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ORIGINAL PAPER
Drought-Responsive Hsp70 Gene Analysis in Populus
at Genome-Wide Level
Esra Nurten Yer
1
&Mehmet Cengiz Baloglu
2
&Ummugulsum Tanman Ziplar
3
&
Sezgin Ayan
1
&Turgay Unver
3
#Springer Science+Business Media New York 2015
Abstract The heat shock protein 70 (Hsp70) family members
are known as molecular chaperones. They play a crucial role
in protecting plant cells and tissues from thermal or abiotic
stress through protein folding and in assembly, stabilization,
activation, and degradation processes. Although many studies
have been performed to identify molecular functions of indi-
vidual family members, there is a limited study on genome-
wide identification and characterizations of Hsps in the
Populus model tree genus. We have identified 34 poplar
Hsp70 genes, which were phylogenetically clustered into
three major groups. Gene structure and motif composition
are relatively conserved in each group. Mainly tandem and
infrequently segmental duplications have a significant role in
poplar Hsp70 gene expansion. The in silico microRNA
(miRNA) and target transcript analyses identified that a total
of 19 PtHsp70 genes were targeted by 27 plant miRNAs.
PtHSP70-14 and PtHSP70-33 are the most targeted by
miR390 and miR414 family members, respectively. For de-
termination of drought response to Hsp70 genes, publicly
available RNA-seq data were analyzed. Poplar Hsp70s are
differentially expressed upon exposure to different drought
stress conditions. Expression analysis of PtHsp70 genes was
also examined under drought stress in drought-sensitive and
drought-resistant Populus clones with quantitative real-time
PCR (qRT-PCR). PtHsp70-16 and PtHsp70-26 genes might
provide adaptation to drought stress for both clones. Because
of high expression responses to drought in only resistant
Populus clone, PtHsp70-25 and PtHsp70-33 genes might be
used for determination of drought-tolerant clones for molecu-
lar breeding studies. This research provides a fundamental
clue for contribution of PtHsp70s to drought tolerance in
poplar.
Keywords Hsp70 .Genome-wide analysis .Phylogenetic
relationships .Gene expression analysis .Drought stress .
Populus trichocarpa
Introduction
Populus (poplar) is a vital genus of trees. Because of their
broad dispersion and usage as a source for paper production
and as a bioenergy source supported by rapid growth and high
genetic diversity, poplar plays a crucial role in economy and
ecosystem (Jansson and Douglas 2007). The species of
Populus presents splendid opportunities to examine stress re-
sponses toward drought which affect not only survival but
also biomass accumulation (Marron et al. 2002; Monclus
et al. 2006). In recent years, there has been an increasing
interest in genotype, transcriptome, and drought response re-
lations in Populus trees (Caruso et al. 2008;Songetal.2012).
Black cottonwood (Populus trichocarpa Torr. & Gray) is a
woody deciduous plant, which lives for long years and culti-
vated basically in western North America. It is the first tree
species whose genome was sequenced and published (Tuskan
et al. 2006). When economic importance of wood and wood
Electronic supplementary material The online version of this article
(doi:10.1007/s11105-015-0933-3) contains supplementary material,
which is available to authorized users.
*Mehmet Cengiz Baloglu
mcbaloglu@gmail.com
1
Silviculture Department, Faculty of Forestry, Kastamonu University,
Kastamonu, Turkey
2
Department of Genetics and Bioengineering, Faculty of Engineering
and Architecture, Kastamonu University, 37100 Kastamonu, Turkey
3
Department of Biology, Faculty of Science, CankırıKaratekin
University, Cankiri, Turkey
Plant Mol Biol Rep
DOI 10.1007/s11105-015-0933-3
products is considered, availability of the poplar genome plays
an important role for molecular processes of growth, ad-
vances, and responses toward environmental changes seen in
trees. The poplar genome also providesa valuable information
for evolutionary comparisons between herbaceous and woody
plants.
A number of researchers (Schlesinger 1990; Schöffl et al.
1998; Kotak et al. 2007;Lund2001; Kampinga and Craig
2010) have reported that heat shock proteins (Hsps) are clas-
sified into five major families based on functions and molec-
ular mass, viz., small Hsps, Hsp60, Hsp70, Hsp90, and
Hsp100. They are highly conserved and well characterized
in a few model plants such as the Arabidopsis, rice, and poplar
(Hu et al. 2009; Krishna and Gloor 2001;Hilland
Hemmingsen 2001; Scharf et al. 2001; Lee et al. 2007;
Zhang et al. 2013). Hsps play a crucial role in maintaining
protein homeostasis, behaving as molecular chaperones and
supporting protein refolding when stress conditions are avail-
able (Vierling 1991;HendrickandHartl1993;Bostonetal.
1996;Hartl1996; Waters et al. 1996; Török et al. 2001). In
recent years, Zhang et al. (2014)showedthatmanyHsp70
genes may play important roles in fiber development process-
es including fiber initiation and elongation in cotton. This
makes Hsps a valuable resource for researchers studying their
response of different stress conditions, their functions for
protecting plants against abiotic stresses, and for development
mechanisms.
Recently, researchers have shown an increased interest in
functional analysis of Hsp70 family members in different or-
ganisms such as Arabidopsis and rice (Lin et al. 2001; Sung
et al. 2001;Wangetal.2014; Sarkar et al. 2013;Jungetal.
2013). Although Hsp90 gene family members in poplar were
identified (Zhang et al. 2013), other Hsps including Hsp70s
still have not been fully characterized in poplar and little is
known about their interactors (Jung et al. 2013). Furthermore,
existing research studies recognize the critical role of Hsp70
genes which enhanced the plant’s tolerance to environmental
stresses. Knockout mutations in Arabidopsis stromal 70-kD
heat shock proteins (cpHSC70-1 and cpHSC70-2)causedfor-
mation of defective phenotypes and decrease in thermo-
tolerance of germinating seeds (Su and Li 2008). A recent
study by Jungkunz et al. (2011) involved in generation of
AtHSP70-15 gene deficient Arabidopsis plants. This resulted
in drastic increase in mortality after heat treatment. So, it can
be concluded that AtHSP70-15 plays an essential role for heat
response. In the same study, overexpression of AtHSP70-1
leads to increase in stress tolerance in Arabidopsis (Jungkunz
et al. 2011). A similar series of experiments to show that
alternation in expression of the Arabidopsis thaliana
cytosolic/nuclear HSC70-1 molecular chaperone directly in-
fluenced development and abiotic stress tolerance was per-
formed by Noel et al. (2007). In another major study, BIP gene
(encoding Hsp70) from A. thaliana was responsible for
development of female gametophyte (Maruyama et al. 2010)
and the same gene in Nicotiana tabacum protected plant
against water stress (Alvim et al. 2001). Researchers conduct-
ed a series of studies related with BIP1/OsBIP3 gene functions
in rice. This gene regulated XA21-mediated immunity (Park
et al. 2010), seed development (Wakasa et al. 2011), and pro-
grammed cell death (Qi et al. 2011). However, the biological
functions of many Hsp70 family members have not yet been
identified in many organisms including poplar.
Omics technologies are very helpful for detection of new
genes and determination of their function (Feist and Palsson
2008). Although the recent developments for gene discovery
studies havesignificantly increased,there is little known about
the genome-wide survey and expression patterns of Hsp70
gene family in poplar. The genome-wide analysis and identi-
fication studies from Arabidopsis (Lin et al. 2001;Sungetal.
2001), rice (Sarkar et al. 2013;Jungetal.2013), cotton
(Zhang et al. 2014), and poplar (Zhang et al. 2015) are a few
examples for Hsp genes. Due to various factors, including a
relatively small genome size, fast-growing industrial materials
and the release of the latest Populus trichocarpa genome se-
quence data, v3.0, gave us an opportunity to identify and
further analyze the poplar Hsp70 gene family. Although the
poplar Hsf and Hsp gene families were previously identified
on a genome-wide level (Zhang et al. 2013; Zhang et al.
