Báo cáo khoa học: Cotton GhMPK2 is involved in multiple signaling pathways and mediates defense responses to pathogen infection and oxidative stress doc

Cotton GhMPK2 is involved in multiple signaling pathways
and mediates defense responses to pathogen infection and
oxidative stress
Liang Zhang
1
, Dongmei Xi
2
, Lu Luo
1
, Fei Meng
1
, Yuzhen Li
1
, Chang-ai Wu
1
and Xingqi Guo
1
1 State Key Laboratory of Crop Biology, Shandong Key Laboratory of Crop Biology, Shandong Agricultural University, Taian, Shandong, China
2 Experimental Center, Linyi University, Linyi, Shandong, China
Introduction
Plants are constantly exposed to a variety of biotic
and abiotic stresses. To survive these challenges, plants
have developed elaborate mechanisms to perceive
external signals and to manifest adaptive responses
with appropriate physiological and morphological
changes [1]. At the molecular level, the perception of
extracellular stimuli and the subsequent activation of
the defense responses require a complex interplay of
signaling cascades, in which reversible protein phos-
phorylation plays a central role [2]. The mitogen-acti-
vated protein kinase (MAPK) cascade is a highly

conserved pathway involved in the phosphorylation of
Keywords
cotton; disease resistance; GhMPK2;
oxidative stress; signaling pathways
Correspondence
X. Guo, State Key Laboratory of Crop
Biology, College of Life Sciences, Shandong
Agricultural University, Taian, Shandong,
271018, China
Fax: +86 538 8226399
Tel: +86 538 8245679
E-mail:
(Received 10 December 2010, revised 15
February 2011, accepted 18 February 2011)
doi:10.1111/j.1742-4658.2011.08056.x
Mitogen-activated protein kinase (MAPK) cascades play important roles in
mediating pathogen responses and reactive oxygen species signaling. In
plants, MAPKs are classified into four major groups (A–D). Previous stud-
ies have mainly focused on groups A and B, but little is known about
group C. In this study, we functionally characterized a stress-responsive
group C MAPK gene (GhMPK2) from cotton. Northern blot analysis indi-
cated that GhMPK2 was induced not only by signaling molecules, such as
ethylene and methyl jasmonate, but also by methyl viologen-mediated oxi-
dative stress. Transgenic tobacco (Nicotiana tabacum) plants that overex-
press GhMPK2 displayed enhanced resistance to fungal and viral
pathogens, and the expression of the pathogenesis-related (PR) genes,
including PR1, PR2, PR4, and PR5, was significantly increased. Interest-
ingly, the transcription of 1-aminocyclopropane-1-carboxylic acid synthase
(ACS) and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) was sig-
nificantly upregulated in transgenic plants, suggesting that GhMPK2 posi-

tively regulates ethylene synthesis. Moreover, overexpression of GhMPK2
elevated the expression of several antioxidant enzymes, conferring on trans-
genic plants enhanced reactive oxygen species scavenging capability and
oxidative stress tolerance. These results increased our understanding of the
role of the group C GhMPK2 gene in multiple defense-signaling pathways,
including those that are involved in responses to pathogen infection and
oxidative stress.
Abbreviations
ABA, abscisic acid; ACC, 1-aminocyclopropane-1-carboxylic acid; ACO, 1-aminocyclopropane-1-carboxylic acid oxidase; ACS,
1-aminocyclopropane-1-carboxylic acid synthase; APX, ascorbate peroxidase; CAT, catalase; CMV, cucumber mosaic virus; CP, coat protein;
DAB, 3,3¢-diaminobenzidine; EREBP, ethylene-responsive element-binding protein; ET, ethylene; GST, glutathione-S-transferase; JA, jasmonic
acid; MAPK, mitogen-activated protein kinase; MeJa, methyl jasmonate; MV, methyl viologen; PR, pathogenesis-related; RbohD, respiratory
burst oxidase homolog; ROS, reactive oxygen species; SA, salicylic acid; SOD, superoxide dismutase; TMV, tobacco mosaic virus.
FEBS Journal 278 (2011) 1367–1378 ª 2011 The Authors Journal compilation ª 2011 FEBS 1367
a wide range of substrates, and it has been suggested
to be the integrative point of multiple pathways [3].
The MAPK cascade consists of three interlinked
protein kinases: a MAPKKK kinase, a MAPKK, and
a MAPK [4]. Signals from extracellular stimuli are
transmitted into the cell and are sensed by downstream
targets via the sequential phosphorylation of a MAP-
KKK, a MAPKK, and a MAPK [5]. The phosphory-
lation and activation of an MAPK can lead to changes
in its subcellular localization and its interaction with
transcriptional effectors, which reprograms gene
expression [6]. Plant genome sequencing projects have
revealed the existence of 20 MAPKs in Arabidopsis [4],
17 in rice [7], and 21 in poplar [8]. The MAPKs can be
classified into four major groups (A, B, C, and D) on
the basis of their sequence homology and the con-

served phosphorylation motifs [4].
In plants, several lines of evidence have revealed that
the MAPK cascades can both positively and negatively
mediate pathogen signal transduction [9]. In tobacco,
salicylic acid (SA)-induced protein kinase and wound-
induced protein kinase, two MAPKs in group A, were
activated by inoculation with the tobacco mosaic virus
(TMV) [10]. Their orthologs in other plant species,
including MPK3 and MPK6 in Arabidopsis (group A
MAPKs), SIMK and SAMK in alfalfa, and
LeMPK1 ⁄ 2(Lycopersicon esculentum SA-induced pro-
tein kinase) and LeMPK3 (L. esculentum wound-
induced protein kinase) in tomato, are all involved in
defense-related signal transduction [11]. In contrast,
transposon inactivation of the Arabidopsis group B
MPK4 gene conferred enhanced disease resistance and
constitutive activation of defense responses, indicating
that MPK4 functions as a negative regulator of
systemic acquired resistance [12]. In rice, group A
OsMAPK5 negatively modulates pathogenesis-related
(PR) gene expression and broad-spectrum disease resis-
tance [13].
Abiotic and biotic stresses are typically associated
with the rapid production of reactive oxygen species
(ROS), including hydrogen peroxide (H
2
O
2
), superox-
ide anion (O

