translation. Previous released gene
sequences with high similarities to present retrotransposon
were retrieved from GenBank databases using BLASTN/P
programs (http://blast.ncbi.nlm.nih.gov) on National Cen-
ter for Biotechnology Information (NCBI). Pairwise se-
quence comparisons and multiple sequence alignments
were conducted using ClustalW (Thompson et al. 1994),
and the results were manually adjusted with GeneDoc 2.7
(Nicholas and Nicholas 1997). Phylogenetic tree was
constructed by the neighbor-joining method with a
500-replicate bootstrap search using MEAG 5 (Tamura
et al. 2011). PlantCARE database (Lescot et al. 2002) was
used for cis-regulatory motif analysis. To reveal if the cis-
regulatory motifs were enriched significantly in GBRE-1
LTR, 20 sequences were collected randomly from cotton
whole-genome sequence and used for significance analysis
(supplemental Table 2). The significance analysis of such
elements was conducted by the one-sample Ttest method
with 95 % confidence interval (supplemental Table 1). If
Pvalue less than or equal to 0.05, it means that this motif
in GBRE-1 was significantly enriched in higher frequency
than by chance at 5 % level.
Results
Discovery of a retrotransposon insertion in the HD1
gene
In a study on the association between HD1 genotype and
trichome phenotype of G. barbadense, the HD1 gene was
cloned and sequenced from plants with different trichome
phenotypes. An unexpected large fragment, approximately
5 kb larger than the expected fragment (Fig. 1a), was
amplified consistently in some G. barbadense varieties
when a pair of primers (contig24f and down2) inside of
HD1 gene was used. By using sub-genome-specific pri-
mers, it was found that the larger fragment was amplified
from the ninth exon of HD1 gene in A-, but not the D-sub-
genome. Sequencing of the large fragments amplified from
H7124 and 1596 revealed an insertion of a 4997 bp in HD1
gene in homologous A-genome. Bioinformatic analysis
showed that the insertion was a Ty1/copia-like retrotrans-
poson (data see next section). We tentatively termed it as
GBRE-1and deposited its nucleotide sequence into Gen-
Bank (accession number: KF740825).
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A total of 53 G. barbadense varieties including the 13
primitive cultivated forms were analyzed. Among them, 34
exhibited the insertion in HD1 of At sub-genome (named
as At-HD1), 11 showed no insertion, and 8 segregated with
or without insertion for different plants. The observation of
their trichome phenotype showed that the plants with the
insertion in At-HD1 genes had no or very few trichome on
their stem, while the plants without insertion were covered
with trichome, which led to the hypothesis that the lack of
At-HD1 accounts for the general lack of stem pubescence
of G. barbadense (data unpublished).
Molecular characterization of GBRE-1
Sequence analysis revealed that GBRE-1was 4997 bp in
length and had the typical retrotransposon structure,
containing a pair of LTRs and a single large open reading
frame (ORF) from nucleotide (nt) positions 498-4607,
which showed the homology to retroviral gag and pol
genes. The pol gene consisted of enzymes protease (PR),
integrase (IN), reverse transcriptase (RVT), and RNase H
(RH) in the order of 50- PR-IN-RVT-RH- 30. According to
this order, GBRE-1was inferred to be a Ty1/copia retro-
transposons (Fig. 2).
LTRs of GBRE-1:The5
0and 30LTR of GBRE-1was
identical, and each was 396 bp long beginning with TG and
ending with CA. The GBRE-1sequence was flanked by
direct repeats of five base pairs (50-GATAT-30) of cotton
genomic sequence (Fig. 2a). They were presumed to be a
duplication of the target site produced by GBRE-1copy
insertion.
Primer binding site (PBS) and polypurine tract (PPT):
The PBS of GBRE-1, which was the binding site of tRNA
primer and necessary for reverse transcription in minus
strand synthesis, was located downstream of the 50LTR end
(Fig. 2a). It contained a sequence of 50-TGGCATCA-
GAGCTACGGTT-30, which was homologous to PBSs of
Art 1,Tnt 1 (Grandbastien et al. 1989), and Tto 1 (Hir-
ochika 1993) with the similarity of 75, 75, and 81.2 %,
respectively (Fig. 2b). The PPT (50-GAGGGGGAG-30) for
plus strand DNA synthesis was located upstream of 30LTR
in GBRE-1(Fig. 2a), and was quite homologous to that of
Tnt 1,AtRE1 (Kuwahara et al. 2000), and copia (Lindauer
et al. 1993) with the similarity of 78.6, 78.6, and 92.9 %,
respectively (Fig. 2b).