2015), we made a detailed study for identification, compari-
son, functionally characterization, and expression analysis of
Hsp70 genes in poplar. In addition, there is a limited study on
genome-wide identification and characterizations of Hsps in
the poplar as well as other plants’genome. Therefore, this
study makes a major contribution to research on function of
Hsp family members. Here, a comprehensive set of Hsp70
genes (34) was identified based on the complete genome se-
quence of poplar. Subsequently, chromosomal localization,
motif analysis, exon–intron organization, homology, and phy-
logenetic analysis were also investigated. Finally, we exam-
ined the expression patterns of Hsp70 family members from
the publicly available transcriptome data and experimental
data. This research serves as a base for future studies and
provides a fundamental clue for exploration into the functions
of this significant gene family. In addition, identified genes
presented here can be used for cloning studies in agricultural
applications
Materials and Methods
Analysis and Identification of Poplar Hsp70 Genes
Different Hsp70 protein members (about 259 amino acid se-
quences) from diverse organisms (A. thaliana,Cucumis
sativus,Glycine max,Hordeum vulgare,Medicago
truncatula,N. tabacum,Oryza sativa,Physcomitrella patens,
Plant Mol Biol Rep
Ricinus cummunis,Solanum lycopersicum,Sorghum bicolor,
Triticum aestivum,Vigna radiate,Vitis vinifera,andZea
mays) at Heat Shock Protein Database Information Resource
(http://pdslab.biochem.iisc.ernet.in/hspir/index.php) (Kumar
et al. 2012) were downloaded to identify potential members
of Poplar Hsp70 proteins. BLASTP at PHYTOZOME v10.3
database (www. phytozome.net) and The Hidden Markov Mod-
el (HMM) search at Pfam database (http://pfam.sanger.ac.uk)
were performed against the poplar genome with default
parameters (Goodstein et al. 2012). Identified poplar HSP70
proteins were also used as query in NCBI BLASTP for
characterization of hypothetical or uncharacterized
proteins in poplar. Redundant sequences were removed
using the decrease redundancy tool (web.expasy.org/
decrease_redundancy). Each non-redundant sequence
was again analyzed to check the presence of Hsp70 do-
mains by SMART (http://smart.emblheidelberg.de)
(Letunic et al. 2012)andPfam(http://pfam.sanger.ac.uk)
searches. Theoretical isoelectric points (pI), molecular
weights, and instability index were calculated using
ProtParam Tool (http://web.expasy.org/protparam).
Physical Location, Gene Structure Classifications,
and Analysis of Poplar Hsp70 Proteins
Specific chromosomal locations, intron numbers, and sizes (bp)
were determined by Phytozome database. The PtHsp70 genes
were plotted on all poplar chromosomes from the short-arm
telomere to the long-arm telomere and finally visualized with
MapChart (Voorrips 2002). Segmental and tandem duplica-
tions were determined by Plant Genome Duplication Database
(PGDD; http://chibba.pgml.uga.edu/duplication/index/blast)
(Tang et al. 2008). In detail, BLASTP search was performed
against all predicted Hsp70 proteins of Populus trichocarpa,
and the first five matches with ≤1e-05 was considered as
potential anchors. Collinear blocks were evaluated by
MCScan, and alignments with ≤1e-10 were selected as
important matches (Tang et al. 2008;Duetal.2013). Tandem
duplications were also characterized as adjacent genes of same
subfamily located within 10 predicted genes apart or within
30 kbp of each other (Du et al. 2013; Shiu and Bleecker
2003). The exon–intron analysis of the PtHsp70 proteins was
generated by Gene Structure Display Server (GSDS) software
(gsds.cbi.pku.edu.cn) (Guo et al. 2007). The coding sequences
and genome sequences were used for prediction of gene struc-
ture of the poplar Hsp70 genes.
Phylogenetic Analysis and Identification of the Conserved
Domains
Phylogenetic analysis was conducted using the neighbor-
joining method with bootstrap analysis for 1000 iterations.
Multiple sequence alignments corresponding to conserved
motif regions, characteristic of the Hsp70 protein members,
were determined by ClustalW with a gap open and gap exten-
sion penalties of 10 and 0.1, respectively (Thompson et al.
1997). The alignment file was firstly imported into MEGA5
(Tamura et al. 2011) and used to construct an unrooted phy-
logenetic tree.
The Multiple EM for motif elicitation (MEME)
(http://meme.nbcr.net/meme3/meme.html) (Bailey et al.
2006) was used to identify motifs in candidate se-
quences. The parameters for the analysis were as fol-
lows: number of repetitions, any; maximum number of
motifs, 20; and optimum width of motif, ≥2and≤300.
Discovered MEME motifs with ≤1e-30 were searched in
the InterPro database with InterProScan (Quevillon et al.
2005).
GO Annotation
The functional annotation of Hsp70 protein sequences and the
analysis of annotation data were performed by using
Blast2GO (http://www.blast2go.com) (Conesa and Götz
2008). First, all identified PtHsp70 amino acid sequences
were introduced into Blast2GO program. Then, functional
annotation was achieved in three steps: (i) BLASTp to find
homologous sequences, (ii) MAPPING to retrieve GO terms
related with the BLAST results, and (iii) ANNOTATION of
Gene Ontology (GO) terms for selection reliable functions to
given amino acid sequences. The program provides the output
defining three categories of GO classification, namely, biolog-
ical processes, cellular components, and molecular functions.
Comparative Physical Mapping of Hsp70 Protein
Member Between Poplar and Other Species
For identification of orthologous relationship between pop-
lar PtHsp70 amino acid sequences and Hsp70s from four
species including Arabidopsis, rice, maize, and grape,
BLASTP search was conducted in Phytozome database
(www.phytozome.net). Hits with ≤1e-5 and at least 80 %
identify were considered significant. Orthologous Hsp70
genes among poplar, Arabidopsis, rice, maize, and grape
were placed on corresponding species chromosomes which
were finally visualized with MapChart.
Estimating the Rates of Synonymous
and Non-synonymous Substitution
For estimation of the synonymous (Ks) and non-synonymous
(Ka) substitution rates, the amino acid sequences belonging to
duplicated protein-encoding PtHsp70 protein members and
orthologous gene pairs between poplar and Arabidopsis,rice,
maize, and grape were firstly aligned with CLUSTALW based
on multiple sequence alignment tool. Then, PAL2NAL
Plant Mol Biol Rep
program (http://www.bork.embl.de/pal2nal) (Suyama et al.
2006) was used for alignment of the amino acid sequences
and their respective original complementary DNA (cDNA)
sequences of PtHsp70 genes. This program converts a multiple
sequence alignment of proteins and the corresponding DNA se-
quences into a codon alignment and finally estimated the synon-
ymous (Ks) and non-synonymous (Ka) substitution rates. Time
(million years ago, Mya) of duplication and divergence of each
Hsp70 genes were also calculated with a formula as T=Ks/2λ
(λ=6.5 × 10 e-9) (Lynch and Conery 2000;Yangetal.2008).
In Silico Analysis of miRNA Targets in PtHsp70 Genes
MicroRNA (miRNA) target analysis helps to understand
miRNA regulatory mechanisms. Previously known plant
pre-miRNA sequences obtained from miRBase v20.0
(http://www.mirbase.org) and plant miRNA database
(http://bioinformatics.cau.edu.cn/PMRD) were utilized
for identification of miRNAs targeting the PtHsp70
genes. Poplar Hsp70 gene targets and plant miRNAs were
characterized by aligning them using the web-based psRNA
Target Server (http://plantgrn.noble.org/psRNATarget) with
default parameters. Alignment between all known plant
miRNAs and its PtHsp70 gene target(s) were evaluated by
the parameters described by Zhang (2005). Further analysis
of the computationally identified miRNA targets were
performed by BLASTX searches with ≤1e-10 against poplar
EST sequences at NCBI database for identification and
confirmation of putative gene homologous.
