ORIGINAL PAPER

Overexpression of PeHSF mediates leaf ROS homeostasisin transgenic tobacco lines grown under salt stress conditions

Zedan Shen • Mingquan Ding • Jian Sun • Shurong Deng • Rui Zhao •

Meijuan Wang • Xujun Ma • Feifei Wang • Huilong Zhang • Zeyong Qian •

Yue Hu • Rui Yu • Xin Shen • Shaoliang Chen

Received: 31 January 2013 / Accepted: 26 July 2013 / Published online: 6 August 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Transcriptional responses of a heat-shock tran-

scription factor from the salt-resistant Populus euphratica,

PeHSF, were assessed in transgenic tobacco seedlings sub-

jected to salinity stress. Moreover, their relevance to NaCl-

induced anti-oxidative defense responses in higher plants

was also explored. Expression of PeHSF was induced by

high-salinity (150 mM NaCl) in leaves and callus cultures of

P. euphratica. The salt-stimulated PeHSF expression in P.

euphratica callus cultures was markedly inhibited by DPI

(an inhibitor of plasma membrane NADPH oxidase) and

LaCl3 (an inhibitor of plasma membrane Ca2?-permeable

channels), indicating the involvement of reactive oxygen

species (ROS) and Ca2? in HSF activation under NaCl stress.

Sequence analysis showed that PeHSF is a typical class A

HSF in higher plants. PeHSF overexpression in tobacco

significantly improved the salt tolerance of transgenic plants,

in term of seed germination and root growth. Compared to

the wild-type plants, the PeHSF-transgenic lines exhibited

an increased capacity to control ROS homeostasis, but not an

enhanced capacity to retain ionic homeostasis under salt

stress. PeHSF expression in tobacco enhanced the activities

of ascorbate peroxidase, glutathione peroxidase, and gluta-

thione reductase, due to upregulated transcriptional levels.

We conclude that PeHSF plays a crucial role in ROS

detoxification under salt stress, rather than in maintenance of

the K?/Na?. It is likely that the salt-induced H2O2 and

cytosolic Ca2? led to transcriptional activation of HSF,

which initiated the transcription of genes encoding antioxi-

dant enzymes in P. euphratica, thus contributing to ROS

homeostasis control under saline conditions.

Keywords Populus euphratica � Heat shock

transcription factor � NaCl � Antioxidant enzyme �H2O2 � Tobacco

Introduction

Salinity leads to reduced plant growth and crop produc-

tivity (Mahajan and Tuteja 2005), and some plants have

developed a signaling network to sense salinity stress. Heat

shock transcription factors (Hsfs) participate in multiple

stress responses, mediating plant responses to heat (Nover

et al. 1996; Swindell et al. 2007), low temperature (Sabehat

et al. 1998), osmotic stress (Sun et al. 2001), salt (Ogawa

et al. 2007), oxidative stress (Banzet et al. 1998; Lee et al.

2000; Volkov et al. 2006), desiccation (Liu et al. 2006),

intense light (Hihara et al. 2001; Rossel et al. 2002),

wounding (Cheong et al. 2002), and heavy metals

(Gyorgyey et al. 1991). Hsfs have a DNA-binding domain

(DBD), containing a helix-turn-helix motif that recog-

nizes and binds the heat shock elements (HSEs) of

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11240-013-0362-7) contains supplementarymaterial, which is available to authorized users.

Z. Shen � M. Ding � J. Sun � S. Deng � R. Zhao � M. Wang �X. Ma � F. Wang � H. Zhang � Z. Qian � Y. Hu � R. Yu �X. Shen � S. Chen (&)

College of Biological Sciences and Technology,

Beijing Forestry University, Box 162, Beijing 100083,

People’s Republic of China

e-mail: Lschen@bjfu.edu.cn

M. Ding

College of Agricultural and Food Science, Zhejiang Agricultural

and Forestry University, Hangzhou 311300, Zhejiang Province,

People’s Republic of China

J. Sun

College of Life Science, Jiangsu Normal University,

Xuzhou 221116, Jiangsu Province, People’s Republic of China

123

Plant Cell Tiss Organ Cult (2013) 115:299–308

DOI 10.1007/s11240-013-0362-7

Hsf-responsive genes (Damberger et al. 1994; Harrison et al.