À
2
), and hydroxyl radicals [14]. ROS are
known to play dual roles, depending on their levels;
moderate accumulation of ROS plays a central role in
the regulation of biological processes, such as hormone
signaling, biotic and abiotic stress responses, and
development [15,16], whereas high concentrations of
ROS can result in oxidative stress and cause irrevers-
ible damage and hypersensitive response-like cell death
[14,17]. MAPKs are key players in ROS signaling. Sev-
eral studies have shown that MAPK signaling path-
ways are not only induced by ROS but can also
regulate ROS production [15,18]. For example, H
2
O
2
activates AtMPK12 in Arabidopsis and MMK3 in
alfalfa [19,20], whereas constitutive expression of Ara-
bidopsis MKK4 or MKK5 results in the generation of
H
2
O
2
[21]. One of the mechanisms that contributes to
ROS-induced pathogen tolerance is the activation of a
large number of enzymatic and nonenzymatic antioxi-
dants, such as glutathione-S-transferases (GSTs),
ascorbate peroxidases (APXs), superoxide dismutases
(SODs), and catalases (CATs) [14]. However, evidence

for the effect of MAPKs on antioxidant gene expres-
sion is lacking.
Previous studies have mainly focused on group A
and B MAPKs; information about group C MAPKs is
relatively limited and has emerged only recently. It has
been shown that Arabidopsis thaliana AtMPK1 and
AtMPK2 are induced by wounding, jasmonic acid
(JA), abscisic acid (ABA), and H
2
O
2
[22–24]. The
expression patterns of pea PsMPK2 in Arabidopsis
revealed that its kinase activity increases in response to
mechanical injury and other stress signals, including
ABA, JA, and H
2
O
2
[23]. Recently, it was shown that
maize ZmMPK7 was induced by ABA and H
2
O
2
, and
that H
2
O
2
may be required for ZmMPK7-mediated

ABA signaling [24]. Increasing evidence has revealed
that group C MAPKs are involved in various signaling
processes and have unique biological functions.
Cotton (Gossypium hirsutum) is one of the oldest
and most important fiber and oil crops. Its growth and
yield are severely inhibited under various biotic and
abiotic stress conditions. To date, few MAPKs have
been identified in cotton, and their function has not
been well documented. In our previous work,
GhMAPK was the first group C MAPK isolated from
cotton, and it was shown to be activated by wounding,
cold, salt, SA, H
2
O
2
, and pathogens [25]. However, its
in vivo function has not been elucidated. In this study,
a more detailed analysis of GhMAPK was carried out
in tobacco. It should be noted that GhMAPK has been
renamed as GhMPK2 on the basis of the nomenclature
for plant MAPKs [4]. Our results showed that
GhMPK2 was strongly induced by ethylene (ET), JA,
and methyl viologen (MV). The ectopic expression of
GhMPK2 in transgenic tobacco plants led to enhanced
resistance to fungi and viruses. This resistance was
most probably attributable to elevated constitutive lev-
els of basal resistance, because uninfected plants
showed upregulated expression of PR and ET biosyn-
thesis genes. Moreover, plants that overexpressed
GhMPK2 showed an increased ability to scavenge

ROS and to tolerate oxidative stress. Thus, this study
suggests a role for GhMPK2 in the defense signaling
pathways in response to both pathogen infection and
oxidative stress.
Cotton GhMPK2 is involved in multiple signaling pathways L. Zhang et al.
1368 FEBS Journal 278 (2011) 1367–1378 ª 2011 The Authors Journal compilation ª 2011 FEBS
Results
Expression of GhMPK2 was upregulated by
oxidative stress and phytohormones
To study the effect of oxidative stress and signaling
molecules on the expression of GhMPK2, total RNA
was extracted from 7-day-old cotton seedlings treated
with 10 lm MV, ET released from 5 mm ethephon or
100 lm methyl jasmonate (MeJA) for northern blot
analysis. As shown in Fig. 1, GhMPK2 expression was
strongly induced at 2 h and then reduced at 4 h in the
presence of MV or MeJA. After ethephon treatment,
GhMPK2 transcripts showed a gradual increase from
2 h to 4 h. Because Ag
+
acts as an ET action repres-
sor by blocking ET signaling [26], 100 lm AgNO
3
alone or 100 lm AgNO
3
with 5 mm ethephon
(ET + Ag
+
) was applied to the cotton seedlings. The
results showed that the expression of GhMPK2 was

significantly increased by AgNO
3
at 2 h, and that this
was followed by a slight decrease at 4 h. However,
when treated with ET + Ag
+
, GhMPK2 mRNA rap-
idly accumulated at 2 h and was almost undetectable
at 4 h. Comparison of the expression patterns after
treatment with ET + Ag
+
and with ET alone revealed
that the induction of GhMPK2 by ethephon was partly
blocked by the addition of Ag
+
. These results suggest
that GhMPK2 might be involved in the oxidative stress
response and in the JA ⁄ ET signaling pathway.
Overexpression of GhMPK2 in tobacco plants
improved viral resistance
To investigate the functional roles of GhMPK2 in
plant defense, we generated transgenic tobacco plants
that constitutively expressed GhMPK2 under the con-
trol of the cauliflower mosaic virus 35S promoter. A
total of 19 independent transgenic lines were obtained
by kanamycin resistance selection, and were confirmed
by genomic PCR detection (data not shown). Three
representative lines (OE1, OE2, and OE3) of T
3
prog-

eny were randomly selected for further investigation.
Northern blot analysis showed that the transgenic lines
accumulated much higher levels of GhMPK2 tran-
scripts than the wild-type. As expected, the myelin
basic protein kinase activity of GhMPK2 was signifi-
cantly increased in the transgenic lines grown under
normal conditions (Fig. 2).
To analyze the viral resistance responses in the
transgenic tobacco plants, 8-week-old plants were inoc-
ulated with TMV and cucumber mosaic virus (CMV).
To ensure consistent comparison of the symptoms
observed on the systemically infected leaves between
the wild-type and OE plants, the sixth or seventh true
leaves were inoculated with TMV or CMV, and the
10th true leaves were used for the determination of
virus accumulation and photography. Fourteen days
after the inoculation, leaf curling, stunting and other
abnormalities appeared in the wild-type plants
(Fig. 3A). However, only slight disease symptoms were
observed on the transgenic plants, and no symptoms
were observed on the mock-inoculated plants. The root
fresh weight of the transgenic plants was much higher
than that of the wild-type plants (Fig. 3B). In addition,
the expression of the viral coat protein (CP) gene was
detected by semiquantitative RT-PCR, and the results
showed that virus accumulation in the transgenic lines
was much lower than in the wild-type plants (Fig. 3C).
Quantitative ELISA analysis revealed that the accumu-
lation of TMV or CMV particles in the wild-type
plants was approximately 1.5-fold higher than the