Homology Modeling of Hsp70 Proteins
All poplar Hsp70 protein sequences were scanned at Pro-
tein Data Bank (PDB) (Berman et al. 2000)byusing
BLASTP to determine the similar sequence and known
best sample which have three-dimensional structure.
Phyre2 database (Protein Homology/Analogy Recognition
Engine; http://www.sbg.bio.ic.ac.uk/phyre2) was used for
prediction of 3D protein structure of Hsp70 proteins
(Kelley and Sternberg 2009). Predicted protein structures
of poplar Hsp70s were evaluated in terms of confidence
level (>90 %) and percentage residue level (80 to 100).
Expression Analysis of the PtHsp70 Genes
in Transcriptome Data
All Illimuna HiSeq readings and Roche454 RNA-Seq
data were obtained from Sequence Read Archive (SRA;
http://www.ncbi.nlm.nih.gov/sra) database at the NCBI
under the following accession numbers: (i) SRP005997 (ex-
periment accession nos. SRX047542 for the control and
SRX047543 for the treatment), (ii) SRP033028 (experiment
accession nos. SRX377987 for the control and SRX472726
for the treatment), and (iii) SRP024267 (experiment accession
nos. SRX297950, SRX297104, SRX29795, and SRX297952
for the control; SRX297957, SRX297955, SRX297954, and
SRX297953 for the moderately dehydrated leaves; and
SRX297964, SRX297963, SRX297962, and SRX297961
for severely dehydrated leaves) (Tang et al. 2013;Tangetal.
2014; Cossu et al. 2014). All readings were downloaded in
raw sequencing data as B.sra^format and converted to Bfastq^
format for Illimuna and Bsff^format for Roche 454 by the
NCBI SRA Toolkit’s fastq-dump command. After discarding
low-quality readings (Phred quality (Q) score <20) and trim-
ming adapters by using FASTX toolkit, all clean readings
were subjected to FastQC analysis for checking reading qual-
ities in terms of per-base sequence qualities, per-sequence
quality scores, per-base nucleotide content, and sequence du-
plication levels. The raw count data were transformed and
normalized using CLC Genomic Workbench version 7.5.
Then, gene expression measurement and hierarchical cluster-
ing heat map were constructed based on log2 RPKM values
by PermutMatrix software (Caraux and Pinloche 2005).
Plant Materials, Growth Conditions, and Treatments
Poplar clones were kindly obtained from Behiçbey Forest
Nursery (Ankara Regional Directorate of Forestry, Ankara,
Turkey). Physiological, morphological, and biochemical re-
sponses of Populus nigra clones to drought stress were previ-
ously evaluated. Clone R and Clone S poplar clones were
determined as drought resistant and drought sensitive, respec-
tively (Yildirim 2013). Plant materials were collected from
nursery clonally propagated 1-year-old Populus nigra clones,
Clone R (drought resistant) and Clone S (drought sensitive),
grown undera natural photoperiod, humidity, and temperature
in the field. These Populus clones were used as a single ma-
ternal plant. Rooted cuttings from these maternal Populus
Clone R and Clone S were cultivated in 20×20 cm
2
pots
containing packaged potting soil, peat moss, and vermiculite
(2:2:1, v/v) in the greenhouse, under controlled environmental
conditions (25 °C day/20 °C night, 16-h light/8-h dark photo-
period) with relative humidity from 55 to 80 %. The trees were
well watered until the drought treatments began. In other
words, they were watered to reach field capacity every day
until uniformly developed trees (with 80–100 cm in height)
were obtained for the water stresstreatments. For control, trees
were normally watered by one to three waterings per day to
compensate field capacity. For drought stress application, soil
relative extractable water (REW) was controlled by water sup-
ply four times a day. Soil REW was maintained for 10 days as
a drought stress. Fully expanded leaves at approximately 6–10
internodes from apex from drought-resistant and drought-
sensitive clones were harvested at 10 days of control and
drought-stress-treated trees (Supplementary Fig. S1). The ex-
perimental design was as follows: 3 clones (biological
Plant Mol Biol Rep
replicates) × 2 treatments (control and drought stress) × 2
clones (drought resistant and drought sensitive).
RNA Isolation and Quantitative Real-Time PCR Analysis
About 150–200 mg leaf samples from control and stress-
treated trees was homogenized with liquid nitrogen. Three
milliliters of pre-heated extraction buffer (2 % [w/v]CTAB,
2%[w/v] PVP, 100 mM Tris/HCl pH 8.0, 25 mM EDTA, 2 M
NaCl, 0.5 g/L spermidine, 2.7 % [v/v] 2-ME) was mixed with
the frozen leaf powder and incubated at 65 °C for 10 min. Two
separate extraction steps with 3 mL ice-cold chloroform/
isoamylalcohol (24:1, v/v) were performed. A total of 0.25 vol-
umes of ice-cold10 M LiCl was added to precipitate RNA and
incubated at 4 °C for 18 h. After centrifugation at 16,000×g,
4 °C, 60 min, the pellets were incubated with 4 mL ice-cold
75 % ethanol at −80 °C for 60 min, followed by centrifugation
at 16,000×g, 4 °C, 20 min. Ethanol was removed from tubes.
Pellets were dried and dissolved in RNase-free water. RNA
concentrations and integrity were determined using a
Multiskan™GO Microplate Spectrophotometer (Thermo
Fisher Scientific, Waltham, MA, USA) and agarose gel elec-
trophoresis, respectively. DNA contamination in samples was
removed with DNase I (Fermentas, Thermo Fisher Scientific,
Waltham, MA, USA) according to the manufacturer’s
instructions.
To examine expression profiles of 13 members of Hsp70
genes in leaf tissues of control and drought-stress-treated pop-
lar clones, quantitative real-time PCR (qRT-PCR) was con-
ducted as previously reported (Turktas et al. 2013)using
SYBR Green I Master Kit (Roche, Germany) on LightCycler
480 Instrument II (Roche, Germany). Primers for the PtHsp70
genes were designed considering the conserved regions of
Hsp70 members. A list of the primers used in qRT-PCR is
presented in Supplementary Table S9. The qRT-PCR was car-
ried out in 96-well optical plates. PCR reactions were per-
formed in a total volume of 20 μL containing 0.1 μLreverse
and forward primers (100 pmol), 2 μL of cDNA, and 10 μL
FastStart SYBR Green I Master Mix, and nuclease-free water
was added up to 20 μL. The 18S rRNA gene was used as the
internal control (Wang et al. 2010; Budak et al. 2013). The
qRT-PCR conditions were set up as follows: preheating at
95 °C for 5 min, followed by 55 cycles of 95 °C for 10 s, 53
or 55 °C for 20 s, and 72 °C for 10 s. The melting curves were
adjusted to 95 °C for 5 s and 55 °C for 1 min and then cooled
to 40 °C for 30 s. All reactions were repeated three times with
triple biological replicates. The expression levels were calcu-
lated as the mean signal intensity across the three replicates.
Relative gene expression was calculated using ΔΔCT values
obtained from the formulas ΔCT=CT target−CT reference
and ΔΔCT=ΔCT treated sample−ΔCT untreated sample
(0-h treatment). For all chart preparations, selected RNA rel-
ative amount was evaluated for gene expression level using
the 2
−
ΔΔCT (Livak and Schmittgen, 2001; Baloglu et al.
2014a). In addition, the standard errors of mean among repli-
cates were calculated. Student’sttest was used to obtain the
statistical significance of the difference between treated sam-
ples and untreated samples (0-h treatment under abiotic
stress). If Pvalues <0.01, we considered the PtHsp70 genes
as differentially expressed genes.