1994; Vuister et al. 1994; Schultheiss et al. 1996). HSEs are

found in the promoter regions of a variety of defense genes

(Rizhsky et al. 2004). Upon stress, inactive Hsf monomers

form a homo-trimer, which can then be further modified by

phosphorylation or conformational changes to activate target

genes (Larson et al. 1988; Lee et al. 2000).

Higher plants possess a large Hsf family, with 18

members in tomato and 34 in soybean (Nover et al. 1996,

2001). The multigene Hsf family appears to benefit plants

that must respond to environmental variations. Different

Hsf family members have distinct roles in responding to

stress and activating target genes (Miller and Mittler 2006),

presumably due to their different structural characteristics.

There are three different classes of Hsfs: Class A, B, and C

(Nover et al. 2001). Class A Hsfs contain a C-terminal

activation domain and are thought to function as tran-

scriptional activators, while Class B and Class C Hsfs lack

a defined activation domain (Nover et al. 1996). Class B

Hsfs are reportedly transcription repressors (Boscheinen

et al. 1997; Czarnecka-Verner et al. 2000, 2004); however,

HsfB1 was recently demonstrated to function as a novel co-

regulator of tomato HsfA1 or HsfA2, enhancing their

transcriptional activity (Bharti et al. 2004). It has been

proposed that Hsfs target heat shock proteins (HSPs) (Ra-

bindran et al. 1993; Milioni et al. 2001). Different types of

Hsfs likely activate various defense pathways, assisting

plant adaptation to adverse environments; however, the

contribution Hsfs to salt tolerance in woody plants is

unknown.

Populus euphratica exhibits higher salt tolerance than

other poplars and is thus considered a model species for

studying tree stress physiology (Chen and Polle 2010).

There are several well-documented molecular and physio-

logical mechanisms underlying the salt tolerance of P.

euphratica (Chen et al. 2001, 2002a, b, 2003; Ottow et al.

2005; Sun et al. 2009a, b; Chen and Polle 2010). Tran-

scriptome analysis reveals that P. euphratica exhibits

permanent activation of control mechanisms for osmotic

adjustment (sugar and sugar alcohols) and ion compart-

mentalization (Ding et al. 2010; Janz et al. 2010). Fur-

thermore, salt stress signals (e.g., extracellular ATP, H2O2,

nitric oxide, and Ca2?) are involved in controlling ionic

homeostasis in P. euphratica (Sun et al. 2010a, b, 2012;

Zhang et al. 2007). These plants also exhibit a high

capacity to maintain anti-oxidant enzyme activity under

NaCl stress (Wang et al. 2007, 2008). Microarray analysis

has shown that salt stress upregulates the expression of a

class A HSF in P. euphratica (our unpublished data).

However, the link between HSF and oxidative stress has

not yet been established in P. euphratica.

The present study aimed to evaluate the transcriptional

response of PeHSF to salinity, and the relevance of this

response to NaCl-induced anti-oxidative defense. We

cloned the PeHSF gene from P. euphratica, and transferred

it to the model species Nicotiana tabacum to investigate the

role of this transcription factor in salinity tolerance. In

wild-type tobacco and PeHSF-overexpressing lines, we

examined ion relations, reactive oxygen species (ROS)

accumulation, and activities of antioxidant enzymes—such

as superoxide dismutase (SOD), catalase (CAT), ascorbate

peroxidase (APX), glutathione peroxidase (GPX), and

glutathione reductase (GR). Real-time PCR was used to

quantify the relative changes in the mRNA expression

levels of these antioxidant enzymes. Moreover, we inves-

tigated the correlations between ROS, Ca2?, and PeHSF

expression in P. euphratica callus cultures.

