BACKGROUND
stresses such as drought, high salinity and fungal infections constitute a major limitation to crop productivity.
plants have developed sophisticated defense mechanisms to deal with diverse unfavorable environmental factors.
the mitogen-activated protein kinase  cascades are conserved pathways through which extracellular stimuli are transduced into intracellular responses in all eukaryotes  <cit> .
plant mapk cascades have been shown to regulate a number of essential biological processes, including growth, development and stress responses  <cit> .
mapk cascades are composed of three interlinked protein kinases: mapkk kinases , mapk kinases  and mapks.
mapks are the terminal components in this cascade, and they are regulated by the dual phosphorylation of the conserved t-x-y motif located in the activation loop by upstream kinases .
there are  <dig> mapk genes identified in arabidopsis, and a similar repertoire of genes have been found in other plants, such as rice , poplar  and grapevine   <cit> .
the mapks can be categorised into four major groups  based on the phylogenetic analyzes of amino acid sequences and phosphorylation motifs .
the tey subtype can be classified into three groups , whereas the tdy subtype is found in the more distant group d  <cit> .
in arabidopsis, three particular mapks in groups a and b  have been extensively studied.
both biochemical and genetic analyzes have been performed for each of these isoforms, which appear to work in multiple signaling pathways and play crucial roles in many distinct processes ranging from stress responses to developmental processes  <cit> .
information about group c mapks has recently emerged.
three members of group c, mpk <dig> and mpk <dig> in arabidopsis and psmpk <dig> in pea , are transcriptionally induced by a variety of stresses  <cit> .
more recently, arabidopsis group c mapks, including mpk <dig>  mpk <dig>  mpk <dig> and mpk <dig>  were reported to be activated by mkk <dig>  and mkk3-mpk <dig> participates in pathogen signaling  <cit> .
cotton ghmpk <dig> may play a role in pathogen resistance, plant growth and development  <cit> .
based on the phylogenetic analysis and pairwise comparison of arabidopsis and rice mapks, it has been proposed that the rice genome contains more mapks with a tdy phosphorylation motif  than with a tey motif .
in contrast, the arabidopsis genome contains more mapks with a tey motif  than with a tdy motif   <cit> .
detailed functional data about tdy mapks was first obtained from a monocot plant.
overexpression of osbwmk <dig>  in tobacco resulted in constitutive pr gene expression and enhanced resistance to fungal and bacterial infections  <cit> .
in maize, zmmpk <dig> is able to interact with a 14-3- <dig> protein, and these data represent the first evidence of the possible involvement of 14-3- <dig> proteins in the regulation of mapk cascades in plants  <cit> .
more recently, arabidopsis mpk <dig>  and mpk <dig>  were found to be preferentially expressed in guard cells, share functional redundancy and function as positive regulators downstream of reactive oxygen species  in guard cell abscisic acid  signaling  <cit> .
moreover, arabidopsis mpk <dig> helps to mediate cortical microtubule functions in plant cells  <cit> .
cotton  is one of the most important fibre and oil crops, and its growth and yield are severely impaired in various biotic/abiotic stress conditions.
the biological significance of cotton group d mapks has not yet been described.
in this study, a cdna clone, ghmpk <dig>  encoding a putative group d mapk gene was isolated and characterised.
our results indicate that the expression of ghmpk <dig> is induced by chemical and biological signals.
ectopic expression of ghmpk <dig> in arabidopsis results in enhanced disease resistance against fungi and bacteria pathogen.
moreover, ghmpk <dig> transgenic plants were obviously more drought-sensitive than control plants.
we deduced that ghmpk <dig> may play important roles in regulating pathogen resistance and drought signaling.