Results and Discussion
Characterization of Hsp70 Protein Coding Sequences
in Poplar
Hsp70 protein sequences belonging to 15 plant genomes—
A. thaliana,C. sativus,G. max,H. vulgare,M. truncatula,
N. tabacum,O. sativa,Physcomitrella patens,R. cummunis,
Solanum lycopersicum,Sorghum bicolor,T. aestivum,Vig n a
radiate,Vitis vin i f era,andZ. mays—were used as queries for
identification of putative poplar Hsp70 genes. We performed
BLAST, hidden Markov model (HMM), and keyword query-
ing searches in relevant databases. Hsp70-related domains
were searched in Pfam and SMART databases for validation
of presence of them. After removingredundant sequences, we
identified 34 putative PtHsp70 genes in the genome of
Populus trichocarpa (Table 1). For convenience, the Hsp70
genes were named from PtHsp70-01 to PtHsp70-34 based on
scientific name of poplar (Populus trichocarpa) and ordered
on the chromosomes from 1 to 19. The particularization of
poplar Hsp70 proteins is listed in Table 1which includes
number of amino acids (length), molecular weight, isoelectric
point (PI), and NCBI annotation. According to the detailed
information, the lengths of PtHsp70 protein sequence ranged
from 99 residues (PtHsp70-30) to 972 residues (PtHsp70-07),
while the isoelectric point (pI) ranged from 4.77 (PtHsp70-22)
to 9.94 (PtHsp70-05).
The protein sequences in the representative genomes of 15
plant species was searched for comparative genomic analyses.
A total of 259 genes encoding Hsp70 proteins were identified
in these selected plant species. The density of PtHsp70 is
about 0.0804 which is higher than in most of the analyzed
plants. A. thaliana (0.2889) and O. sativa (0.0941) are plant
species that showed the highest density when com-
pared to ratio of the number of PtHsp70 to genome size
(Supplementary Table S1). Although individual Hsp70 genes
have been identified in different plant species such as maize
(Rochester et al. 1986), barley (Chen et al. 1994), and pea
(Dhankher et al. 1997), identification of this family genes on
genomic level has been firstly performed in Arabidopsis which
contains 18 Hsp70 genes (Lin et al. 2001;Sungetal.2001). In a
recent study, characterization of 32 Hsp70 genes from rice was
announced (Rouard et al. 2011;Sarkaretal.2013;Jungetal.
2013). We also found similar gene numbers in poplar genome
Plant Mol Biol Rep
Tab l e 1 A catalog of 34 Poplar Hsp70 proteins
ID Phytozome
identifier
Physical position on poplar genome Protein
length
(aa)
pI Molecular
weight
(Da)
Instability
index
Stable or
unstable
Phylogeny
group
NCBI
Accession
No.
NCBI BLASTP
annotation
Score E-value
Chromosome Start
position
(bp)
End
position
(bp)
PtHsp70-1 Potri.001G042600.1 Chr01 3,098,600 3,100,784 655 5.40 71881.3 34.33 Stable III c XP_002332067.1 Predicted protein
[Populus trichocarpa]
1347 0.0
PtHsp70-2 Potri.001G042700.1 Chr01 3,102,549 3,104,642 655 5.34 71903.3 34.49 Stable III c XP_002332049.1 Predicted protein
[Populus trichocarpa]
1350 0.0
PtHsp70-3 Potri.001G087500.1 Chr01 6,920,967 6,924,312 666 5.05 73510.3 29.46 Stable III c XP_002299448.1 Bip isoform A
family protein
[Populus trichocarpa]
1352 0.0
PtHsp70-4 Potri.001G180100.1 Chr01 1,558,9912 15,595,439 852 5.24 94160.8 42.92 Unstable III b XP_002299641.1 Heat shock protein 70
[Populus trichocarpa]
1758 0.0
PtHsp70-5 Potri.001G285100.1 Chr01 2,916,1925 29,164,031 282 9.94 30559.3 41.80 Unstable II XP_002300314.2 Hypothetical protein
POPTR_0001s29170g
[Populus trichocarpa]
580 0.0
PtHsp70-6 Potri.001G285500.1 Chr01 2,917,7059 29,180,511 683 5.56 73209.9 35.91 Stable II XP_002300311.2 Heat shock protein 70
[Populus trichocarpa]
1389 0.0
PtHsp70-7 Potri.001G289800.2 Chr01 2,956,3381 29,577,092 972 6.55 111276.3 61.34 Unstable I XP_002300284.2 Hypothetical protein
POPTR_0001s29710g
[Populus trichocarpa]
2010 0.0
PtHsp70-8 Potri.002G098500.1 Chr02 7,108,528 7,109,134 184 8.15 20434.6 42.11 Unstable II XP_002301061.2 Hypothetical protein
POPTR_0002s09870g
[Populus trichocarpa]
385 5e-137
PtHsp70-9 Potri.003G006300.1 Chr03 522,434 526,644 706 5.24 75343.9 29.89 Stable I XP_002331133.1 Predicted protein
[Populus trichocarpa]
1418 0.0
PtHsp70-10 Potri.004G224400.1 Chr03 522,434 526,644 766 5.72 82079.7 32.90 Stable I XP_006385039.1 Stromal 70-kDa heat
shock-related
family protein
[Populus trichocarpa]
1554 0.0
PtHsp70-11 Potri.003G055800.1 Chr03 8,248,685 8,254,158 858 5.32 94625.4 39.98 Stable III b XP_002304187.1 Heat shock protein 70
[Populus trichocarpa]
1778 0.0
PtHsp70-12 Potri.003G143600.1 Chr03 1,600,0920 16,004,484 666 5.10 73466.2 29.28 Stable III c XP_002303672.1 BiP isoform A
family protein
[Populus trichocarpa]
1353 0.0
PtHsp70-13 Potri.003G184000.1 Chr03 1,902,5261 19,027,423 651 8.16 72153.2 31.23 Stable III c XP_002303859.2 Heat shock protein
70 cognate
[Populus trichocarpa]
1343 0.0
PtHsp70-14 Potri.004G016700.2 Chr04 1,118,346 1,123,819 757 5.09 85169.2 42.62 Unstable III b XP_002305580.2 Hypothetical protein
POPTR_0004s01640g
[Populus trichocarpa]
1562 0.0
PtHsp70-15 Potri.006G022100.1 Chr06 1,560,362 1,567,426 899 5.34 100119.3 38.96 Stable III b XP_002308826.1 Hypothetical protein
POPTR_0006s02290g
[Populus trichocarpa]
1821 0.0
PtHsp70-16 Potri.008G054000.1 Chr08 3,189,756 3,192,879 648 5.14 71265.7 34.29 Stable III c XP_002311161.1 Heat shock protein 70
[Populus trichocarpa]
1336 0.0
Plant Mol Biol Rep
Tab l e 1 (continued)
ID Phytozome
identifier
Physical position on poplar genome Protein
length
(aa)
pI Molecular
weight
(Da)
Instability
index
Stable or
unstable
Phylogeny
group
NCBI
Accession
No.