average value for the transgenic lines at 21 days post-
inoculation (Fig. 3D). These results indicate that
overexpression of GhMPK2 confers enhanced viral
resistance in transgenic plants.
Fig. 1. Northern blot analysis of GhMPK2 expression induced by
stresses and hormone signals. One-week-old cotton seedlings
were treated with 10 l
M MV, ET released from 5 mM ethephon,
100 l
M MeJA, and 100 lM AgNO
3
alone or 100 lM AgNO
3
with
5m
M ethephon. a-
32
P-labeled GhMPK2 cDNA was used as the
probe. Ethidium bromide-stained rRNA was included as a loading
control. The gene expression level at 2 h after treatment with dis-
tilled water served as the control (C).
Fig. 2. GhMPK2 expression analysis in wid-type and T
1
transgenic
tobacco plants. Three representative lines (OE1, OE2, and OE3)
grown under normal conditions were selected for northern blot
analysis and in-gel kinase activity assay. Ethidium bromide-stained
rRNA and Coomassie Brilliant Blue (CBB)-stained blots were
included as the loading controls. WT, wild-type.
L. Zhang et al. Cotton GhMPK2 is involved in multiple signaling pathways

FEBS Journal 278 (2011) 1367–1378 ª 2011 The Authors Journal compilation ª 2011 FEBS 1369
Overexpression of GhMPK2 conferred enhanced
resistance to fungal pathogens
To determine the effect of GhMPK2 overexpression on
fungal resistance, tobacco plants were challenged with the
Fusarium oxysporum and Phytophthora parasitica patho-
gens. Ten days postinoculation, wild-type leaves exhibited
wilting and yellowing with necrotic lesions, and the stems
displayed significant black shank. However, the trans-
genic plants showed less severe or no disease symptoms
(Fig. 4A,B). Typically, the pathogen mainly invades
plants at the root tips, so close attention was paid to the
growth phenotype of the roots. The roots of the trans-
genic plants developed much better than those of the
wild-type plants, with reduced growth inhibition and only
partial secondary root browning. These results indicate
that overexpression of GhMPK2 greatly enhances resis-
tance to fungal pathogens in transgenic tobacco plants.
Overexpression of GhMPK2 activated disease
response and ET biosynthesis genes in
transgenic tobacco plants
To elucidate the possible mechanisms of enhanced path-
ogen resistance in transgenic plants, the expression
levels of several disease-responsive genes, including PR1a,
PR2 (b-1,3-glucanase), PR4, PR5 (osmotin), SAR8.2l,
and CBP20, were determined by northern blot analysis.
As shown in Fig. 5, the transcripts of these genes were
significantly upregulated in the transgenic plants, with
the exception of that of CBP20, whose accumulation
was not obviously altered. Notably, PR1a and PR5 are

the marker genes for SA signaling, and PR4 is the mar-
ker gene for MeJA signaling. Thus, we propose that the
GhMPK2-dependent activation of the PR genes plays a
key role in enhanced disease resistance in transgenic
plants, which might be related to SA-dependent and
MeJA-dependent signaling pathways.
In addition, we examined the expression of two key
enzymes that are involved in ET biosynthesis, 1-amino-
cyclopropane-1-carboxylic acid (ACC) synthase (ACS)
and ACC oxidase (ACO), which catalyze the conver-
sion of S-adenosyl-l-methione into ACC, and the oxi-
dative cleavage of ACC to form ET, respectively [27].
As shown in Fig. 5, the transcriptional levels of ACS
and ACO were significantly increased in transgenic
plants without any stress treatment. The data indicate
that GhMPK2 positively regulates ET synthesis in
plants, suggesting a possible role for GhMPK2 in the
ET signaling pathway.
A
B
CD
Fig. 3. Enhanced viral resistance against
TMV and CMV in transgenic tobacco plants
overexpressing GhMPK2. (A) Leaf and root
symptoms of tobacco plants infected with
TMV and CMV at 14 days postinoculation
(dpi). Mock: mock inoculation. (B) The root
weight of the transgenic plants and the con-
trol plants at 14 days postinoculation. Differ-
ent letters above the columns indicate

significant differences (P < 0.01) according
to Duncan’s multiple range test. (C) Expres-
sion analysis of virus CP genes by semi-
quantitative RT-PCR. (D) Virus accumulation
as determined by ELISA. Data in (B) and (D)
are the mean ± standard error from three
independent experiments.
Cotton GhMPK2 is involved in multiple signaling pathways L. Zhang et al.
1370 FEBS Journal 278 (2011) 1367–1378 ª 2011 The Authors Journal compilation ª 2011 FEBS
GhMPK2 regulated the accumulation of ROS
Plants respond to pathogen attack by activating various
defense responses. The production of ROS often follows
pathogen invasion, and plays a critical role in defense
responses [28]. To examine whether the enhanced dis-
ease resistance of the transgenic plants is associated
with ROS accumulation, tobacco plants were inoculated
with viruses or treated with NaCl, poly(ethylene glycol),
and H
2
O
2
. The accumulation of H
2
O
2
and O
À
2
, which
are the major ROS, was determined by 3,3¢-diam-

inobenzidine (DAB) and Nitro Blue tetrazolium (NBT)
staining, respectively. Under normal growth conditions,
no obvious H
2
O
2
was detected in either wild-type or
transgenic plants. After TMV or CMV infection for
10 days, DAB staining of the third upper systemic
leaves showed significantly reduced H
2
O
2
production in
transgenic plants as compared with wild-type plants
(Fig. 6A). After treatment with NaCl, poly(ethylene gly-
col), and H
2
O
2
, the accumulation of H
2
O
2
was remark-
ably lower in the transgenic lines than in the wild-type
plants at 4 h (Fig. 6B). Similarly, NBT staining showed
less O
À
2