Coding region of GBRE-1: The coding region was
4110 bp long and consisted of one ORF encoding 1369
amino acids including the conserved domains of gag,
PR, IN, RVT, and RH (Fig. 3). The RNA-binding
motif (CX
2
CX
4
HX
4
C) is a characteristic feature of gag
Fig. 1 Agarose gel (1 %) electrophoresis pattern of PCR products of
HD1 gene. The large fragment with an inserter was labeled by red box
and the normal fragments by white box. MDNA marker (DS 15000,
Dongsheng, China); 1 G. barbadense variety H7124; 2 G. barbadense
1596; 3and 4two G. barbadense varieties without retrotransposon
insertion
Fig. 2 a The structure of GBRE-1.LTR long terminal repeat, GAG
group antigen, Zn zinc knuckle, PR protease, IN integrase, RVT
reverse transcriptase, RH RNase H; the primer binding site for minus
strand DNA synthesis (PBS) and the polypurine tract (PPT) are
underlined. bComparison of the PBS and PPT between GBRE-1and
other similar retrotransposons. Different shading levels, from black
(100 %), gray ([80 %), grayish ([60 %), to unshaded (\60 %),
stand for different degrees of residue conservation (Nicholas and
Nicholas 1997)
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(Peterson-Burch and Voytas 2002). The gag domain in
GBRE-1was CYNCNKYCHFSYEC (Fig. 3a). PR is re-
quired to release the other functional components from the
polyprotein, and integrase is responsible for the insertion of
retroelement cDNA into the host genome. Three PR do-
mains and GKGY domain of IN (Peterson-Burch and
Voytas 2002) were found in GBRE-1(Fig. 3b, c). RVT is
responsible for the generation of cDNA copy of the
retroelement from genomic mRNA (Telesnitsky and Goff
1997) and is the most conserved retroelement coding re-
gion (Xiong and Eickbush 1988). The RVT contained
seven conserved motifs in other retrotransposons (Fig. 3d)
(Xiong and Eickbush 1990). The RH, containing conserved
residues D10, E48, D70, and D134 (Malik and Eickbush
2001), was also identified in GBRE-1(Fig. 3e). Interest-
ingly, the end of ORF overlapped with 30LTR, sharing six
base pairs of TGTTAG (Fig. 2a), which has not yet been
reported in other retrotransposons.
Phylogenetic relationship of GBRE-1with other
retrotransposons
To investigate the evolutionary relationship between
GBRE-1and other similar retrotransposons from plants, a
phylogenetic tree was constructed based on the deduced
amino acid sequences of the RVT domain in GBRE-1and
other retrotransposons (Fig. 4). GBRE-1formed a cluster
with Art 1 of A. thaliana and radi5fr of Raphanus sativus
(Ito et al. 2013) firstly, and then grouped with Tpv2-6of
Phaseolus vulgaris (Garber et al. 1999).
GBRE-1had higher similarity to Art 1,radi5fr, and
Tpv2-6than others, with amino acid identities of 71.3, 70.4,
and 63.2 % in the conserved RVT domain, respectively.
The lower similarity of GBRE-1to other copia-type
retrotransposons was 36–45 % (Table 1). The radi5fr is a
member of ONSEN transposon family from Brassicaceae
(Ito et al. 2013). Comparative search of the RVT domain of
GBRE-1against NCBI protein sequence databases with
BLAST-P program showed the high similarities with 32
putative RVT sequences from ONSEN retrotransposon
family. This analysis indicated that GBRE-1was ho-
mologous with Art 1,ONSEN, and Tpv2-6, and all of them
belonged to a same super family.
Fig. 3 The features of the GBRE-1coding region. aThe RNA-binding motif of GAG. bThree motifs of protease. cThe GKGY domain of
integrase. dSeven motifs of reverse transcriptase. eRNase H domain
Fig. 4 Comparison of the reverse transcriptase encoded by GBRE-1
and other plant retrotransposons. Neighbor-joining tree based on 247
amino acids of conserved reverse transcriptase domain from GBRE-1
(labeled by black triangle) and other retrotransposons (GenBank
accession number see Table 1). Bootstrap values are indicated at the
branches
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Cis-regulatory motifs in the LTR of GBRE-1
To identify the putative cis-regulatory motifs participating
in the transcription, the LTR sequence of GBRE-1was
analyzed using the PlantCARE database (Lescot et al.