Materials and methods

Plant materials and treatments

One-year-old Populus euphratica Oliver (P. euphratica)

seedlings were obtained from the Xinjiang Uygur Auton-

omous Region of China. They were planted in individual

10-L pots containing loam soil, and placed in a greenhouse

at Beijing Forestry University. The potted plants were

irrigated two to three times per week according to evapo-

ration demand, and watered with 1 L full-strength Hoa-

gland’s nutrient solution every 2 weeks. The greenhouse

temperature was maintained at 20–25 �C, with a 16-h

photoperiod (7:00 AM–11:00 PM). Photosynthetically

active radiation was 150–600 lmol m-2 s-1 during the

2-month culture period. Seedlings were subjected to

4 weeks of NaCl stress by watering with 2 L of 150 mM

NaCl in full-strength Hoagland’s nutrient solution on days

1, 8, 15, and 23. For RNA extraction, upper mature leaves

(leaf index numbers 4–20 from shoot apex) were sampled

on days 2, 9, 22, and 29 after salt treatment.

P. euphratica callus cultures were started from shoots,

as described by Sun et al. (2010a, b). The calluses were

grown in Murashige and Skoog (1962) solid medium

(2.5 % sucrose, pH 5.7) supplemented with 0.25 mg L-1

benzyl adenine (BA) and 0.50 mg L-1 naphthalene acetic

acid (NAA). Callus cultures were raised in the dark at

25 �C, and subcultured every 15 days. Ten days after

transplantation, the calluses were sampled for salt and

inhibitor treatments on fresh propagation medium. P. eu-

phratica callus cultures were subjected to 150 mM NaCl,

with or without 100 lM DPI (diphenylene iodonium, an

inhibitor of PM NADPH oxidase) and/or 5 mM LaCl3 (an

inhibitor of PM Ca2? channels). Control callus cultures

were not subjected to the application of NaCl or inhibitors.

For RNA extraction, calluses were harvested after 1 and

12 h of salt treatment.

300 Plant Cell Tiss Organ Cult (2013) 115:299–308

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Full-length PeHSF gene cloning and sequence analysis

Total RNA was isolated from P. euphratica leaves using

TRIzol reagent (Invitrogen) according to the manufac-

turer’s instructions. First-strand cDNA was synthesized

from 1 lg total RNA using the SuperScriptTM First Strand

Synthesis System. The 1437-nt fragment containing the

PeHSF ORF was amplified using specific primers (forward,

50-ATG GAT GGT TCA CAG AGT AA-30; reverse, 50-TCT GAC ATT ATG ACC TAT AC-30), and PCR prod-

ucts were cloned into the pMD18-T vector for sequencing.

The poplar putative amino acid sequences were aligned

using the multiple sequence alignment function of the

DNAMAN software. The phylogenetic tree was created

using the neighborhood-joining bootstrap (bootstrap NJ

Tree) function of the MEGA program (version 5.0).

The full-length PeHSF cDNA contained a 1437-bp open

reading frame encoding a putative protein of 479 amino

acids (Fig. S1). Phylogenetic analysis revealed PeHSF to

be a typical class A HSF (Doring et al. 2000; Fig. S2). The

putative PeHSF amino acid sequence contains a DNA-

binding domain (DBD), an adjacent domain with heptad

hydrophobic repeats (HR-A/B) involved in oligomeriza-

tion, nuclear localization sequences (NLS), two aromatic

hydrophobic acidic (AHA)-type activation domain, and a

nuclear export sequence (NES; Fig. S1). The putative Pe-

HSF protein from P. euphratica was highly homologous to

RcHsfA4 at position 20–120, which functions as a DNA-

binding domain (Fig. S1). Therefore, PeHSF can be clas-

sified as an HSFA4 with an E-Value of 9.3e-176 (http://

www.cibiv.at/services/hsf/).

Yeast one-hybrid assay

To construct the yeast-hybrid vector, the 1335-bp PeHSF

ORF was amplified using specific primers (forward, 50-CATG GAG GCC GAATTC-ATG GAT GGT TCA CAG

AGT AA-30; and reverse, 50-GC AGGTCGACGGATCC-

TCA TGA TCT TTC AGC TGT CGC AATG-30). Purified

PCR products were then fused in-frame with the GAL4BD

in the pGBKT7 vector by cloning into EcoRI and BamHI

sites. The recombinant plasmids were transformed into the

yeast strain Y2HGold using the YeastmakerTM Yeast

Transformation System 2. Yeast cells containing pGBKT7

were used as negative controls. The strains were tested

using synthetic defined (SD) media lacking histidine and

tryptophan (SD/-T-H), followed by X-a-Gal analysis.