NCBI BLASTP
annotation
Score E-value
Chromosome Start
position
(bp)
End
position
(bp)
PtHsp70-17 Potri.008G054600.1 Chr08 3,220,568 3,223,132 648 5.09 71173.6 32.77 Stable III c XP_002312089.1 Heat shock protein
70 cognate
[Populus trichocarpa]
1334 0.0
PtHsp70-18 Potri.008G054800.1 Chr08 3,232,998 3,235,127 482 5.86 54080.1 47.53 Unstable III c XP_002312091.1 Shock protein 70 cognate
[Populus trichocarpa]
1005 0.0
PtHsp70-19 Potri.008G054900.1 Chr08 3,236,615 3,238,878 651 5.29 72101.0 37.83 Stable III c XP_002312092.1 Heat shock protein
70 cognate
[Populus trichocarpa]
1342 0.0
PtHsp70-20 Potri.009G079700.1 Chr09 7,632,192 7,635,660 682 5.56 73261.0 38.42 Stable II XP_002313955.1 Heat shock protein 70
[Populus trichocarpa]
1384 0.0
PtHsp70-21 Potri.010G088600.1 Chr10 11,243,707 11,247,110 572 5.31 62378.8 40.02 Unstable I XP_002315776.1 Heat shock protein 70
[Populus trichocarpa]
1177 0.0
PtHsp70-22 Potri.010G205500.1 Chr10 19,638,188 19,640,721 124 4.77 13550.7 28.82 Stable III a XP_002332590.1 Predicted protein
[Populus trichocarpa]
254 2e-87
PtHsp70-23 Potri.008G054700.1 Chr10 19,646,950 19,649,921 648 5.13 71116.4 32.62 Stable III c XP_002312090.1 Heat shock protein
70 cognate
[Populus trichocarpa]
1336 0.0
PtHsp70-24 Potri.010G205700.1 Chr10 19,646,950 19,649,921 648 5.12 71139.4 33.29 Stable III c XP_002332589.1 Predicted protein
[Populus trichocarpa]
1335 0.0
PtHsp70-25 Potri.010G205800.1 Chr10 19,657,381 19,660,151 649 5.09 71044.4 32.89 Stable III c XP_006378715.1 Hypothetical protein
POPTR_0010s21280g
[Populus trichocarpa]
1335 0.0
PtHsp70-26 Potri.010G206600.1 Chr10 19,697,773 19,700,832 648 5.09 71131.6 35.33 Stable III c XP_002316294.1 Heat shock protein 70
[Populus trichocarpa]
1334 0.0
PtHsp70-27 Potri.011G139100.2 Chr11 16,158,448 16,163,780 770 5.41 85904.8 46.27 Unstable III b XP_002317001.1 Hypothetical protein
POPTR_0011s14240g
[Populus trichocarpa]
1601 0.0
PtHsp70-28 Potri.012G017600.1 Chr12 1,650,883 1,654,098 668 5.13 73406.1 28.63 Stable III c XP_002317789.2 BiP isoform A
family protein
[Populus trichocarpa]
1354 0.0
PtHsp70-29 Potri.013G018000.1 Chr13 1,178,017 1,181,121 660 5.13 73463.5 30.74 Stable III c XP_002318993.2 Heat shock protein
70 cognate
[Populus trichocarpa]
1340 0.0
PtHsp70-30 Potri.013G041500.1 Chr13 2,920,427 2,920,819 99 6.39 11005.4 22.35 Stable III c XP_006375837.1 Hypothetical protein
POPTR_0013s03880g
[Populus trichocarpa]
205 5e-71
PtHsp70-31 Potri.014G114600.1 Chr14 8,938,972 8,939,227 131 9.40 14870.6 43.85 Unstable I XP_002320948.1 Hypothetical protein
POPTR_0014s10990g
[Populus trichocarpa]
271 7e-94
PtHsp70-32 Potri.015G078000.1 Chr15 10,312,625 10,315,085 282 9.64 30173.0 44.80 Unstable II XP_002321606.2 Hypothetical protein
POPTR_0015s08910g
[Populus trichocarpa]
575 0.0
Plant Mol Biol Rep
with 34 Hsp70 genes which is in contrast to Zhang et al.’s
(2015) findings. Greenphyl phylogenomics database
(GreenPhyl v4) (Rouard et al. 2011) also indicates the same
Hsp70 gene numbers for poplar. This database contains a cat-
alogue of gene families based on gene predictions of plant
genomes. So, it can be concluded that we found an exact
number of Hsp70 genes based on Pfam, SMART domain
searches, and GreenPhyl v4 and Phytozome v10.3 databases.
Chromosomal Distribution, and Tandem and Segmental
Duplications
The position of all 34 Hsp70 genes was mapped on chromo-
somes of poplar (Fig. 1and Supplementary Fig. S2). The
distribution of the Hsp genes on chromosomes was not uni-
form. Some chromosomes and chromosomal regions have
high density of the Hsp70 genes than other regions. Chromo-
some 1 (20.5 %) contained the highest number of Hsp70 gene
among all chromosomes. Conversely, ten chromosomes
(chromosome 2, 4, 6, 9, 11, 12, 14, 15, 16, and 19) possessed
only one Hsp70 gene (2.94 %) and showed the lowest density.
The exact position (in bp) of each PtHsp70 on poplar chro-
mosome is indicated in Table 1. Gene distribution pattern on
chromosomes revealed that PtHsp70 genes located on chro-
mosomes 10 and chromosomes 8 and 13 appear to be congre-
gate at the lower end and upper end of the arms, respectively
(Fig. 1).
It is known that segmental and tandem duplication has
played a role in the evolution and expansion of gene families
in plants (Cannon et al. 2004). Tandem and segmental dupli-
cation of PtHsp70 gene members was also determined
(Supplementary Tables S2 and S3). The highest numbers of
tandem duplication were observed in chromosome 10. Several
direct tandem repeats were found on chromosome 1
(PtHsp70-01,PtHsp70-02,PtHsp70-03), chromosome 3
(PtHsp70-12,PtHsp70-13), and chromosome 8 (PtHsp70-
16,PtHsp70-17)(Fig.1).
Events of gene duplication occur frequently and cause evo-
lution of related genes in organisms (Mehan et al. 2004). Ear-
lier studies show that two genome-wide duplication events
called as eurosid and salicoid have occurred in poplar genome.
This resulted in a series of chromosomal reorganizations that
involve reciprocal tandem/terminal fusions and translocations
(Tuskan et al. 2006). Overall, there are 20 segmental duplicat-
ed poplar Hsp70 genes detected, which equals to approxi-
mately 59 % (20/34) of total PtHsp70 genes (Supplementary
Table S3). The most surprising aspect of the data is high seg-
mental duplicated ratio which is firstly shown for Hsp70 genes
in different organisms including poplar. In rice, two segmental
duplicates of Hsp70 genes (cHsp70-1 with cHsp70-6, and
cHsp70-7 with uHsp70-2) were observed (Sarkar et al.,
2013). This demonstrated that tandem and segmental duplica-
tions have a significant role in poplar HSP70 gene expansion.
Tab l e 1 (continued)
ID Phytozome
identifier
Physical position on poplar genome Protein
length
(aa)
pI Molecular
weight
(Da)
Instability
index
Stable or
unstable
Phylogeny
group
NCBI
Accession
No.
NCBI BLASTP
annotation
Score E-value
Chromosome Start
position
(bp)
End
position
(bp)
PtHsp70-33 Potri.016G019800.2 Chr16 1,078,329 1,085,012 881 5.44 98330.6 36.86 Stable III b XP_002322555.2 Hypothetical protein
POPTR_0016s02100g
[Populus trichocarpa]
1786 0.0
PtHsp70-34 Potri.019G077900.1 Chr19 11,174,655 11,176,280 291 6.85 31665.6 41.94 Unstable I XP_006371453.1 Hypothetical protein
POPTR_0019s10660g
partial
[Populus trichocarpa]
592 0.0
Plant Mol Biol Rep
This interprets that evolution of those genes might have
proceeded quickly thorough specific gene duplications or
through integration into genomic region following a reverse
transcription (Lecharny et al. 2003).
Phylogenetic Classification of PtHsp70 and Identification
of Domain Conservation, and Gene Structure
An elaborative phylogenic analysis was conducted to under-
stand the evolutionary distinction of domain structure in
Hsp70 proteins. The phylogenetic tree was constructed using
34 PtHsp70 proteins through neighbor-joining (NJ) method.
The phylogenetic analysis classified all PtHsp70 into three
main clusters (clusters I to III) comprising of 6, 5, and 23
proteins, respectively (Fig. 2). Cluster III was then further split
into three subgroups (subgroups IIIa, IIIb, and IIIc). Because a
good count of the internal branches were found to have high
bootstrap values, it was clear by bootstrap analysis of 1000
replicates. A great number of internal branches also had high
bootstrap values, demonstrating statistically reliable pairs of
potential homologous derivation. Construction of a phyloge-
netic tree has been also performed for functional prediction of
Hsp70 proteins in other species like Arabidopsis and rice.