RESULTS
cloning and characterisation of the full-length ghmpk <dig> cdna
based on the conserved region of plant group d mapk genes, the degenerate primers mp <dig> and mp <dig> were designed and synthesized to clone the internal conserved region of mapks from cotton, and a putative mapk fragment  was cloned.
next, rapid amplification of cdna ends-pcr  was performed, and the full length sequence was retrieved.
a 3'-end fragment of about  <dig> bp and a 5'-end fragment of about  <dig> bp were obtained with the specific primers.
the full-length cdna sequence was pcr amplified using the gene-specific primers , and it showed a high degree of homology to group d mapks especially arabidopsis atmpk <dig>  according to the nomenclature for plant mapks, the novel cotton mapk gene was named ghmpk <dig>  <cit> , and is the first group d mapk identified in cotton.
the full-length cdna sequence of ghmpk <dig>  was  <dig> bp with a  <dig> bp 5' untranslated region  and a  <dig> bp 3' utr.
the cdna contains a  <dig> bp open reading frame  that is predicted to encode a protein of  <dig> amino acids with a predicted molecular mass of  <dig>  kda and an isoelectric point  of  <dig> .
a blast search  and multi-alignment analysis revealed that ghmpk <dig> is highly related to other group d mapks, sharing a homology of  <dig> % to atmpk <dig>   <dig> % to tamapk <dig>   <dig> % to osmpk16- <dig> and  <dig> % to zmmpk <dig> .
moreover, as with other known plant group d mapks, ghmpk <dig> has the same family signature of  <dig> conserved subdomains, an activation loop , a phosphorylation motif  in the t-loop, and an extended c-terminal region relative to groups a, b and c. additionally, ghmpk <dig> also lacks a c-terminal cd domain, a feature conserved in mapks belonging to the other groups   <cit> .
to reveal the evolutionary relationship of ghmpk <dig> with other mapks from various plant species, we constructed a phylogenetic tree using amino acid sequences derived from the genbank database .
our results indicate that ghmpk <dig> has high similarity with atmpk <dig> and a close genetic relationship with many monocot  group d mapks, such as osmpk16- <dig>  osmpk <dig>  zmmpk <dig> and tamapk <dig>  which suggests that these genes may have a similar function across species including dicotyledons and monocotyledons.
genomic structure and southern blot analysis of ghmpk16
to isolate a genomic ghmpk <dig> clone, two pairs of specific primers, fl1/z <dig> and fl2/z <dig>  were designed based on the ghmpk <dig> cdna sequence, and the cotton genomic dna was used as the template to generate two pcr fragments.
these two fragments were further linked together through their overlapping region and the full-length ghmpk <dig> genomic sequence of  <dig> bp  was deduced.
a comparison between the ghmpk <dig> genomic and cdna sequences indicated that ten introns were present in the gene .
interestingly, exon  <dig> was only  <dig> bp, intron  <dig> did not contain a 3'-splice acceptor ag signal, and intron  <dig> did not contain the conserved 5'-splice donor gt signal.
a comparative analysis of the homologous genes from arabidopsis thaliana, oryza sativa and vitis vinifera was performed .
this analysis indicated that the size of the exons was conserved, but the length of the introns for each member was different.
to date, the most noticeable difference in ghmpk <dig> was the number of exons.
ghmpk <dig> has  <dig> exons, and the others have  <dig>
southern blots were used to investigate the genomic organization of the ghmpk <dig> gene.
genomic dna was completely digested with ecor v, xba i, hind iii and ecor i and hybridised to the 3' partial sequence of ghmpk <dig>  which contains no restriction sites of ecor v, hind iii and ecor i and only one xba i site presents in the probe region.
as shown in figure  <dig>  only one band was observed for the ecor v, hind iii and ecor i digestions, and two bands were observed for the xba i digestion.
these results imply that there is a single ghmpk <dig> gene in the cotton genome.
subcellular localization of ghmpk16
to reveal the cellular localization of ghmpk <dig>  a reporter gene encoding gfp was fused to ghmpk <dig> and placed under the control of the camv35s promoter, and immunoblot analysis indicated that ghmpk16::gfp was an integrated fusion protein .
the biolistic transformation system was used for a transient assay in onion epidermal cells.
the nuclear localization of gfp-conjugated ghmpk <dig> was confirmed in individual transgenic cells by gfp fluorescence, using dapi staining to detect the nuclei and interference contrast images to detect whole-cell structures.
as shown in figure 3b, the 35s-ghmpk16::gfp construct localized to the nucleus, and the 35s-gfp control construct showed gfp signals in both the cytoplasm and the nucleus.
in addition, a program that predicts the subcellular localization of proteins  predicted that ghmpk <dig> is localized in the nucleus with an expected accuracy of nearly 74%.
these results indicate that the ghmpk <dig> protein is likely localized in the nucleus.