accumulation in the leaves of the transgenic
lines (Fig. 6B). The transgenic plants produced a lower
amount of ROS, suggesting that overexpression of
GhMPK2 either inhibited ROS production or effectively
scavenged excess ROS.
GhMPK2-overexpressing plants displayed
increased tolerance to oxidative stress
Because GhMPK2-overexpressing tobacco plants
showed reduced ROS accumulation in response to bio-
tic and abiotic stresses (Fig. 6), the possible protective
A
B
Fig. 4. Enhanced fungal resistance against pathogenic F. oxyspo-
rum and P. parasitica in GhMPK2 transgenic tobacco plants. (A)
Symptoms of the leaves, stems and roots of the tobacco plants
inoculated with F. oxysporum at 10 days postinoculation. (B) Symp-
toms of the leaves and stems of tobacco plants inoculated with
P. parasitica at 10 days postinoculation. WT, wild-type.
A
B
Fig. 5. Expression of the defense-related genes and ET biosynthe-
sis genes in transgenic and wild-type plants. (A) The mRNA levels
of defense-related genes and ET biosynthesis genes in transgenic
and wild-type tobacco plants without any stress, analyzed by north-
ern blot. Ethidium bromide-stained rRNA was included as a loading
control. (B) The relative expression levels of the defense-related
genes and ET biosynthesis genes in transgenic and wild-type
tobacco plants. Transcriptional levels of these genes in transgenic
tobacco are indicated relative to the level of wild-type tobacco,
taken as 1, referring to the transcripts of CBP20 in the same sam-

ples. WT, wild-type.
L. Zhang et al. Cotton GhMPK2 is involved in multiple signaling pathways
FEBS Journal 278 (2011) 1367–1378 ª 2011 The Authors Journal compilation ª 2011 FEBS 1371
role of GhMPK2 against oxidative stress was evaluated
by testing the tolerance of the plants to MV. In 1 ⁄ 2
Murashige–Skoog medium without addition of MV,
no difference was observed between the wild-type and
transgenic lines. However, a large variation in the ger-
mination rate occurred in the presence of MV
(Fig. 7A,B). Four days after sowing, 5 lm MV severely
inhibited the germination of the wild-type seeds, which
had a germination rate of only 14%, whereas the
transgenic seeds displayed a high tolerance to MV,
achieving a 32% germination rate. At a higher dose
(10 lm MV), the remarkable protection against MV
was still observed in the GhMPK2-overexpressing lines,
with an approximately 20% higher germination rate
than in the wild-type on day 4, a 16% higher rate on
day 6, and a 13% higher rate on day 8. Measurements
of the root length and fresh weight revealed a similar
pattern (Fig. 7C,D). These data suggest that the over-
expression of GhMPK2 may improve tolerance to oxi-
dative stress in transgenic plants during seed
germination.
Oxidative stress tolerance in transgenic plants was
further studied by testing the tolerance of leaf disks
from 8-week-old plants to exogenous MV. As shown
in Fig. 7E, the discs incubated in water without MV
showed no abnormalities. After incubation in different
concentrations of MV for 72 h, symptoms of bleaching

or chlorosis appeared in the leaf disks from both wild-
type and transgenic plants. However, MV treatment
led to more severe damage in the wild-type plants.
This result was further confirmed by measuring the
chlorophyll content in the leaf disks after MV treat-
ment (Fig. 7F). These results indicate that overexpres-
sion of GhMPK2 confers enhanced tolerance to
oxidative stress during the vegetative stage.
Expression of antioxidant enzymes was
upregulated in transgenic tobacco plants
Plants have evolved antioxidant defense systems to dis-
pose of excess ROS and to maintain cellular ROS
homeostasis [17]. To investigate the possible underlying
mechanisms of the enhanced oxidative stress tolerance
in transgenic plants, the expression of genes that encode
ROS-scavenging enzymes, such as MnSOD, CAT1,
APX, and GST, as well as the ROS producer, respira-
tory burst oxidase homolog (RbohD), was determined
by northern blot analysis. The mRNA levels of CAT1
and APX, and particularly of MnSOD and GST, were
greatly upregulated in the transgenic plants, whereas
RbohD transcripts showed no obvious difference
between the transgenic and wild-type plants (Fig. 8A,B).
Moreover, after treatment with NaCl or poly(ethylene
glycol) 6000, the total activities of the antioxidant
enzymes SOD, CAT and APX in the transgenic plants
were significantly higher than in the wild-type (Fig. 8C).
These results indicate that the enhanced oxidative stress
tolerance in GhMPK2-overexpressing plants is conferred
by upregulation of the expression of multiple antioxi-

dant enzymes, and suggest that GhMPK2 may be
involved in the regulation of ROS network pathways.
Discussion
The role and significance of group C MAPKs in
response to biotic and abiotic stresses have only
recently begun to emerge [22–24]. In the present study,
we describe the characterization of the cotton
GhMPK2 gene, which belongs to the group C MAPK
family. Our results suggest that GhMPK2 plays impor-
tant roles in disease resistance responses and oxidative
stress tolerance by triggering the expression of defense-
related genes and antioxidant genes, respectively. The
findings not only extend our knowledge of the biologi-
A
B
Fig. 6. Analysis of ROS accumulation in wild-type and transgenic
plants in response to abiotic and abiotic stresses. (A) Virus infec-
tion-induced H
2
O
2
accumulation detected by DAB staining. (B) Abi-
otic stress-induced H
2
O
2
and O
À
2
accumulation detected by DAB