2002). A large number of light-responsive elements were
identifiedintheLTRofGBRE-1, including ACE, Box-4,
G-Box, and LAMP-element (Fig. 5b). Several stress-re-
sponsive elements, including LTR, TC-rich repeats, and
TCA-element, were also found (Fig. 5b). TCA-element is
a binding site of salicylic acid inducible proteins, which is
highly conserved in many stress-inducible genes (Pas-
tuglia et al. 1997). Many cis-regulatory motifs in this LTR
suggested that the GBRE-1promoter may play a core role
in response to light and defense. Besides, it showed that
such putative elements were significantly enriched in
higher frequency (P=0.05) (Fig. 5b, supplemental
Tables 1 and 2).
In addition, 7 CAAT-Box (1 in sense strand and 6 in
antisense strand) and an endosperm expression element
Skn-1 motif were found in the LTR (Fig. 5), indicating the
potential expression of GBRE-1. Moreover, 50UTR Py-rich
stretch (Daraselia et al. 1996) conferred high transcrip-
tional levels of GBRE-1retrotransposon.
Transcriptional activity of GBRE-1in G.hirsutum
The possible expression of GBRE-1in G.hirsutum was
studied by searching the Gossypium EST database for the
presence of GBRE-1transcripts. There were 5 hits recov-
ered (GenBank accession nos. DW495444, DW495445,
ES817858, EX165537, and DT464682) from G. hirsutum
ESTs, indicating that GBRE-1was transcribed in meris-
tematic region, root, stem, ovule, and fiber.
In order to verify the expression level of GBRE-1in G.
hirsutum, the transcriptional activity in different tissues of
G. hirsutum, including leaf, stem, petal, root, ovules at 10
and 18 days post anthesis (DPA), and fiber at 18 DPA,
was investigated by RT-PCR. The PCR was repeated
three times (supplemental Fig. 1). It was found that the
GBRE-1sequence was represented in the population of
total RNA, meaning that the retrotransposon was tran-
scribed in all of these tissues under normal plant condi-
tion (Fig. 6).
To examine the heat activation of GBRE-1in G. bar-
badense and G. hirsutum, its transcripts in the cotyledon
and in the leaf were measured by RT-PCR when they were
planted under heat-stressed (37 °C) and non-heat-stressed
(25 °C) conditions. The relative transcript abundance was
estimated by brightness of the band when the RT-PCR
products were visualized by agarose gel electrophoresis
(Fig. 7). In G. barbadense, no significant effect of heat
treatment on GBRE-1transcription was detected, while in
G. hirsutum, the levels of the retrotransposon transcripts
were showed significant increase under 37 °C treatment in
both cotyledon and leaf (Fig. 7).
GBRE-1in diploid ancestor genomes of tetraploid
cottons
When whole retrotransposon sequences including LTR and
ORF were used to query G. raimondii (taxid: 29730) and
G. arboreum (taxid: 29729), whole-genome shotgun contig
sequence (wgs) from NCBI to investigate the existence of
Table 1 Similarity of reverse
transcriptase sequences of
GBRE-1 to other plant
retrotransposon
Name GenBank accession number Host organism Group Amino acid
identity (%)
Art 1 Y08010 Arabidopsis thaliana copia 71.3
AtRE1 AB021265 Arabidopsis thaliana copia 38.9
BARE-1 Z17327 Hordeum vulgare copia 41.3
CIRE1 AM040263 Citrus sinensis copia 40.9
FaRE1 FJ871121 Fragaria x ananassa copia 43.7
Osser X69552 Volvox carteri copia 36.8
radi5fr JX975169 Raphanus sativus copia 70.4
RIRE1 D85597 Oryza australiensis copia 44.1
SORE-1 AB370254 Glycine max copia 42.9
Sto-4 AF082133 Zea mays copia 40.1
Ta1-3 X13291 Arabidopsis thaliana copia 40.1
Tnt 1 X13777 Nicotiana tabacum copia 41.3
Tpv2-6 AJ005762 Phaseolus vulgaris copia 63.2
Tto 1 D83003 Nicotiana tabacum copia 41.7
GE-gypsy DX395000 Gossypium exiguum gypsy 9.8
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GBRE-1, and no LTR-homologous sequences were de-
tected. However, a number of ORF-homologous sequences
were found. Removing the sequences with the query cov-
erage less than 60 %, G. arboreum had four times more
homologous sequences than G. raimondii (25 vs 6,
Table 2). The similarity of the best hits in G. arboreum was
slightly higher than that in G. raimondii (78 vs 75 %).