expression pattern of ghmpk <dig> under diverse abiotic and biotic stresses in cotton
to determine whether ghmpk <dig> expression is triggered by abiotic stresses, cotton seedlings were exposed to low and high temperatures, mannitol, nacl and wounding treatments.
ghmpk <dig> showed a slightly response to low and high temperatures .
ghmpk <dig> transcripts accumulated within  <dig> h following mannitol treatment, and the induction was still present  <dig> h after treatment .
as shown in figure 4e, ghmpk <dig> expression increased and reached the maximum at  <dig> h after the nacl treatment.
wounding rapidly and transiently enhanced the expression level of ghmpk <dig> .
taken together, the expression profiles indicate that ghmpk <dig> is induced by various abiotic stresses.
to explore the roles of ghmpk <dig> in plant biotic stresses, cotton seedlings were inoculated with pathogens, including xanthomonas campestris pv.
malvacearum , colletotrichum gossypii  and fusarium oxysporum f. sp.
vasinfectum .
as shown in figure  <dig> , infection by all of these pathogens elevated the transcription level of ghmpk <dig>  although the induction kinetic was variable.
there was no significant change in ghmpk <dig> transcript level without pathogen treatment during  <dig> days .
these results indicate that ghmpk <dig> may be intimately involved in the plant pathogen defense response.
additionally, we examined the response of ghmpk <dig> to exogenously applied salicylic acid , methyl jasmonate  and aba, which are plant signaling molecules involved in plant defense signaling pathways.
the expression of ghmpk <dig> was induced by all of these signal molecules .
furthermore, the solvent control  did not significantly induce the expression of ghmpk <dig>
ghmpk <dig> promoter analysis
inverse-pcr  was used to obtain a  <dig> bp fragment of the 5' flanking region upstream of the transcriptional start site, as determined by the ghmpk <dig> cdna sequence.
in order to find cis-acting elements, the plantcare databases were analyzed.
sequence analysis revealed that the ghmpk <dig> promoter contains tata and caat motifs located at nucleotides - <dig> and - <dig> relative to the transcriptional start site, respectively, which is characteristic of eukaryotic gene promoters.
as shown in figure  <dig>  the  <dig> bp promoter region contains several motifs probably related to pathogen and drought signals, such as a box-w <dig> element , an ere element , two tca-element  and a mbs element .
this suggests that these putative cis-acting elements are responsible for enhanced expression of ghmpk <dig> during stress conditions.
enhanced resistance of ghmpk <dig> transgenic plants to pathogenic infections
in order to further explore the function of ghmpk <dig> in plant defense, the coding sequence of ghmpk <dig> was cloned into the plant binary vector pbi <dig> and transformed into arabidopsis.
a total of  <dig> independent transgenic lines were obtained by kanamycin-resistance selection and confirmed by pcr .
rna blot analysis was performed to determine the transgenic expression levels in  <dig> randomly selected lines .
two representative lines  exhibiting different expression levels were selected and their t <dig> transgenic plants used to evaluate disease resistance.
to analyze bacterial resistance responses in wild-type and ghmpk16-overexpressing plants, 3-week-old plants were inoculated with p. solanacearum and monitored daily for the appearance of typical disease symptoms.
p. solanacearum caused more severe chlorotic symptoms in the wild-type plants compared to the transgenic plants  <dig> d after inoculation .
as shown in figure 6c, the bacterial titres in the inoculated leaves of the transgenic plants were significantly reduced as compared to those in wild-type plants at 3- and 6-day post-inoculation, which is consistent with the observed symptoms.
the rate of bacterial growth in the inoculated leaves was lower for transgenic line # <dig> than for transgenic line # <dig>  these results indicate that ghmpk <dig> can enhance resistance to p. solanacearum in a dose-dependent manner in transgenic plants.
in order to better understand the role of ghmpk <dig> in disease resistance in transgenic plants, three marker genes associated with pathogen responses  were selected, and the transcript levels of those genes were analyzed by reverse transcription-pcr .
the expression of the three marker genes, especially atpr <dig> and atpdf <dig> , was more rapidly and strongly induced by the p. solanacearum infection in transgenic plants than in wild-type plants .