staining and NBT staining, respectively. WT, wild-type.
Cotton GhMPK2 is involved in multiple signaling pathways L. Zhang et al.
1372 FEBS Journal 278 (2011) 1367–1378 ª 2011 The Authors Journal compilation ª 2011 FEBS
cal function of group C MAPKs, but also provide new
insights for further exploration of the significance of
GhMPK2 in the regulation of plant defense responses.
It has been previously established that the signaling
molecules SA, ET and JA play important roles in the
regulation of the complex defense mechanisms [29]. SA
is an essential signaling molecule that induces systemic
acquired ressitance and is implicated in resistance to
biotrophic pathogens [29–31]. ET and JA are typically
associated with the defense responses to necrotrophic
pathogens and herbivorous insects [29,32]. In Arabid-
opsis, AtMPK4 responds to the balance between SA
A
B
C
E
F
D
Fig. 7. Overexpression of GhMPK2 confers increased tolerance to oxidative stress. (A) Seed germination in the presence of the indicated
MV concentrations. (B) Germination rates of the wild-type and OE lines shown in (A). Different letters above the columns indicate significant
differences (P < 0.05) according to Duncan’s multiple range test. (C, D) The phenotypes of the plants treated with MV are shown in (C), and
their corresponding relative root lengths and fresh weights are shown in (D). Different letters above the columns indicate significant differ-
ences (P < 0.05) according to Duncan’s multiple range test. (E) Leaf disks from wild-type and transgenic plants were infiltrated with different
concentrations of MV (0, 5 and 10 l
M). (F) Relative chlorophyll contents in the leaf disks after MV treatments. The data in (B), (D) and (F) are
the mean ± standard error from three independent experiments. WT, wild-type.
L. Zhang et al. Cotton GhMPK2 is involved in multiple signaling pathways

FEBS Journal 278 (2011) 1367–1378 ª 2011 The Authors Journal compilation ª 2011 FEBS 1373
and MeJA through the EDS1–PAD4 module, and
regulates the SA-mediated and JA ⁄ ET-mediated
defence responses [33]. Like AtMPK4, GhMPK2 might
be involved in the crosstalk between the SA-mediated
and JA ⁄ ET-mediated pathogen defense signaling path-
ways. Generally, the gene expression pattern is an indi-
cation of gene function. A remarkable increase in the
expression of GhMPK2 was observed in the cotton
seedlings treated with exogenous ET and JA (Fig. 1).
Our previous report showed that the transcriptional
levels of GhMPK2 could be greatly upregulated by SA
treatment [25]. These results imply that GhMPK2 may
play roles in both plant defense responses and in the
regulation of certain components of multiple stress-sig-
naling pathways. Consistent with this hypothesis,
sequence analysis of the GhMPK2 promoter (GenBank
accession no. HM150999), using the PLACE and
PlantCARE databases, revealed the existence of an
SA-responsive element, as-1, and an MeJA-responsive
cis-acting regulatory element (CGTCA motif ⁄ TGACG
motif) (data not shown). More direct evidence was
obtained from functional analysis of ectopically
expressed GhMPK2 in Nicotiana tabacum. As shown in
Figs 3 and 4, the transgenic plants displayed enhanced
resistance to both viruses and fungi. In addition, the
expression of the marker genes from various pathways
(PR1a and PR5 for SA signaling; PR4 for MeJA sig-
naling) was greatly elevated (Fig. 5). Furthermore,
A

C
B
Fig. 8. Overexpression of GhMPK2 acti-
vates antioxidant enzymes in transgenic
tobacco plants. (A) The mRNA levels of oxi-
dative stress-related genes in the transgenic
and wild-type tobacco plants analyzed by
northern blot analysis. The ethidium bro-
mide-stained rRNA was included as a load-
ing control. (B) The relative expression
levels of the oxidative stress-related genes
in the transgenic and wild-type tobacco
plants. Transcriptional levels of these genes
in transgenic tobacco are indicated relative
to the level of wild-type tobacco, taken as 1,
referring to the transcripts of CAT1 in the
same samples. (C) The total activities of the
antioxidant enzymes SOD, CAT and APX in
the tobacco plants when treated with NaCl
or poly(ethylene glycol) 6000. Data are the
means ± standard errors of three indepen-
dent experiments. Different letters above
the columns indicate significant differences
(P < 0.05) according to Duncan’s multiple
range test. FW, fresh weight; WT,
wild-type.
Cotton GhMPK2 is involved in multiple signaling pathways L. Zhang et al.
1374 FEBS Journal 278 (2011) 1367–1378 ª 2011 The Authors Journal compilation ª 2011 FEBS
GhMPK2 positively regulated ET synthesis in plants,
as shown by the significant increase in expression of

the ET biosynthesis genes ACS and ACO (Fig. 5).
Thus, it is reasonable to speculate that GhMPK2 may
inhibit the pathogens by influencing both the SA-medi-
ated and the JA ⁄ ET-mediated defense responses.
In plants, ROS have been implicated in the damag-
ing effects of various environmental stresses. How-
ever, cells have evolved strategies to utilize ROS in
multiple biological pathways [34]. In Arabidopsis,
H
2
O
2
activates the MKK3–MPK7 module, which
induces target genes, such as PR1, and therefore acti-
vates the defense responses [35]. As shown by Naka-
gami et al. [36], Arabidopsis MPK4 is the downstream
target of MEKK1, and MEKK1–MPK4 can maintain
ROS homeostasis by regulating the expression of a
group of redox-related genes. In the present study,
our results suggest that GhMPK2 may play key roles
in ROS homeostasis and that ROS-mediated injury
may be effectively alleviated by the induction of
GhMPK2. On the one hand, GhMPK2 was strongly
induced in response to MV, which can cause continu-
ous formation of O
À
2
(Fig. 1), suggesting that ROS
participate in the activation of GhMPK2. On the
other hand, GhMPK2 was demonstrated to regulate

ROS production. As shown in Fig. 6, GhMPK2 trans-
genic lines accumulated much lower amounts of ROS
(mainly H
2
O
2
and O
À
2
) in the presence of NaCl,
poly(ethylene glycol), and H
2
O
2
, which can trigger
the excessive accumulation of ROS. Consistent with
this finding, the transgenic plants developed signifi-
cantly better than the wild-type plants during seed
germination and the vegetable growth stage when
treated with MV (Fig. 7). Further analysis revealed
that the increase in oxidative stress tolerance was
achieved through the constitutive upregulation of
multiple antioxidant enzymes in the GhMPK2 trans-
genic lines (Fig. 8). Therefore, the excessive accumula-
tion of ROS may induce the expression of GhMPK2
and lead to the direct or indirect upregulation of
antioxidant genes, which will result in the scavenging
of excessive ROS and the maintenance of the ROS at
moderate levels. This study provides direct evidence
of a link between MAPK and antioxidant genes.