Fig. 5 The putative cis-regulatory motifs in the LTR of GBRE-1.
aThe positions of predicted cis-regulatory motifs. Each motif was
indicated by different colors.bFunctional categories, numbers of
promoter motifs, and significance analysis with randomly collected
cotton genomic sequences
Fig. 6 Transcriptional activity of GBRE-1in different tissues of G.
hirsutum demonstrated by RT-PCT. EF1agene transcripts were used
as an internal control. 1leaf, 2stem, 3petal, 4root, 510 dpa ovule, 6
18 dpa ovule, 718 dpa fiber
Fig. 7 Levels of GBRE-1transcripts in plants subjected to 25 °Cor
37 °C quantified by RT-PCR, with EF1agene transcripts as an
internal control. aEffect of heat stress and the relative transcript
abundance in cotyledon; beffect of heat stress and the relative
transcript abundance in leaf. The italic letters (aand b)inhistogram
indicated the significant difference of the relative abundance between
two transcripts
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However, when GBRE-1was blasted against the G. hir-
sutum TM1_Mira454_transcripts, an LTR sequence (Gen-
Bank accession no. GALV01020191) was retrieved with
the similarity of 99 % and coverage of 100 %. Although
the ORF did not match in whole length, three hits from G.
hirsutum TM1_Mira454_transcripts database (GenBank
accession nos. GALV01021973, GALV01037816, and
GALV01024046) showed very high similarity (99 %),
suggesting the existence of the same retrotransposon in G.
hirsutum.
To confirm the existence of LTR and reveal its sequence
diversity in different cotton species, the amplifications of a
fragment of 665 bp including RH-LTR were tried in 10
diploid and 2 tetraploid cotton species (Table 3) with EF1a
as a control to ensure the quality of DNA. No targeted
fragment was amplified in all tried accessions of diploid
A-genome species (4 G. herboceum and 2 G. arboreum),
while in D-genome species, the RH-LTR fragment was
successfully amplified in 4 out of 18 accessions (D3-k, D4,
D7 and D5). These 4 PCR products together with those
from H7124 and TM-1 were cloned into pMD18-T vector
and sequenced.
A total of 35 plasmids were sequenced, and the results
showed slight difference in RH region, which may be
caused by DNA sequencing error. Comparatively, LTR
region showed the more variation than RH. According to
four stable divergent sites, these sequences could be ap-
proximately divided into two groups, D3 and D7 as one
group, and D4, D5, H7124, and TM-1 as another group. In
addition, a 41 bp fragment appeared to be absent in two
sequences from D7 (Fig. 8a).
In accordance to the divergence mentioned above,
phylogenetic analysis showed two major clades, D3 and D7
as one, and D4, D5, H7124, and TM-1 as the other
(Fig. 8b). It was obvious that sequences from H7124 and
TM-1 formed a cluster with those from D4 and D5, which
meant that the GBRE-1from G. hirsutum and G. bar-
badense evolved from G. aridum or G. raimondii.