these results demonstrate that the overexpression of ghmpk <dig> activates defense-related gene expression when plants are challenged with p. solanacearum.
as shown in figure  <dig>  ghmpk16-overexpressing plants were evaluated for their resistance to fungal pathogens .
detached leaves were inoculation with c. nicotianae and a. alternate, and after  <dig> days, the lesions in the wild-type leaves were larger than those in the transgenic leaves, and line # <dig>  which displayed the highest level of ghmpk <dig> expression, exhibited the least severe disease symptoms.
to quantify the lesions, the diameters of the disease spots were measured, and the data were consistent with the visual observations.
these results indicate that ghmpk <dig> can enhance resistance to infections by pathogenic fungi in transgenic plants.
after inoculating whole plants with c. nicotianae and a. alternate, the expression of atpr <dig>  atpr <dig> and atpdf <dig>  was observed at 3- and 6-day post-infection in both the wild-type and transgenic plants, and the expression patterns of the three marker genes were similar to those observed with the p. solanacearum challenge .
reduced drought tolerance and rapid h2o <dig> accumulation in ghmpk16-overexpressing plants
compared with wild-type plants, the ghmpk16-overexpressing plants exhibited obvious drought sensitivity.
as shown in figure 8a, the leaves began to turn yellow and curl in both the 3-week-old wild-type and transgenic plants after a 15-day drought treatment.
additionally, drought treatment led to more serious damage in the transgenic plants than in the wild-type plants.
under normal conditions, no overt morphological differences were observed between wild-type and transgenic plants .
we tested the seed germination capacity on 1/ <dig> ms agar medium with  <dig> mm mannitol to mimic drought conditions and found that the germination percentage was higher for wild-type plants than for transgenic plants .
the root lengths of both wild-type and transgenic plants were used as indicators of drought stress tolerance.
root growth was inhibited by mannitol treatment to a greater extent in transgenic seedlings than in wild-type seedlings .
these results suggest that the overexpression of ghmpk <dig> in arabidopsis results in reduced drought tolerance.
one of the important responses of plants under osmotic stress is the accumulation of reactive oxygen species .
among the different ros, only h2o <dig> can cross plant membranes and thus directly function in cell-to-cell signaling.
therefore, we examined endogenous h2o <dig> accumulation in wild-type and transgenic plants in response to mannitol treatment.
mannitol  was used to treat one-week-old seedlings, and the leaves were collected after  <dig>   <dig>   <dig> and  <dig> h. staining with  <dig> '-diaminobenzidine  revealed different levels of h2o <dig> production in the leaves of wild-type and transgenic plants after osmotic stress treatment.
specifically, h2o <dig> accumulated in transgenic plants at a significantly faster speed than that in wild-type plants .
these results suggest that the overexpression of ghmpk <dig> leads to the rapid production of ros or ineffective scavenge of excess ros after osmotic stress.
discussion and
CONCLUSIONS
in this study, we isolated and characterised ghmpk <dig>  a gene whose product belongs to the family of group d mapks.
ghmpk <dig> is the first characterized mapk gene from cotton belonging to the group d mapks.
the corresponding protein is characterized by the presence of a tdy activation motif in its t-loop, the lack of a cd domain and an extended c-terminal region compared to the tey subtypes of mapks .
these structural differences suggest that group d mapks may function differently from other mapks.
the subcellular localization of group d mapks has not been established.
osbwmk <dig>  a group d mapk from rice, is found in the nucleus and mediates pathogenesis-related gene expression by activating the oserebp <dig> transcription factor  <cit> .
intriguingly, the alternative splicing of osbwmk <dig> generates three different transcriptional variants that produce proteins with different subcellular localizations  <cit> .
in arabidopsis, atmpk <dig> is located in the cytoplasm  <cit> .
analysis of the subcellular localization of a gfp-tagged ghmpk <dig> protein in transiently transformed onion epidermal cells revealed that ghmpk <dig> is likely localized in the nucleus .
therefore, we speculated that ghmpk <dig> may act as a transcriptional activator by activating the expression of a set of target genes in the nucleus.
gene expression patterns are usually an indicator of gene function.
notably, ghmpk <dig> can be induced by various pathogens, including x. campestris pv.
malvacearum, r.solani and c. gossypii.