X-a-Gal was directly added to the culture media to test

positive colonies (blue, direct visualization on the plate).

The transcriptional activity of PeHSF was tested with a

yeast one-hybrid assay. In contrast to the negative control,

the pGBKT7-PeHSF strain grew on SD media lacking

histidine and tryptophan (SD/-T-H; Fig. S3a). Moreover,

the pGBKT7-HSF strain also turned blue in the X-a-Gal

analysis (Fig. S3b). These results confirmed the transcrip-

tional activity of the PeHSF protein.

Overexpression of PeHSF in transgenic tobacco lines

To construct the 35S:PeHSF overexpression vector, a

1437-nt fragment containing the PeHSF ORF was ampli-

fied from P. euphratica cDNA using specific primers

(forward, 50-ATG GAT GGT TCA CAG AGT AA-30;reverse, 50-TCT GAC ATT ATG ACC TAT AC-30), and

then cloned into the pMD18-T vector. Next, the PeHSF

ORF was subcloned into the EcoRI and BamHI sites of the

pCABIA2300 vector driven by the 35S promoter. The

35S:PeHSF-2300 construct was introduced into Agrobac-

terium tumefaciens strain LBA4404, and then transformed

into wild-type N. tabacum using an Agrobacterium-medi-

ated method. The blank pCABIA2300 vector was intro-

duced into wild-type tobacco plants as a control. Ten lines

(#1–#10) of transgenic tobacco were obtained. The seeds of

T1 transformants were screened on half-strength MS agar

medium supplemented with 100 mg/L kanamycin. Four

positive transgenic lines #1, #2, #6, and #10 were con-

firmed by semi-PCR and real-time PCR using PeHSF-

specific primers (see below).

Among T1 progeny, the segregation ratio of kanamycin-

resistant (KanR) to kanamycin-sensitive (KanS) seedlings

ranged from 2.7 to 3.47 (Fig. S4a; Table S1), close to the

typical Mendelian ratio of 3:1, suggesting that the selected

transgenic tobacco lines (#2 and #6) harbored a single copy

of the transgene. We also examined the continuation of the

transgene expression. T2 seedlings of lines #2 and #6 could

grow on MS medium containing kanamycin (Fig. S4b), and

real-time PCR analysis showed PeHSF expression in both

lines (Fig. S4c). Wild-type and transgenic plants (T2) on

MS agar medium were exposed to short-term (24 h) and

long-term (10-15 days) of NaCl stress (100 mM). Seed

germination, root growth, ion relations, hydrogen peroxide

level, activity of antioxidant enzymes and gene expression

were examined at indicated time. The tobacco plants were

grown in a growth chamber at 25 ± 1 �C with relative

humidity of 50–60 %, a light intensity of 50 lmol m-2 s-1,

and a photoperiod of 16 h light/8 h dark.

Real-time PCR analysis

To examine the salt-induced expression of PeHSF in P. eu-

phratica, total RNA was isolated from P. euphratica leaves

and callus cultures grown under normal and saline condi-

tions. RNA was extracted with the Plant RNA Kit (QBio

Technologies Inc., Beijing, China) following the manufac-

turer’s introductions. The RNA was then treated with RNase-

free DNase (Promega, Madison, WI, USA), and first-strand

Plant Cell Tiss Organ Cult (2013) 115:299–308 301

123

cDNA was prepared as described above. One microliter of

cDNA product was used for SYBR Green-based real-time

PCR analysis in a Real-Time PCR System (MJ option2; Bio-

Rad, Hercules, CA, USA). Each sample was run in triplicate.