Table 2 GBRE-1 homolog amplified from two diploid ancestor species of tetraploid cotton
Locus Sequences in Gossypium raimondii Sequences in Gossypium arboreum
Chr. GenBank accession Contig From To Identity (%) GenBank accession Contig From To Identity (%)
1 ALYE01001082 1082 9981 14,081 73 AYOE01001919 3837 42,779 46,557 78
AYOE01002693 5385 81,012 85,096 75
2 ALYE01001885 481 14,903 18,995 75 AYOE01006555 5229 108,295 112,064 73
3 – – – – – AYOE01009349. 5157 20,807 24,902 76
4 ALYE01004155 185 42,319 45,732 73 AYOE01010696 2449 30,217 34,341 78
AYOE01011464 3985 187,487 191,585 77
AYOE01011483 4023 46,282 50,079 77
5 ALYE01006407 997 93,139 96,929 75 – – – – –
6 – – – – – AYOE01017064 4891 43,374 47,523 77
AYOE01015403 1569 112,832 116,930 77
7 – – – – – AYOE01018329 1179 16,294 19,312 76
AYOE01019096 2713 3661 7772 77
AYOE01019258 3037 67,138 71,241 75
8 – – – – – AYOE01022301 2463 20,059 23,635 76
AYOE01022642 3145 24,729 28,488 74
AYOE01023626 5113 43,341 47,453 76
9 – – – – – AYOE01025528 2109 73,436 77,550 77
AYOE01026397 3847 25,546 28,857 76
AYOE01026237 3527 58,871 61,887 77
10 ALYE01013011 274 86,220 89,333 75 AYOE01027109 95 43,313 46,592 78
AYOE01028528 2933 118,608 122,725 76
AYOE01028662 3201 14,321 18,185 76
AYOE01029679 5235 93,659 97,325 77
11– –––– – –– – –
12 ALYE01016254 545 8136 11,487 74 AYOE01033488 1169 39,136 43,262 77
AYOE01036718 7629 242,073 245,118 72
13 – – – – – AYOE01039852 6229 237,474 241,573 76
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Table 3 Amplification of the fragment RH-LTR in GBRE-1 from different Gossypium species
Species Genome Genome size (pg/1C) Accession/cultivar Geographical origin RH-LTR EF1a
G. herbaceum A1 1.705 A1-108 – 0 1
A1-111 – 0 1
A1-172 – 0 1
A1-180 – 0 1
G. arboreum A2 1.749 208083.1 – 0 1
A2-142 – 0 1
G. trilobum D8 0.87 D8-7 Mexico 0 1
D8-8 Mexico 0 1
G. klotzschianum D3-k 0.9 D3-k-57 Ecuador 0 1
D3-k-58 Ecuador 0 1
D3-k-59 Ecuador 1 1
G. armourianum D2-1 0.875 D2-1-7 Mexico 0 1
D2-1-9 Mexico 0 1
G. harknessii D2-2 0.93 D2-2-4 Mexico 0 1
G. turneri D10 0.93 D10-1 Mexico 0 1
G. aridum D4 0.94 D4-5 Mexico 1 1
G. lobatum D7 0.955 D7-4 Mexico 0 1
208082.07 Mexico 1 1
G. raimondii D5 0.9 D5-3 Peru 0 1
D5-6 Peru 0 1
D5-8 Peru 0 1
D5 American 1 1
G. hirsutum (AD)2 2.464 TM-1 China 1 1
G. barbadense (AD)2 2.505 H7124 China 1 1
0no band detected by agarose (1 %) gel electrophoresis, 1one targeted band
Fig. 8 Comparison of the RH-LTR sequences of GBRE-1from
different Gossypium species, amultiple alignment of RH-LTR
sequences which presented difference. Four stable divergent sites
were labeled by red arrows.bNeighbor-joining tree based on all
sequenced RH-LTR sequences. Bootstrap values are indicated at the
branches
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Discussion
Each cotton genome contains many transposable elements,
especially the retrotransposon (Hawkins et al. 2006), which
account for a large proportion of many other plant gen-
omes. Transposable elements have played important roles
in genome plasticity and evolution by bringing dramatic
changes in genome size, structure, and gene expression
(Hawkins et al. 2006; Kumar and Bennetzen 1999; Zedek,
et al.). GBRE-1, a Ty1/copia-like LTR retrotransposon
identified in the genome of G. barbadense, is an ideal
example that illustrates the functions of transposable ele-
ment in speciation and domestication of cotton. In the
current study, G. arboreum and G. raimondii were found to
have considerable difference in numbers of GBRE-1ORF
homolog, with the numbers of GBRE-1ORF homolog in G.
arboreum being four times that of G. raimondii. This result
was in accordance to the genome size variation between G.
arboreum and G. raimondii (1697 Mb vs 885 Mb). The
best G. arboreum hit of GBRE-1ORF has higher similarity
than that of G. raimondii (78 vs 75 %), suggesting that this
retrotransposon is still active, at least the latest activity in
G. arboreum occurred more frequently than in G. rai-
mondii. Our finding also supported the hypothesis that the
Gossypium genome size variation is largely attributable to
the insertion and accumulation of transposable elements
(Hawkins et al. 2006).
