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,
Trang 1and mediates defense responses to pathogen infection and oxidative stress
Liang Zhang1, Dongmei Xi2, Lu Luo1, Fei Meng1, Yuzhen Li1, Chang-ai Wu1and Xingqi Guo1
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: xqguo@sdau.edu.cn
(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.
Trang 2a 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
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
MPK4gene 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 (H2O2),
superox-ide anion (O2), 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, H2O2
activates AtMPK12 in Arabidopsis and MMK3 in alfalfa [19,20], whereas constitutive expression of Ara-bidopsis MKK4 or MKK5 results in the generation of
H2O2 [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 H2O2 [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 H2O2 [23] Recently, it was shown that maize ZmMPK7 was induced by ABA and H2O2, and that H2O2 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, H2O2, 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
Trang 3Expression 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 AgNO3
alone or 100 lm AgNO3 with 5 mm ethephon
(ET + Ag+) was applied to the cotton seedlings The
results showed that the expression of GhMPK2 was
significantly increased by AgNO3 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 T3
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 m M ethephon,
100 l M MeJA, and 100 l M AgNO3alone or 100 l M AgNO3with
5 m 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.
Trang 4Overexpression 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 oxysporumand 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
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.
Trang 5GhMPK2 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 H2O2 The accumulation of H2O2 and O2, 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 H2O2 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 H2O2production in transgenic plants as compared with wild-type plants (Fig 6A) After treatment with NaCl, poly(ethylene gly-col), and H2O2, the accumulation of H2O2was remark-ably lower in the transgenic lines than in the wild-type plants at 4 h (Fig 6B) Similarly, NBT staining showed less O2 accumulation in the leaves of the transgenic lines (Fig 6B) The transgenic plants produced a lower amount of ROS, suggesting that overexpression of GhMPK2either 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 abiobio-tic 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.
Trang 6role 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 H2O2 and O2 accumulation detected by DAB
staining and NBT staining, respectively WT, wild-type.
Trang 7cal function of group C MAPKs, but also provide new
insights for further exploration of the significance of
GhMPK2in 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
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.
Trang 8and 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.
Trang 9GhMPK2 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,
H2O2 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 O2 (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 H2O2 and O2) in the presence of NaCl,
poly(ethylene glycol), and H2O2, 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 28C 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 AgNO3 treatment, 100 lm AgNO3alone or 100 lm AgNO3in 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 T3lines were used in the experiments
Northern blot analyses
With an RNeasy Mini Kit (Qiagen, MD, USA), 20 lg of 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+
Trang 10membranes, 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 T3OE
tobacco plants were inoculated by irrigation with F
oxy-sporum and P nicotianae Tucker spore suspensions
(106conidiaÆmL)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 harvestnoninoculat-ed at the specifinoninoculat-ed times for
mRNA and protein extraction
Analysis of ROS scavenging on the basis of H2O2
and O22 staining
For H2O2staining, 3-week-old tobacco seedlings were
trea-ted with 200 mm NaCl, 15% poly(ethylene glycol) 6000 and
100 mm H2O2for 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 25C in the
dark After staining, the leaves were soaked in 95% ethanol
overnight to remove the chlorophyll In addition, the
accumulation of H2O2 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 H2O2 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),
(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)