It has been reported that plant MAPKs can phos-
phorylate transcription factors, such as ET-responsive
element-binding proteins (EREBPs) and WRKYs,
which then triggers the expression of the PR genes
[9,37]. EREBPs, which are implicated in the ET signal-
ing pathway, can bind to the GCC box DNA motif
(AGCCGCC) of the promoters of several PR genes,
such as PR1a, PR2, PR4, and PR5, in tobacco [38].
OsEREBP1 has been demonstrated to be phosphory-
lated as the substrate by a MAPK in rice, BWMK1
[39]. In addition, WRKYs can specifically recognize
W-box elements that are conserved in the promoters of
many PR genes (PR1, PR2, PR3, and PR5), and there-
fore induce the expression of these defense genes
[40,41]. In this study, the mRNA levels of these PR
genes were all upregulated in the transgenic plants.
Therefore, we speculate that EREBPs and WRKYs
may play important roles downstream of GhMPK2 in
pathogen defense signaling.
On the basis of these observations, we propose that
there are at least two regulatory pathways for
GhMPK2, one of which responds to pathogens and the
other of which is involved in oxidative stress responses.
GhMPK2 may serve as a crosstalk point between biotic
and abiotic stress responses. Further investigation is
required for a more comprehensive understanding of the
functional roles and mechanisms of action of GhMPK2
in plant defense. As we learn more about MAPKs
and their regulation, the design of efficient strategies for
crop improvement should become possible.

Experimental procedures
Plant materials, growth conditions, and
treatments
Cotton (G. hirsutum L. cv. lumian 22) seeds were surface-ster-
ilized and germinated on 1 ⁄ 2 Murashige–Skoog medium. The
seedlings were grown in a growth chamber under greenhouse
conditions of 28 °C with a 16 h light ⁄ 8 h dark cycle. For the
MV, MeJA, and ET treatments, 7-day-old cotton seedlings
were sprayed with 10 lm MV, 100 lm MeJA, and 5 mm ethe-
phon (ET-releasing chemical), respectively. For the AgNO
3
treatment, 100 lm AgNO
3
alone or 100 lm AgNO
3
in combi-
nation with 5 mm ethephon was applied to the seedlings.
Vector construction and genetic transformation
The GhMPK2 cDNA (GenBank accession no. DQ132852)
was inserted into the binary vector pBI121 under the control
of the cauliflower mosaic virus 35S promoter via BamHI and
SacI sites. The recombinant plasmid was electroporated into
Agrobacterium tumefaciens (strain LBA4404) for tobacco
transformation with the leaf disk method, and the transfor-
mants were screened for kanamycin (100 mgÆL
)1
) resistance.
The transgenic T
3
lines were used in the experiments.

Northern blot analyses
With an RNeasy Mini Kit (Qiagen, MD, USA), 20 lgof
total RNA was extracted according to the manufacturer’s
instructions, and was separated on a 1% agarose–
formaldehyde gel. RNA was transferred onto Hybond-N
+
L. Zhang et al. Cotton GhMPK2 is involved in multiple signaling pathways
FEBS Journal 278 (2011) 1367–1378 ª 2011 The Authors Journal compilation ª 2011 FEBS 1375
membranes, and northern blot hybridizations were per-
formed as previously described [25].
In-gel kinase activity assay
The in-gel kinase activity assay was performed as previously
described [21], with a slight modification. Extracts contain-
ing 40 lg of protein were electrophoresed on 12%
SDS ⁄ polyacrylamide gels with 0.25 mgÆmL
)1
myelin basic
protein embedded in the separating gel as a kinase substrate.
Pathogen infection
For virus infection, 8-week-old tobacco plants were inocu-
lated with 100 lL of TMV and CMV suspension inoculum
(TMV and CMV in 50 mm phosphate buffer, pH 7.2) by
rubbing the fully expanded true leaves with wet carborun-
dum, and then immediately rinsing with deionized water.
Tobacco plants inoculated with 100 lL of buffer were used
as controls. For inoculation with pathogenic fungi, F. oxy-
sporum f. sp. vasinfectum and P. parasitica var. nicotianae
Tucker were cultured on potato dextrose agar (PDA) med-
ium at 25° C for 15 days, and the conidia were then sus-
pended in 1% glucose. Eight-week-old wild-type and T

3
OE
tobacco plants were inoculated by irrigation with F. oxy-
sporum and P. nicotianae Tucker spore suspensions
(10
6
conidiaÆmL
)1
), respectively. The inoculated plants were
maintained under greenhouse conditions.
ELISA detection of virus accumulation
Virus accumulation was determined at both the mRNA
and protein levels by semiquantitative RT-PCR and
ELISA, respectively. For the ELISA, TMV and CMV CPs
were used to prepare the polyclonal antiserum. A
1 : 5000 (v ⁄ v) dilution of horseradish peroxidase-conjugated
goat anti-(rat IgG) (Dingguo, Beijing, China) was used to
detect the antibody against CP. Absorbance was measured
at 492 nm with an ELISA plate reader. Upper noninoculat-
ed true leaves were harvested at the specified times for
mRNA and protein extraction.
Analysis of ROS scavenging on the basis of H
2
O
2
and O
2
2
staining
For H

2
O
2
staining, 3-week-old tobacco seedlings were trea-
ted with 200 mm NaCl, 15% poly(ethylene glycol) 6000 and
100 mm H
2
O
2
for 4 h by smearing the leaves with a cotton
bud. The leaves were then collected and incubated in a
DAB solution (1 mgÆmL
)1
, pH 3.8) for 12 h at 25 °Cinthe
dark. After staining, the leaves were soaked in 95% ethanol
overnight to remove the chlorophyll. In addition, the
accumulation of H
2
O
2
in 8-week-old tobacco plants that
had been inoculated with TMV and CMV for 10 days was
evaluated by DAB staining. For superoxide detection,
tobacco leaves were treated with 200 mm NaCl, 15%
poly(ethylene glycol) 6000 and 100 mm H
2
O
2
for 4 h. The
leaves were then collected and incubated in a Nitro Blue