ACT7 (a housekeeping gene in P. euphratica; NCBI Ref Seq

acc. XM_002322628) was used as an internal control. ACT7

forward and reverse primers were 50-CAC ACT GGA GTG

ATG GTT GG-30 and 50-ATT GGC CTT GGG GTT AAG

AG-30, respectively. PeHSF forward and reverse primers

were 50-AACGCCCAGTTATCTCGGTGAT-30 and 50-AG

TCAGGAAGTGCTGCCAGAA-30, respectively. The PCR

running conditions were: 95 �C for 5 min, followed by 34

cycles of 94 �C for 30 s, 55 �C for 30 s, and 72 �C for 30 s,

with a final step of 72 �C for 10 min.

Real-time PCR was also performed on selected transgenic

tobacco lines with higher PeHSF expression. EF1a (a

housekeeping gene in tobacco; GenBank acc. D63396) was

used as an internal control. EF1a forward and reverse primers

were 50-GCT GTG AGG GAC ATG CGT CAA A-30 and 50-GTA GTA GAT ATC GCG AGT ACC ACC A-30, respec-

tively (Wang et al. 2013). Real-time quantification was based

on Ct values and the expression data was normalized to the

EF1a expression level. For semi-PCR, the PCR products were

separated in 2 % agarose gels, stained with ethidium bromide,

and then photographed under UV illumination.

Tissue ion analysis

Wild-type and transgenic seedlings were oven-dried (65 �C

for 4 days), ground, and passed through a 1.0-mm sieve for

mineral analysis. Na?, K?, Ca2? were analyzed as previ-

ously described (Chen et al. 2003; Wang et al. 2013).

H2O2 measurement

The H2O2-specific fluorescent probe H2DCF-DA (green;

Molecular Probe) was used to measure H2O2 (Leshem et al.

2007; Sun et al. 2010b). Roots were treated with 50 lM

H2DCF-DA (prepared in a Mes–KCl buffer, pH 5.7) for

5 min at room temperature in the dark. Then, the H2DCF-

DA-loaded plants were washed three to four times with the

basic solution. DCF-dependent fluorescence was measured

with a fluorescence microscope.

Activity and transcriptional analyses of ROS-

scavenging enzymes

Transgenic seedlings and wild-type tobacco (0.2 g) were

harvested, ground to a fine powder in liquid N2, and then

immediately homogenized in 2 mL of 50 mM potassium

phosphate buffer (pH 7.0) containing 1 mM ethylenediami-

netetraacetic acid (EDTA) and 1 % polyvinylpyrrolidone

(PVP; Jiang and Zhang 2003). The homogenate was centri-

fuged at 10,000g for 20 min at 4 �C, and the supernatant was

used to examine the antioxidant enzymes CAT, GR, APX, and

SOD, as described by Wang et al. (2008). Total GPX activity

was measured using the Cellular Glutathione Peroxidase

Assay Kit (Sigma), and the transcript levels of the antioxidant

enzymes were quantified by real-time PCR using gene-specific

primers (described in Supplementary Table S2).

Data analysis

Data were subjected to analysis of variance. Differences

between means were evaluated with Duncan’s multiple

range test. Unless otherwise stated, differences were con-

sidered statistically significant when P \ 0.05.

Results

Salt-induced PeHSF expression in P. euphratica

To clarify whether PeHSF was transcriptionally responsive

to high salt stress in P. euphratica leaves, we used real-

time PCR to examine the PeHSF transcript abundance

during NaCl treatment (150 mM, 29 days). The PeHSF

transcript level significantly increased by threefold on the

first day of salt treatment, followed by a gradual decline,

reaching a level similar to that in control plants at the end

of the experiment (Fig. 1a).

Pharmacological experiments showed that PeHSF

expression in NaCl-treated P. euphratica callus cultures

was regulated by H2O2 and Ca2?. The PM NADPH oxidase

inhibitor DPI and the PM Ca2?-permeable channel blocker

LaCl3 each had a marked inhibitory effect on salt-induced

PeHSF expression at the two measured time-points (1 and

12 h; Fig. 1b).