Members of the Hsp70 proteins have been separated into
two large groups with seven subgroups in Arabidopsis. In
Arabidopsis, members of the Hsp70 and the Hsp110, which
is a subfamily of Hsp70 superfamily and structurally very
similar to Hsp70, constituted of two large groups (Lin et al.
2001). Phylogenetic tree analysis of rice Hsp70 proteins indi-
cated that four well-supported clades, called as A, B, C, and D,
were separated with each other (Sarkar et al. 2013). These
results agree with the findings of other studies, in which dif-
ferent domains including endoplasmic reticulum (ER) reten-
tion signal (HDEL sequence), and classical cytoplasmic
Hsp70 characteristic C-terminal signal (EEVD sequence)
were shown in rice and Arabidopsis. In our study, we also
characterized ER Hsp70 protein members in clusters IIIa and
IIIb. In addition, cytoplasmic poplar Hsp70 proteins
(PtHsp70-01, PtHsp70-02, PtHsp70-13, PtHsp70-16,
PtHsp70-17, PtHsp70-23, PtHsp70-24, PtHsp70-25, and
PtHsp70-26) mainly cluster on cluster IIIc. So, it can be con-
cluded that certain members of groups were separated from
their clusters.
To check reliability of the phylogeny, motif compositions
were also examined. MEME software was used to determine
motifs through complete amino acid sequences of HSP70 pro-
teins. Based on domain compositions of Hsp70, a total of 15
distinct motifs were identified. Conserved amino acid compo-
sitions of identified motifs are shown in Supplementary
Table S4. The majority of the closely correlated items have
common motif composition, providing potential functional
similarity among the Hsp70 proteins (Supplementary
Fig. S3). For instance, all Hsp70 genes that are tandem dupli-
cated (PtHsp70-01,PtHsp70-02,PtHsp70-03,PtHsp70-12,
PtHsp70-13,PtHsp70-16,PtHsp70-17,PtHsp70-19,
PtHsp70-23,PtHsp70-24,PtHsp70-25,PtHsp70-26,
Fig. 1 Physical locations of the poplar Hsp70 genes. The chromosome numbers (numbered 1–19) are shown at the top of each chromosome (Chr;
represented as bars). Tandem duplicated genes on a particular chromosome are indicated in the box. Chromosomal distances are given in Mbp
Plant Mol Biol Rep
PtHsp70-28) were found in the cluster IIIc. Those genes
demonstrate that there were 15 motifs conserved among
these sequences. In addition, other Hsp70 proteins in
the phylogenetic tree also have similar motif structure.
However, this type of motif sequence conservation or
variation between the proteins specifies a functional
equivalence or diversification in respect to the different
biological functions (Puranik et al. 2012). On the other
hand, certain motifs were also defined and clustered into
different clades. They might be species specific for
poplar. Other than the Hsp70 domain region, Hsp70
proteins also contain some additional conserved motifs
that may demonstrate possible function sites or take part
in activation of the Hsp70 protein functions. The results
obtained from this study also match those observed in
earlier studies. In the rice, Sarkar et al. (2013)found
thatC-terminusandATPbindingdomainhashighmotif
similarity. They also indicated similar motif composition
in closely related Hsp70 proteins in the phylogenetic
tree (Sung et al. 2001). In the beginning of the N-ter-
minal, a highly conserved ATP-binding motif (GID) was
also indicated in Arabidopsis (Sung et al. 2001). This
motif was the same as the ATP-binding motif of
Arabidopsis Hsp70 proteins and defined as motif 2 in
our study.
We also analyzed the exon–intron organization of 34
poplar Hsp70 genes to gather some insight information
for gene structure (Supplementary Fig. S4). We found a
total of six Hsp70 genes without intron, which equals to
17.64 % of overall PtHsp70 genes. The maximum
intron numbers was observed in PtHSP70-07 gene with
23 introns. Examination of the intron–exon organization
indicated that family members of Hsp70 within the
same cluster shared similar gene structures in respect
to intron number or exon length. Especially, PtHsp70
genes found in cluster 3b and 3c showed similar
exon–intron patterns. The present findings seem to be
consistent with those of Sung and colleagues (2001)
Fig. 2 Phylogenetic tree of
poplar Hsp70 proteins. The
sequences were aligned by
CLUSTALW at MEGA5 and the
unrooted phylogenetic tree was
deduced by neighbor-joining
method. The proteins were
classified into three distinct
clusters. Each family was
assigned a different color
according to well-known
members in other species
Plant Mol Biol Rep
who found that the intron–exon structure of the
Arabidopsis Hsp70 genes differed from proteins which
were targeted to different subcellular locations. This also
accords with earlier observations, which showed that
Hsp70 genes in rice showed similar intron–exon
arrangement in their respective phylogenetic clades
(Sarkar et al. 2013).
Gene Ontology Annotation
For determination of the functional annotation of identified
Hsp70 genes, blast2GO Gene Ontology package was used
(Conesa et al. 2005). The GO slim analysis demonstrated the
putative involvement of 34 Hsp70 proteins in diverse biological
processes, molecular function, and cellular localization (Fig. 3
andSupplementaryTableS5). A total of 11 and 4 categories
were determined for biological process and molecular function,
respectively. The highest represented categories in biological
processes were biological regulation, response to stimulus/
abiotic stimulus, and cellular/developmental processes. Al-
though they are fewer in number, conditions related to
secondary response to metal ion, response to reactive oxygen
species, and response to biotic stimulus and protein folding
were also observed. Mostly represented categories in molecular
functions were binding activity, transcription factor activity,
and enzyme regulator activities. Cellular localization prediction
indicated that 17 Hsp70 proteins were localized in the cell and
its sections including cytoplasm, membrane, cell wall, cytosol,
and nucleus. Remaining poplar Hsp70 proteins were found in
organelle such as chloroplast, apoplast, Golgi apparatus, and
endoplasmic reticulum (Fig. 3and Supplementary Table S5).
Orthologous Relationships of Hsp70 Genes
Between Poplar and Other Species
Physically mapped PtHsp70 genes were compared with those
in chromosomes of Arabidopsis, rice, maize, and grapevine
for comparative mapping to obtain orthologous relationships
of Hsp70s (Supplementary Fig. S5). When compared to these
organism’s genomes, specific orthologous relationships could
be derived on an average for 45 % proteins for the identified
34 PtHsp70 protein-encoding genes in poplar. Maximum
Fig. 3 Gene Ontology (GO) distributions for the Hsp70 proteins. The Blast2Go program provides the gene ontology terms under three categories
including biological processes, molecular functions, and cellular component
Plant Mol Biol Rep
orthology of PtHsp70 genes annotated on the poplar chromo-
somes was obtained with maize (53 %), followed by rice
(47 %), Arabidopsis (45 %), and grapevine (35 %). These
findings further support the idea of chromosomal rearrange-
ments which are mainly responsible for shaping the distribu-
tion and organization of PtHsp70 genes in poplar,
Arabidopsis, rice, maize, and grapevine genomes. According
to these data, we can infer that comparative mapping can
provide a useful information for understanding the evolution-
ary process of Hsp70 genes among poplar and other plant
species. The present results are also significant for isolation
and cloning of similar Hsp70 genes from poplar, using the
map-based genomic information of other related plant species
for genetic enhancement.
Duplication and Divergence Rate of the PtHsp70 Genes
Some gene families which are composed of multiple copies of
genes could possibly evolve primarily through tandem dupli-
cation and infrequently large-scale segmental duplications.