tetrazolium solution (0.1 mgÆmL
)1
) for 24 h at room tem-
perature in the dark. After staining, the leaves were soaked
in 95% ethanol overnight for chlorophyll removal. Seed-
lings treated with water were used as controls.
Expression analysis of defense-related genes in
transgenic tobacco plants
To study the possible effects of GhMPK2 overexpression in
tobacco on defense-related genes, PR1a (X06361), PR2
(M60460), PR4 (X58546), osmotin (M29279), SAR8.2l
(NTU96152), pathogen-inducible and wound-inducible anti-
fungal protein gene (CBP20, S72452), ACS (AJ005002),
ACO (AB012857), GST (D10524), MnSOD (AB093097),
CAT1 (NTU93244), APX (AF443182) and RbohD
(AJ309006) were used for northern blot analyses.
Enzyme activity assays
Tobacco seedlings were treated with 200 mm NaCl and 15%
poly(ethylene glycol) 6000 for 4 days, and 0.5 g of leaves
was then collected for SOD, CAT and APX measurements,
which were performed as previously described [42].
Analysis of the response of transgenic plants to
oxidative stress
To observe the growth performance of tobacco plants
under oxidative stress conditions, the wild-type and trans-
genic seeds were surface-sterilized and germinated on 1 ⁄ 2
Murashige–Skoog medium supplemented with different
concentrations of MV (0, 5 and 10 lm). Leaf disks 1.3 cm
in diameter were detached from healthy and fully expanded
tobacco leaves of wild-type and transgenic plants at the

same age. The disks were floated in solutions of various
concentrations of MV (0, 5 and 10 lm) for 72 h, and then
immersed in 80% acetone for 48 h to extract the chloro-
phyll; the disks were then subjected to spectrophotometric
measurement of chlorophylls a and b. The experiment was
repeated at least twice, with five leaf disks each, for each of
the transgenic lines.
Acknowledgements
This work was financially supported by the Genetically
Modified Organisms Breeding Major Projects of China
(2009ZX08009-092B) and the National Natural Science
Foundation of China (Grant no. 30970225).
Cotton GhMPK2 is involved in multiple signaling pathways L. Zhang et al.
1376 FEBS Journal 278 (2011) 1367–1378 ª 2011 The Authors Journal compilation ª 2011 FEBS
References
1 Bohnert HJ, Nelson DE & Jensen RG (1995) Adapta-
tions to environmental stresses. Plant Cell 7, 1099–1111.
2 Colcombet J & Hirt H (2008) Arabidopsis MAPKs:
a complex signalling network involved in multiple
biological processes. Biochem J 413, 217–226.
3 Jonak C, Okresz L, Bogre L & Hirt H (2002) Complex-
ity, cross talk and integration of plant MAP kinase sig-
nalling. Curr Opin Plant Biol 5, 415–424.
4 MAPK Group (2002) Mitogen-activated protein kinase
cascades in plants: a new nomenclature. Trends Plant
Sci 7, 301–308.
5 Zhang S & Liu Y (2001) Activation of salicylic acid-
induced protein kinase, a mitogen-activated protein
kinase, induces multiple defense responses in tobacco.
Plant Cell 13, 1877–1889.

6 Fiil BK, Petersen K, Petersen M & Mundy J (2009)
Gene regulation by MAP kinase cascades. Curr Opin
Plant Biol 12, 615–621.
7 Reyna NS & Yang Y (2006) Molecular analysis of the
rice MAP kinase gene family in relation to Magnaporthe
grisea infection. Mol Plant Microbe Interact 19, 530–
540.
8 Nicole MC, Hamel LP, Morency MJ, Beaudoin N,
Ellis BE & Seguin A (2006) MAP-ping genomic
organization and organ-specific expression profiles of
poplar MAP kinases and MAP kinase kinases. BMC
Genomics 7, 223–245.
9 Pitzschke A, Schikora A & Hirt H (2009) MAPK
cascade signalling networks in plant defence. Curr Opin
Plant Biol 12, 421–426.
10 Zhang S & Klessig DF (1998) Resistance gene N-medi-
ated de novo synthesis and activation of a tobacco
mitogen-activated protein kinase by tobacco mosaic
virus infection. Proc Natl Acad Sci USA 95, 7433–
7438.
11 Ren D, Yang KY, Li GJ, Liu Y & Zhang S (2006)
Activation of Ntf4, a tobacco mitogen-activated protein
kinase, during plant defense response and its involve-
ment in hypersensitive response-like cell death. Plant
Physiol 141, 1482–1493.
12 Petersen M, Brodersen P, Naested H, Andreasson E,
Lindhart U, Johansen B, Nielsen HB, Lacy M, Austin
MJ, Parker JE et al. (2000) Arabidopsis map kinase 4
negatively regulates systemic acquired resistance. Cell
103, 1111–1120.

13 Xiong L & Yang Y (2003) Disease resistance and abi-
otic stress tolerance in rice are inversely modulated by
an abscisic acid-inducible mitogen-activated protein
kinase. Plant Cell 15, 745–759.
14 Kovtun Y, Chiu WL, Tena G & Sheen J (2000) Func-
tional analysis of oxidative stress-activated mitogen-acti-
vated protein kinase cascade in plants. Proc Natl Acad
Sci USA 97, 2940–2945.
15 Pitzschke A & Hirt H (2009) Disentangling the com-
plexity of mitogen-activated protein kinases and reactive
oxygen species signaling. Plant Physiol 149, 606–615.
16 Xue T, Li X, Zhu W, Wu C, Yang G & Zheng C
(2009) Cotton metallothionein GhMT3a, a reactive oxy-
gen species scavenger, increased tolerance against abi-
otic stress in transgenic tobacco and yeast. J Exp Bot
60
, 339–349.
17 Mittler R (2002) Oxidative stress, antioxidants and
stress tolerance. Trends Plant Sci 7, 405–410.
18 Asai S & Yoshioka H (2008) The role of radical burst
via MAPK signaling in plant immunity. Plant Signal
Behav 3, 920–922.
19 Jammes F, Song C, Shin D, Munemasa S, Takeda K,
Gu D, Cho D, Lee S, Giordo R, Sritubtim S et al.
(2009) MAP kinases MPK9 and MPK12 are preferen-
tially expressed in guard cells and positively regulate
ROS-mediated ABA signaling. Proc Natl Acad Sci USA
106, 20520–20525.
20 Nakagami H, Kiegerl S & Hirt H (2004) OMTK1, a
novel MAPKKK, channels oxidative stress signaling