Seed germination, root growth, and salinity tolerance

To determine the role of the transcriptional factor in the salt

tolerance of P. euphratica, the PeHSF gene was trans-

formed into wild-type tobacco. Four positive transgenic

plant lines were subjected to semi-quantitative RT-PCR and

real-time PCR to check transgene expression (Fig. 2). The

two transgenic lines with higher PeHSF transcription levels

(#2 and #6, Fig. 2) were used for salt tolerance tests. Seeds

from wild-type tobacco and transgenic lines #2 and #6 (T2

generation) were germinated on MS medium without or

with NaCl (100 mM). Under NaCl stress, the two transgenic

lines showed significantly higher germination rates than

WT plants (Figs. 3a, S5a). Moreover, root length was less

reduced by salt treatment in the salinized #2 and #6 plants

than in WT plants (Figs. 3b, S5b).

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Ion relations

To determine the role of PeHSF in ionic homeostasis

control, we examined Na?, K?, and Ca2? levels in the

leaves of control and salt-treated tobacco plants. In WT and

transgenic plants, NaCl significantly increased Na? but

reduced K? and Ca2? (Fig. 4). Consequently, the K?/Na?

and Ca2?/Na? ratios markedly declined with increasing

duration of salt exposure (Fig. 4). However, the salt

accumulation did not significantly differ between WT and

transgenic lines during the period of salt stress (Fig. 4).

H2O2 levels in roots

In saline environments, it is crucial for plants to control

ROS levels. We used the H2O2-sensitive fluorescent probe

H2DCF-DA to measure the salt-induced H2O2 in WT plants

and PeHSF-transgenic lines. The salt treatment caused a

significant rise of H2O2 in the roots of WT and transgenic

lines over the observation period (3, 24 h, and 15 days;

Fig. 5). However, the root H2O2 level with NaCl treatment

was 30–50 % lower in transgenic lines than in wild-type

plants (Fig. 5).

Activity of antioxidant enzymes and gene transcription

Under salt stress, antioxidant enzymes scavenge excess ROS

to maintain a redox state in P. euphratica (Wang et al. 2007,

2008; Sun et al. 2010a). We measured the activities of anti-

oxidant enzymes, such as superoxide dismutase (SOD),

catalase (CAT), ascorbate peroxidase (APX), glutathione

reductase (GR), and glutathione peroxidase (GPX), and

found that NaCl treatment in WT and transgenic lines

Fig. 1 PeHSF expression in P. euphratica leaves and callus cultures

under short-term and long-term of NaCl treatment. a PeHSF expres-

sion in P. euphratica leaves after long-term salt treatment (150 mM

NaCl, 29 days). Control plants were not treated with NaCl. b PeHSF

expression in P. euphratica callus cultures after short-term salt

treatment (150 mM NaCl, 12 h), with or without DPI (100 lM),

LaCl3 (5 mM), or DPI (100 lM) ? LaCl3 (5 mM). Control callus

cultures were not treated with pharmacological agents or NaCl. In

both a and b, the actin gene was used as an internal reference. Each

column is the mean of four individual samples, and bars represent the

standard error of the mean. Columns labeled with different letters are

significantly different, with a P value of \0.05 between treatment

times (a) or between treatments at the two measured time-points (1

and 12 h) (b)

Fig. 2 Real-time PCR analysis of wild-type (WT) tobacco and

transgenic lines (#1, #2, #6, and #10) under normal growth conditions.

a Real-time PCR analysis. PeHSF expression was normalized to

expression of the housekeeping gene elongation factor 1a (EF1a).

Each column shows the mean of four individual plants, and bars

represent the standard error of the mean. Columns labeled with

different letters significantly differ (P \ 0.05) between WT and

transgenic lines. b Semi-quantitative RT-PCR analysis of wild-type

(WT) tobacco and transgenic lines (#1, #2, #6, and #10) under normal

growth conditions

Plant Cell Tiss Organ Cult (2013) 115:299–308 303

123

increased the activities of APX, GR, and GPX, but not of

SOD and CAT, over the observation period (15 days;

Fig. 6). Compared to WT, the transgenic plant lines #2 and

#6 exhibited significantly higher antioxidant enzyme activ-

ities on day 1 (APX and GPX) and day 15 (APX, GR, and

GPX; Fig. 6).