Gene duplications including segmental or tandem have been
reported in many plant TF gene families such as bZIP, NAC,
MBF, and bHLH as well as HSPs (Nijhawan et al. 2008;
Baloglu et al. 2014b;Wangetal.2011; Kavas et al. 2015;
Puranik et al. 2012; Cannon et al. 2004; Jain et al. 2007).
Therefore, we investigated relation of Darwin’s positive selec-
tion in divergence and duplication of Hsp70 genes to under-
stand family expansion of the important family members. To
elaborate this, non-synonymous (Ka) versus synonymous
(Ks) substitution rate ratios (Ka/Ks) were predicted for 13
tandem and 20 segmentally duplicated gene pairs, as well as
between orthologous gene pairs of PtHsp70 with those of
grapevine (seven pairs), Arabidopsis, rice, and maize (for each
five pairs) (Fig. 4). Ka/Ks ratios for tandem duplication
differed from 0.02 to 3.01, with an average of 1.07
(Supplementary Table S2), while Ka/Ks for segmentally
duplicated gene pairs varied from 0.0096 to 0.1682 with an
average of 0.07 (Supplementary Table S3). These results pro-
vide further support for the hypothesis that duplicated
PtHsp70 genes are under strong purifying selection pressure
because their Ka/Ks ratios were below 1 (i.e., <1). Further-
more, duplication action of these tandemly and segmentally
duplicated genes can be estimated to have arisen from 0–4and
2–60 Mya, respectively (Fig. 4). It can be seen from the data in
Supplementary Table S6 that the maximum and minimum Ka/
Ks values among the orthologous gene pairs of poplar Hsp70
with grapevine (0.08) and poplar Hsp70 with rice–maize (for
each 0.02) were obtained, respectively. Even though synony-
mous substitution rates between rice–poplar and maize–pop-
lar Hsp70 genes were the same, previous divergence was es-
timated around 24–77 Mya from rice–poplar, when compared
to maize–poplar Hsp70 genes (23–134 Mya). Estimated tan-
dem and segmental duplication period (average of 1.25 and
16.03 Mya, respectively) for poplar Hsp70 genes can be used
for evolutionary studies with –Arabidopsis (22.5 Mya), –rice
(45 Mya), –grapevine (9.8 Mya), and –maize (67.7 Mya)
orthologous Hsp70 gene pairs. There were significant differ-
ences between tandem (Ka/Ks=1.07) and segmental (Ka/Ks=
0.07) duplication events of HSP70 gene pairs. Tandemly du-
plicated genes showed more recent duplication events (aver-
age 1.25 Mya), whereas those estimations for segmentally
duplicated gene pairs were average of 16.03 Mya. This study
therefore suggested that tandem duplication events in poplar
Hsp70 genes have played a more predominant role in evolu-
tion than segmental duplication events. It can be concluded
that combination of information about tandem and segmental
duplications in poplar and different organisms help us to un-
derstand evolution and maintenance of members of the Hsp70
gene family.
Identification of miRNAs Targeting HSP70T transcripts
We implemented the grading schema based on miRU to score
the complementarity between miRNA and their target tran-
script (Zhang 2005). The maximum expectation which is the
threshold of the score and an UPE defined as maximum ener-
gy to unpair the target site are two important parameters for
determination of targets. The maximum expectation threshold
value was adjusted to 3.0. A miRNA/target site pair has been
discarded if its score is greater than the threshold. The acces-
sibility of messenger RNA (mRNA) target site to miRNAwas
determined to be one of the important factors involved in
target recognition. The psRNATarget server uses RNA for
calculation of target accessibility, which is represented by
the energy needed to disassociate secondary structure around
target. The lesser energy means the higher possibility of small
RNA binding and cleavage to target mRNA. There were 19
PtHsp70 genes (PtHsp70-03,PtHsp70-04,PtHsp70-07,
PtHsp70-08,PtHsp70-11,PtHsp70-12,PtHsp70-14,
PtHsp70-15,PtHsp70-16,PtHsp70-18,PtHsp70-19,
PtHsp70-21,PtHsp70-23,PtHsp70-24,PtHsp70-26,
PtHsp70-27,PtHsp70-28,PtHsp70-32,PtHsp70-33) targeted
by 27 plant miRNAs were found in poplar genome through
psRNATarget: A Plant Small RNATarget Analysis Server. On
the other hand, certain plant miRNAs did not demonstrate any
gene target. PtHsp70-14 and PtHsp70-33 are the most abun-
dant transcripts among the target genes, which were targeted
by all 27 plant miRNAs (Supplementary Table S7). The
miR390, one of most abundant identified miRNA in different
species, regulated several auxin-responsive factors through
TAS3-derived tasiARFs (Axtell et al. 2006)andtargetedto
PtHsp70-14 in our study. It is therefore likely that connections
exist between functions of PtHsp70-14 gene (protein folding
and oxidation–reduction process) and miR390. Most of the
targets identified in our study were responsible for plant
growth, development, metabolism, and defense responses to
Plant Mol Biol Rep
environmental changes. For example, PtHsp70-33 plays im-
portant roles in oxidation–reduction process, protein folding,
and response to heat/hydrogen peroxide. The miR414, whose
target was PtHsp70-33, has been also a widely found miRNA
in our study. The miR414 primarily targets transcriptional
regulators and transcription factors such as bZIP, WRKY,
MYB, B3 family transcription factors, scarecrow, heat shock
proteins, and TCP (Guleria and Yadav 2011;Eulgemetal.
2000;Gurley2000; Jakoby et al. 2002; Suo et al. 2003;
Romanel et al. 2009). It can thus be suggested that identifica-
tion of miRNAs and their targets play a crucial roles for un-
derstanding of Hsp70 gene family functions.
Homology Modeling of HSP70 Proteins
BLASTP search was conducted against PDB to build the ho-
mology pattern. A total of 28 Hsp70 proteins (PtHsp70-01-02-
03-05-06-08-09-10-12-13-14-16-17-18-19-20-21-22-23-24-
25-26-27-28-29-30-32-34) with a higher homology were se-
lected. Detection rate was used for estimation of homology
modeling in Phyre2, which employs the alignment of hidden
Markov models through HMM-HMM search (Söding 2005)
in order to remarkably improve accuracy of the alignment.
The intensive mode of Phyre 2 utilizes the multi-template
modeling to achieve a higher accuracy. In addition, it inte-
grates a new ab initio folding simulation termed as Poing
(Jefferys et al. 2010) to model areas of proteins without any
significant homology for known structures. All 28 PtHsp70
proteins were modeled at >90 % reliability, and the residue
percentage varied from 80 to 100 (Fig. 5and Supplementary
Table S8). The secondary structures were predominantly con-
stituted of αhelices and have rare incurrence of βsheets.
Thus, all suggested protein structures are assessed to be highly
reliable which offers a preliminary basis for understanding the
molecular function of PtHsp70 proteins.
Differential Expression Patterns of Hsp70 Genes
in Populus
One of the fundamental objectives of a gene expression pro-
filing on a genomic scale is to identify the genes that are
differentially expressed within the organism being examined.
This can provide useful clues for the functions of these genes.
To acquire information about the drought response of
PtHsp70 genes in poplar, a RNA-Seq approach was imple-
mented to data sets obtained from SRA database. Following
normalization and transformation analysis, PtHsp70 genes
were scored from the highest to the lowest based on their
differential expression under control and drought stress
conditions. Cossu et al. (2014)usedIlluminasequencingtech-
nology to obtain a global view of the molecular responses of
poplar hybrid to drought. In that study, hybrids between
Populus deltoides (L155-079, female) and Populus nigra
(71077-2-308, male) were utilized with three treatments (con-
trol, C; moderate, D1; and severe drought, D2) for tran-
scriptome analysis. According to high-throughput tag se-
quencing analysis, we found some PtHsp70 genes (PtHsp70-
01-02-03-05-06-20 and 21) whose expression was increased
in at least one of the D1 and D2 drought stress treatments
(Fig. 6a). Expression level of remaining poplar Hsp70 genes
decreased after moderate or severe drought stress application.