rna blot analyzes revealed that the expression of ghmpk <dig> could be induced by signal molecules such as sa, meja, and aba .
these results imply that ghmpk <dig> may play a role in plant defense responses and in the regulation of certain components of multiple stress-signaling pathways.
consistent with this hypothesis, sequence analysis of the ghmpk <dig> promoter revealed several motifs, such as box-w <dig> element, ere element and tca-element, related to motifs with important roles in defense signaling.
recently, a variety of mapk genes have been identified and subsequently explored by both genetic and biochemical approaches.
increasing evidence has shown that the mapk cascade is an important pathway in pathogen-triggered signal transduction  <cit> .
signal molecules such as sa, jasmonic acid  and ethylene , regulate distinct sets of pathogenesis-related  genes in different pathogen defense pathways.
sa and meja are involved in two major defense signaling pathways, the sa-dependent and ja/et-dependent defense mechanisms, which act against different types of pathogens  <cit> .
in arabidopsis, atmpk <dig> responds to the balance between sa and meja through the eds1/pad <dig> module and regulates the sa and ja/et-related defense responses  <cit> .
when ghmpk <dig> is ectopically expressed in arabidopsis, the transgenic arabidopsis show enhanced disease resistance against bacteria  and fungi  .
pr <dig> is known as a marker gene for the sa signaling pathway, and atpr <dig> and atpdf <dig>  are markers for the ja/et signaling pathway.
along with the enhanced disease resistance in ghmpk16-overexpressing plants, the expression of the examined defense-related genes atpr <dig>  atpr <dig>  and atpdf <dig>  was elevated.
thus, it is reasonable to speculate that ghmpk <dig> may be involved in the crosstalk between the sa- and ja/et- mediated pathogen defense pathways.
ghmpk <dig> showed a response to various abiotic stresses, such as low or high temperatures, mannitol and nacl .
interestingly, transgenic plants overexpressing ghmpk <dig> exhibited obvious drought sensitivity .
ghmpk <dig> overexpression resulted in a significant reduction in both germination percentage and root growth after mannitol treatment, and seedling growth was more severely inhibited by drought stress in ghmpk16-overexpressing plants than in wild-type plants.
aba responds to the environmental signals to protect plants from abiotic stresses, such as cold, drought and salt stresses  <cit> .
the expression of ghmpk <dig> was strongly induced by exogenous aba and the ghmpk16-overexpressing plants were sensitive to drought tolerance comparing with wild-type arabidopsis, suggesting that ghmpk <dig> may be involved in aba signaling and caused negative function for drought tolerance.
dab staining revealed that h2o <dig> rapidly accumulation in ghmpk16-overexpressing transgenic plants after drought treatment .
excessive ros can injure plants.
ghmpk <dig> accelerates the accumulation of h2o <dig>  and this may be the reason that drought causes more serious damage in the transgenic plants.
in arabidopsis, mpk <dig> and mpk <dig> function downstream of ros to regulate guard cell aba signaling positively  <cit> .
guard cell-specific inhibition of arabidopsis mpk <dig> expression causes abnormal stomatal responses to aba and h2o <dig>  <cit> .
more physiological, biochemical and molecular experiments are needed to elucidate the mechanism of ghmpk <dig> in response to drought stress.
on the basis of these observations, we propose that ghmpk <dig> functions in at least two signaling pathways, one that responds to pathogens and another that is involved in drought stress.
ghmpk <dig> may serve as a point for crosstalk between biotic and abiotic stress response signaling.
further studies on the function and regulation of ghmpk <dig> are required.