During the evolutionary process, LTRs play critical
roles in bringing retrotransposons to jump around. The
detected expression of GBRE-1in G. hirsutum and G.
barbadense under normal plant condition (Figs. 6,7)
suggests that GBRE-1is a transcriptionally active retro-
transposon which is ubiquitous in all tissues types of cot-
ton. In addition, the abundant transcriptional regulatory
elements existed in the LTR also suggest that this LTR is a
functionally active promoter.
Besides the function of LTR, the activity of a retro-
transposon also depends on environment. A few studies
indicated that the retrotransposons have adapted to their
host genomes through the evolution of promoters that
mimic the stress-induced plant genes (Beguiristain et al.
2001). For example, Tnt1 subfamilies in Nicotiana taba-
cum showed different stress-associated expression patterns
(Beguiristain et al. 2001). In addition, the expression of
Retrolyc1 subfamilies in two different Lycopersicon spe-
cies was defined by their LTR regulatory regions (Araujo
et al. 2001). Similarly, the existence of stress-responsive
elements in the GBRE-1LTR indicates that its transcrip-
tional level might be regulated by the LTR under the en-
vironmental stresses, such as high temperature, salinity,
drought, salicylic acid, UV, and pathogens. A cis-acting
element annotated to be related to low-temperature re-
sponsiveness existed in LTR of GBRE-1indicates that the
activation of GBRE-1may be due to the temperature stress.
This inference has been supported indirectly by the current
results from G. barbadense and G. hirsutum (Fig. 7). The
expressions of GBRE-1in two tetraploid species were
different after 37 °C treatment. The transcriptional level of
GBRE-1was increased by heat treatment in G. hirsutum,
but not in G. barbadense. It was predicted that the retro-
transposons in these two species might be from two sub-
families and also be different in LTR regions. However,
sequencing results revealed no significant difference be-
tween their LTR sequences, indicating that a retrotrans-
poson might have different performance in different cotton
species, or microenvironment of cultivars might affect its
activity when responding to stress. According to the ana-
lysis of LTR sequences, GBRE-1could be approximately
divided into two groups (Fig. 8), and their differences have
been formed in diploid cotton. The abundant light-re-
sponsive cis-regulatory elements are identified in the LTR
of GBRE-1, which may be responsible for adapting to light
changes from low latitudes to high latitudes during the
propagation of the host cotton. These elements were related
to stress and light, conferring the ancient retrotransposon
high adaptability to the environment.
The effect of GBRE-1insertion within HD1 gene on
trichome growth and its different activities in response to
heat stress observed in two tetraploid cotton species are
reminiscent of what was found for ONSEN family in
Brassicaceae (Ito et al. 2013). ONSEN is a heat-stressed
activated LTR retrotransposon (Ito et al. 2011). Its ex-
pressions under heat condition in several Brassicaceae
species were also different (Ito et al. 2013). ONSEN in-
sertion can trigger gene network modification (Ito et al.
2011,2013). In our work, phylogenetic analysis showed
that GBRE-1was highly close to the members of ONSEN
retrotransposon family (Fig. 4). All these suggest that
GBRE-1is a very important retrotransposon like ONSEN
and worthy of further study. The regulatory mechanism of
LTR promoter responding to different stresses is also an-
other interesting research issue in next stage.
Author contribution statement Yuefen Cao did the
most of the relevant experiments and data analysis. Yurong
Jiang analyzed the GBRE-1transcriptional activity in G.
hirsutum. Mingquan Ding designed primers. Shae He dis-
covered the insertion in HD1 gene and cloned its sequence
firstly. Hua Zhang cloned the LTR region. Junkang Rong
leads the group and wrote the paper.
Acknowledgments We thank Jing Xu and Yuting Zhang for help
with RNA extraction. This work was supported by the Genetically
Modified Organisms Breeding Major Projects (2009ZX05009-028B),
National Natural Science Foundation of China (31301372) to
Y. Jiang, Initial Foundation of ZAFU (2010FR042), and State
Key Laboratory of Cotton Biology Open Fund (CB2014B04) to
J. Rong.
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Conflict of interest The authors declare that they have no conflict
of interest.
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