The transcript levels of genes encoding antioxidant

enzymes were examined via real-time PCR analysis. The

expressions of these genes closely resembled the pattern

of their activity profiles (Figs. 6, S6). Salt treatment sig-

nificantly enhanced transcription of GR, GPX, and APX

on days 1 and 15, and these effects were more pro-

nounced in transgenic lines compared to in wild-type

tobacco (Fig. S6). NaCl treatment did not change the

expressions of the SOD and CAT genes over the obser-

vation period (Fig. S6).

Discussion

PeHSF overexpression enhances salt tolerance

of tobacco

Phylogenetic tree and sequence analyses revealed that the

transcription factor PeHSF is a class A HSF. PeHSF

Fig. 3 Salt tolerance of wild-type (WT) tobacco, vector control

(VC), and PeHSF-transgenic lines (#2 and #6). Seeds of wild-type,

vector control, and transgenic lines were germinated on MS medium,

with or without 100 mM NaCl. Survival rates and root length were

measured after 10 days of salt treatment. Each column shows the

mean of three independent experiments, and bars represent the

standard error of the mean. Columns labeled with different letters

significantly differ (P \ 0.05) between WT, VC, and PeHSF

transgenic lines. N.S., no significant difference

Fig. 4 Effects of NaCl (100 mM, 15 days) on K?, Na?, and Ca2?

concentrations and on the K?/Na? and Ca2?/Na? ratios in wild-type

(WT) tobacco and transgenic seedlings (#2 and #6). Each column

shows the mean of four individual plants, and bars represent the

standard error of the mean

304 Plant Cell Tiss Organ Cult (2013) 115:299–308

123

contains an N-terminal DNA-binding domain, an adjacent

domain with heptad hydrophobic repeats involved in

oligomerization, AHA motifs, and nuclear localization and

nuclear export sequences (Figs. S1, S2; Doring et al. 2000).

It has previously been determined that the binding of

transcription factor HSF to the HSE promoter sequences

controls the expression of Hsf-responsive genes (Damber-

ger et al. 1994; Harrison et al. 1994; Vuister et al. 1994;

Schultheiss et al. 1996). Here we confirmed the

transcriptional activity of PeHSF by yeast one-hybrid assay

(Fig. S3), and the rapid induction of PeHSF by NaCl

suggested that PeHSF may contribute to salinity tolerance

in the salt-resistant poplar (Fig. 1).

Under saline conditions, the transgenic plants that

overexpressed PeHSF had higher germination rates and

root lengths than wild-type tobacco (Figs. 2, 3, S4-S5). Our

results were consistent with those of Ogawa et al. (2007),

who confirmed that over-expression of the transcription

Time after NaCl treatment

0 h 3 h 24 h 15 d

WT

#2

#6

A

B

Fig. 5 Salt-induced H2O2

levels in roots of wild-type

(WT) tobacco and transgenic

lines (#2 and #6) after short-

term (100 mM, 24 h) or long-

term (100 mM, 15 days) NaCl

treatment. a Representative

confocal images of NaCl-

induced H2O2 production in WT

tobacco and transgenic lines.

b Mean H2DCF-DA

fluorescence intensity. Each

column shows the mean of 8–10

independent seedlings, and bars

represent the standard error of

the mean. *Significant

difference (P \ 0.05) between

WT and transgenic lines

Plant Cell Tiss Organ Cult (2013) 115:299–308 305

123

factor Arabidopsis HsfA2 gene confers salt and osmotic

stress tolerance. HSFA1 proteins in Arabidopsis were also

identified as being involved in the expression of several

heat shock genes induced by H2O2, salt, and mannitol (Liu

et al. 2011). In the present study, transgenic tobacco lines

overexpressing PeHSF exhibited increased capacity to

control ROS homeostasis, but not enhanced capacity to

retain ionic homeostasis under NaCl stress (Figs. 4, 5).

Moreover, under saline conditions, the PeHSF-transgenic

lines typically showed higher activities of antioxidant

enzymes (GR, GPX and APX) compared to in the wild-

type tobacco (Fig. 6), implying that PeHSF contributed to

the regulation of antioxidant defenses under NaCl stress.