We also examined RNA-seq data from Tang et al. (2014)who
investigated leaf transcriptome derived from Populus
trichocarpa seedlings grown in normal condition (control;
well watered) and drought stress (D1; water-limited). Based
on their transcriptome data, PtHsp70-03-04-07-10-16-20-21-
32 and 33 genes were induced by drought stress (Fig. 6b). In
addition, we realized that some PtHsp70 genes including
PtHsp70-03-20 and 21 had similar expression patterns for
both studies. In other words, these genes were significantly
upregulated in different poplar species such as poplar hybrids
Fig. 4 Time of duplication and
divergence (MYA) of Hsp70
genes. This is based on
synonymous substitution rate
(Ks), which estimated using
duplicated Hsp70 gene pairs of
poplar and orthologous Hsp70
gene pairs between poplar and
Arabidopsis or rice or maize or
grapevine
Plant Mol Biol Rep
Fig. 5 Predicated 3D structures of Hsp70 proteins. The structure of 28 Hsp70 proteins with >90 % confidence level is shown
Fig. 6 Heat maps of the differentially expressed Hsp70 genes under
control and different drought stress conditions. Three different images
constructed based on studies. aCossu et al. 2014 and C: control (plant
85_4), D1: moderate drought stress (plant 85_12), and D2: severe
drought stress (plant 85_42); bTang et al. 2014 C: control (well
watered) and D1: drought stress (water-limited); and cTang et al. 2013
C: control (well-watered), and D: moderate drought stress summarize
expression pattern of 34 PtHsp70. Note that expression values mapped
to a color gradient from low (plain green) to high expression (dark red)
Plant Mol Biol Rep
(between Populus deltoides and Populus nigra)andPopulus
trichocarpa. Lastly, Tang et al. (2013) investigated the re-
sponses of the Populus euphratica to soil water deficit using
pyrosequencing approach. According to high-throughput se-
quencing data, only three PtHsp70 genes (PtHsp70-12-23 and
26) gave the response to drought stress with increase in their
expression levels after moderate drought stress application
(Fig. 6c). It can be concluded that PtHsp70s genes are differ-
entially expressed upon exposure to different drought stress
conditions and different Populus species such as hybrid (be-
tween Populus deltoides and Populus nigra), Populus
trichocarpa,andPopulus euphratica.
Drought Stress Responses of Hsp70 Genes in Populus
In order to reveal the responses of poplar Hsp70 genes to
drought stress, we analyzed the expression profiles of
PtHsp70s in leaf tissues of two Populus nigra L. clones,
Clone S (drought sensitive) and Clone R (drought resistant),
with qRT-PCR. A total of 13 PtHsp70 genes including
PtHsp70-03-04-09-10-12-16-17-20-23-24-25-26-33 were se-
lected for expression analysis. Based on the literature search,
highly expressed Hsp70 genes under the drought stress were
determined for quantitative real-time PCR (Neill et al. 1999;
Cho and Choi 2009; Song et al. 2009; Cohen et al. 2010).
Expression of PtHsp70-03,PtHsp70-04,PtHsp70-09,
PtHsp70-10,PtHsp70-12,PtHsp70-17,PtHsp70-20,
PtHsp70-23,andPtHsp70-24 was found to be repressed. All
downregulated PtHsp70 genes shared similar expression pat-
terns in both controls of Clone S and Clone R. However, a
substantial difference between the susceptible and resistant
controls was observed. The transcript concentrations of all
downregulated PtHsp70 genes in control samples of Clone
R are higher than Clone S ones. This result may be explained
by the fact that all downregulated PtHsp70 genes in Clone R
(PtHsp70-03,PtHsp70-04,PtHsp70-09,PtHsp70-10,
PtHsp70-12,PtHsp70-17,PtHsp70-20,PtHsp70-23,
PtHsp70-24) might contribute to different biological process-
es as molecular chaperones under normal condition. In addi-
tion, PtHsp70-16 and PtHsp70-26 were viewed to be upreg-
ulated in both leaf tissues of poplar, i.e., compared to control
samples of Clone S and Clone R, an increased response at the
transcription level of PtHsp70-16 and PtHsp70-26 genes was
observed after drought stress treatment. So, it is possible to
Fig. 7 Expression profiles of 13 PtHsp70 genes in leaf tissues of two
Populus nigra L. clones, Clone S (drought sensitive) and Clone R
(drought resistant) with qRT-PCR. Three biological replicates each with
three technique replicates were performed and bars represent standard
error of the mean for the replicates. SC: clone S-control; ST:cloneS-
drought stress treatment; RC: clone R-control; RT: clone R-drought
stress treatment
Plant Mol Biol Rep
hypothesize that PtHsp70-16 and PtHsp70-26 genes might
likely play a role in drought stress response for both sensitive
and resistant Populus nigra L. clones.
Although PtHsp70-25 and PtHsp70-33 were downregulat-
ed in Clone S, they were induced in Clone R (Fig. 7).
PtHsp70-25 and PtHsp70-33 genes might be used for deter-
mination of drought-tolerant clones for molecular breeding
studies because high expression responses to drought stress
for these genes were only observed in Clone R, resistant poplar
clone. So, it can be also suggested that PtHsp70-25 and
PtHsp70-33 genes might provide adaptation to drought stress
for resistant poplar clone. The expression pattern of PtHsp70-
16,PtHsp70-26, and PtHsp70-33 genes detected by qRT-PCR
is generally consistent with the RNA-seq results. For example,
PtHsp70-16 and PtHsp70-33 genes were induced after
drought treatment for both Populus nigra, in our study, and
Populus trichocarpa in leaf transcriptome study (Tang et al.
2014)(Fig.6b). In addition, increase in gene expression level
of PtHsp70-26 gene was observed in Populus nigra and
Populus euphratica for our study and study performed by
Tang et al. (2013)(Fig.6c), respectively. Genotypic variation
in the expression response of these genes to drought stress is an
interesting observation in this study. The different expression
patterns of PtHsp70s imply that PtHsp70 members in different
Populus species may be involved in response to drought stress.
Understanding the plant responses at molecular level is
crucial to improve the stress tolerance and productivity. In this
study, we measured the expression of 13 PtHsp70 genes to
analyze their possible drought-responsive roles. Differential
expression profiles of the Hsp70 genes under drought stress
suggest that some other genes functioning in water deficiency
might also be regulated by this family. In other biotic and
abiotic stress studies, similar results were reported (Neill
et al. 1999; Cho and Choi 2009; Song et al. 2009; Cohen
et al. 2010).
In a recent study, expression patterns of rice OsctHsp70
genes including Os05g38530,Os01g62290,Os03g16920,
and Os03g16860 were significantly upregulated by salt and
drought treatments, indicating that they might have roles in
various abiotic stress responses (Jung et al. 2013). These re-
sults match those observed in our study, in which PtHsp70-16
and PtHsp70-26 genes were induced with the same pattern
under the drought stress conditions for both poplar clones.
The present findings seem to be consistent with recent re-
search which found an increase in expression level of
PtHsp90 group I, PtHsp90-1a,PtHsp90-1b,andPtHsp90-3
in both the Soligo and Carpacio poplar genotypes under
drought stress conditions (Zhang et al. 2013). The results of
these studies indicate that some other genes might also be
regulated by water deficiency. These differentially
expressed gene families including PtHsp70 gene mem-
bers deserve further investigation into their potential role
in different abiotic stresses.
Acknowledgments This work was financially supported by the
Kastamonu University Scientific Research Project Management Coordi-
nation Unit under Grant No: KÜBAP-01/2014-09. We would like to
thank Dr. Tuncay PORSUK from Director of Inner Anatolia Forestry
Research Institute for his help in providing the Populus nigra L. clones.
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