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

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-

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

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

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-

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

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

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

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

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

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

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

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.

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

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

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.

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

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.

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