Our results suggest that PeHSF plays a crucial role in ROS

detoxification, rather than in the maintenance of K?/Na?

under salt stress.

PeHSF expression contributes to ROS homeostasis

control in salinized plants

Under NaCl stress, PeHSF-transgenic tobacco plants dis-

played increased transcription of GR, GPX, and APX (Fig.

S6), and the gene expressions closely resembled their

activity profile, suggesting that the elevated antioxidant

enzyme activity in transgenic plants was due to their

transcript abundance (Figs. 6, S6). Our data indicate that

the genes encoding antioxidant enzymes are likely the

downstream targets of PeHSF. Previous studies have

demonstrated a link between Hsfs and antioxidant

enzymes. AtHsfA2 modulates APX1 expression and

enhances tolerance to heat and oxidative stress in Arabid-

opsis (Li et al. 2005). Moreover, high-level overexpression

of AtHsfA2 induces increased APX2 expression and salt/

osmotic stress tolerance (Ogawa et al. 2007). HsfA1b is

involved in regulating the expression of APX2, which

encodes ascorbate peroxidase to scavenge H2O2 in the

cytosol (Panchuk et al. 2002). It was recently demonstrated

that expression of the glutaredoxin family member At-

GRXS17 enhances thermotolerance in an agriculturally

important crop by mediating the anti-oxidative and HSP/

HSF systems (Wu et al. 2012).

Our present pharmacological studies revealed that the

PM NADPH oxidase inhibitor DPI and the PM Ca2?-per-

meable channel blocker LaCl3 each markedly decreased

PeHSF in salt-stressed P. euphratica plants (Fig. 1), indi-

cating the involvement of both H2O2 and Ca2? in HSF

activation. HSF is generally a direct sensor of H2O2 in

Drosophila and human cells (Zhong et al. 1998; Ahn and

Thiele, 2003). Volkov et al. (2006) found that HSF–HSE

binding activation is H2O2-dependent. These data indicate

that Arabidopsis class A HSF (AtHSFA1a or AtHSFA1b)

is a subject of oxidative stress activation (Volkov et al.

Fig. 6 Effects of NaCl (100 mM, 15 days) on total activities of

superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase

(APX), glutathione reductase (GR), and glutathione peroxidase

(GPX) in wild-type (WT) tobacco and transgenic lines (#2 and #6).

Each column shows the mean of three individual seedlings, and bars

represent the standard error of the mean. *Significant difference

(P \ 0.05) between WT and transgenic lines

306 Plant Cell Tiss Organ Cult (2013) 115:299–308

123

2006). Furthermore, HsfA4a is a candidate for a sensor

molecule involved in H2O2 perception in Arabidopsis

(Davletova et al. 2005), and Ca2? and calmodulin modulate

DNA-binding activity of Hsf in maize (Li et al. 2004).

Our previous studies have repeatedly shown that salt

stress results in an early H2O2 burst and elevation of

cytosolic Ca2? ([Ca2?]cyt) in P. euphratica callus cells

(Sun et al. 2010a, b, 2012). P. euphratica plants rapidly up-

regulated anti-oxidant enzyme activity upon NaCl expo-

sure (Wang et al. 2007, 2008). Overall, based on our

present data, we conclude that the salt-induced H2O2 and

[Ca2?]cyt led to transcriptional activation of HSF, which

initiated transcription of target genes encoding antioxidant

enzymes in P. euphratica, thus contributing to ROS

homeostasis control. However, future research is needed to

explore how HSFs respond to salt-elicited signals and

regulate the expression of downstream target genes.

Acknowledgments This research was supported jointly by the

National Natural Science Foundation of China (Grant Nos. 31270654,

31170570, and 31200470), the Key Grant Project of the Chinese Min-

istry of Education (Grant No. 2013001), the Beijing Natural Science

Foundation (Grant No. 6112017), the Program of Introducing Talents of

Discipline to Universities (111 Project, Grant No. B13007), Funda-

mental Research Funds for the Central Universities (Grant Nos.

JC2011-2 and TD-2012-04), the Foundation for the Supervisors of

Beijing Excellent Doctoral Dissertations (Grant No. YB20081002201).

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