through direct MAPK interaction. J Biol Chem 279,
26959–26966.
21 Ren D, Yang H & Zhang S (2002) Cell death mediated
by MAPK is associated with hydrogen peroxide pro-
duction in Arabidopsis. J Biol Chem 277, 559–565.
22 Ortiz-Masia D, Perez-Amador MA, Carbonell J & Mar-
cote MJ (2007) Diverse stress signals activate the C1
subgroup MAP kinases of Arabidopsis. FEBS Lett 581,
1834–1840.
23 Ortiz-Masia D, Perez-Amador MA, Carbonell P,
Aniento F, Carbonell J & Marcote MJ (2008) Charac-
terization of PsMPK2, the first C1 subgroup MAP
kinase from pea (Pisum sativum L.). Planta 227, 1333–
1342.
24 Zong X, Li D, Gu L, Li D, Liu L & Hu X (2009) Ab-
scisic acid and hydrogen peroxide induce a novel maize
group C MAP kinase gene, ZmMPK7, which is respon-
sible for the removal of reactive oxygen species. Planta
229, 485–495.
25 Wang M, Zhang Y, Wang J, Wu X & Guo X (2007)
A novel MAP kinase gene in cotton (Gossypium
hirsutum L.), GhMAPK, is involved in response to
diverse environmental stresses. J Biochem Mol Biol 40,
325–332.
26 Jung JY, Shin R & Schachtman DP (2009) Ethylene
mediates response and tolerance to potassium depriva-
tion in Arabidopsis. Plant Cell 21, 607–621.
27 Kim CY, Liu Y, Thorne ET, Yang H, Fukushige H,
Gassmann W, Hildebrand D, Sharp RE & Zhang S
(2003) Activation of a stress-responsive mitogen-acti-

vated protein kinase cascade induces the biosynthesis of
ethylene in plants. Plant Cell 15, 2707–2718.
28 Kotchoni SO & Gachomo EW (2006) The reactive oxy-
gen species network pathways: an essential prerequisite
L. Zhang et al. Cotton GhMPK2 is involved in multiple signaling pathways
FEBS Journal 278 (2011) 1367–1378 ª 2011 The Authors Journal compilation ª 2011 FEBS 1377
for perception of pathogen attack and the acquired
disease resistance in plants. J Biosci 31, 389–404.
29 Spoel SH, Johnson JS & Dong X (2007) Regulation of
tradeoffs between plant defenses against pathogens with
different lifestyles. Proc Natl Acad Sci USA 104, 18842–
18847.
30 Leon-Reyes A, Spoel SH, De Lange ES, Abe H,
Kobayashi M, Tsuda S, Millenaar FF, Welschen RA,
Ritsema T & Pieterse CM (2009) Ethylene modulates
the role of NONEXPRESSOR OF PATHOGENESIS-
RELATED GENES1 in cross talk between salicylate
and jasmonate signaling. Plant Physiol 149, 1797–1809.
31 Spoel SH & Dong X (2008) Making sense of hormone
crosstalk during plant immune responses. Cell Host
Microbe 3, 348–351.
32 Bari R & Jones JD (2009) Role of plant hormones in
plant defence responses. Plant Mol Biol 69, 473–488.
33 Brodersen P, Petersen M, Bjorn Nielsen H, Zhu S,
Newman MA, Shokat KM, Rietz S, Parker J & Mundy
J (2006) Arabidopsis MAP kinase 4 regulates salicylic
acid- and jasmonic acid ⁄ ethylene-dependent responses
via EDS1 and PAD4. Plant J 47, 532–546.
34 Apel K & Hirt H (2004) Reactive oxygen species:
metabolism, oxidative stress, and signal transduction.

Annu Rev Plant Biol 55, 373–399.
35 Doczi R, Brader G, Pettko-Szandtner A, Rajh I,
Djamei A, Pitzschke A, Teige M & Hirt H (2007) The
Arabidopsis mitogen-activated protein kinase kinase
MKK3 is upstream of group C mitogen-activated
protein kinases and participates in pathogen signaling.
Plant Cell 19, 3266–3279.
36 Nakagami H, Soukupova H, Schikora A, Zarsky V &
Hirt H (2006) A mitogen-activated protein kinase
kinase kinase mediates reactive oxygen species homeo-
stasis in Arabidopsis. J Biol Chem 281, 38697–38704.
37 Park JM, Park CJ, Lee SB, Ham BK, Shin R & Paek
KH (2001) Overexpression of the tobacco Tsi1 gene
encoding an EREBP ⁄ AP2-type transcription factor
enhances resistance against pathogen attack and
osmotic stress in tobacco. Plant Cell 13, 1035–1046.
38 Ohme-Takagi M & Shinshi H (1995) Ethylene-inducible
DNA binding proteins that interact with an ethylene-
responsive element. Plant Cell 7, 173–182.
39 Cheong YH, Moon BC, Kim JK, Kim CY, Kim MC,
Kim IH, Park CY, Kim JC, Park BO, Koo SC et al.
(2003) BWMK1, a rice mitogen-activated protein
kinase, locates in the nucleus and mediates pathogene-
sis-related gene expression by activation of a transcrip-
tion factor. Plant Physiol 132, 1961–1972.
40 Zheng Z, Mosher SL, Fan B, Klessig DF & Chen Z
(2007) Functional analysis of Arabidopsis WRKY25
transcription factor in plant defense against Pseudomo-
nas syringae. BMC Plant Biol 7, 2–15.
41 Yu D, Chen C & Chen Z (2001) Evidence for an

important role of WRKY DNA binding proteins in the
regulation of NPR1 gene expression. Plant Cell 13,
1527–1540.
42 Yang L, Tang R, Zhu J, Liu H, Mueller-Roeber B,
Xia H & Zhang H (2008) Enhancement of stress toler-
ance in transgenic tobacco plants constitutively express-
ing AtIpk2b, an inositol polyphosphate 6- ⁄ 3-kinase
from Arabidopsis thaliana . Plant Mol Biol 66, 329–343.
Cotton GhMPK2 is involved in multiple signaling pathways L. Zhang et al.
1378 FEBS Journal 278 (2011) 1367–1378 ª 2011 The Authors Journal compilation ª 2011 FEBS