 stresses such as drought, high salinity and fungal infections constitute a major limitation to crop productivity. plants have developed sophisticated defense mechanisms to deal with diverse unfavorable environmental factors. the mitogen-activated protein kinase cascades are conserved pathways through which extracellular stimuli are transduced into intracellular responses in all eukaryotes. plant mapk cascades have been shown to regulate a number of essential biological processes, including growth, development and stress responses. mapk cascades are composed of three interlinked protein kinases: mapkk kinases, mapk kinases and mapks. mapks are the terminal components in this cascade, and they are regulated by the dual phosphorylation of the conserved t-x-y motif located in the activation loop by upstream kinases. there are mapk genes identified in arabidopsis, and a similar repertoire of genes have been found in other plants, such as rice, poplar and grapevine. the mapks can be categorised into four major groups based on the phylogenetic analyzes of amino acid sequences and phosphorylation motifs. the tey subtype can be classified into three groups, whereas the tdy subtype is found in the more distant group d. in arabidopsis, three particular mapks in groups a and b have been extensively studied. both biochemical and genetic analyzes have been performed for each of these isoforms, which appear to work in multiple signaling pathways and play crucial roles in many distinct processes ranging from stress responses to developmental processes. information about group c mapks has recently emerged. three members of group c, mpk and mpk in arabidopsis and psmpk in pea, are transcriptionally induced by a variety of stresses. more recently, arabidopsis group c mapks, including mpk mpk mpk and mpk were reported to be activated by mkk and mkk3-mpk participates in pathogen signaling. cotton ghmpk may play a role in pathogen resistance, plant growth and development. based on the phylogenetic analysis and pairwise comparison of arabidopsis and rice mapks, it has been proposed that the rice genome contains more mapks with a tdy phosphorylation motif than with a tey motif. in contrast, the arabidopsis genome contains more mapks with a tey motif than with a tdy motif. detailed functional data about tdy mapks was first obtained from a monocot plant. overexpression of osbwmk in tobacco resulted in constitutive pr gene expression and enhanced resistance to fungal and bacterial infections. in maize, zmmpk is able to interact with a 14-3- protein, and these data represent the first evidence of the possible involvement of 14-3- proteins in the regulation of mapk cascades in plants. more recently, arabidopsis mpk and mpk were found to be preferentially expressed in guard cells, share functional redundancy and function as positive regulators downstream of reactive oxygen species in guard cell abscisic acid signaling. moreover, arabidopsis mpk helps to mediate cortical microtubule functions in plant cells. cotton is one of the most important fibre and oil crops, and its growth and yield are severely impaired in various biotic/abiotic stress conditions. the biological significance of cotton group d mapks has not yet been described. in this study, a cdna clone, ghmpk encoding a putative group d mapk gene was isolated and characterised. our results indicate that the expression of ghmpk is induced by chemical and biological signals. ectopic expression of ghmpk in arabidopsis results in enhanced disease resistance against fungi and bacteria pathogen. moreover, ghmpk transgenic plants were obviously more drought-sensitive than control plants. we deduced that ghmpk may play important roles in regulating pathogen resistance and drought signaling.  cloning and characterisation of the full-length ghmpk cdna based on the conserved region of plant group d mapk genes, the degenerate primers mp and mp were designed and synthesized to clone the internal conserved region of mapks from cotton, and a putative mapk fragment was cloned. next, rapid amplification of cdna ends-pcr was performed, and the full length sequence was retrieved. a 3'-end fragment of about bp and a 5'-end fragment of about bp were obtained with the specific primers. the full-length cdna sequence was pcr amplified using the gene-specific primers, and it showed a high degree of homology to group d mapks especially arabidopsis atmpk according to the nomenclature for plant mapks, the novel cotton mapk gene was named ghmpk, and is the first group d mapk identified in cotton. the full-length cdna sequence of ghmpk was bp with a bp 5' untranslated region and a bp 3' utr. the cdna contains a bp open reading frame that is predicted to encode a protein of amino acids with a predicted molecular mass of kda and an isoelectric point of. a blast search and multi-alignment analysis revealed that ghmpk is highly related to other group d mapks, sharing a homology of % to atmpk % to tamapk % to osmpk16- and % to zmmpk. moreover, as with other known plant group d mapks, ghmpk has the same family signature of conserved subdomains, an activation loop, a phosphorylation motif in the t-loop, and an extended c-terminal region relative to groups a, b and c. additionally, ghmpk also lacks a c-terminal cd domain, a feature conserved in mapks belonging to the other groups. to reveal the evolutionary relationship of ghmpk with other mapks from various plant species, we constructed a phylogenetic tree using amino acid sequences derived from the genbank database. our results indicate that ghmpk has high similarity with atmpk and a close genetic relationship with many monocot group d mapks, such as osmpk16- osmpk zmmpk and tamapk which suggests that these genes may have a similar function across species including dicotyledons and monocotyledons. genomic structure and southern blot analysis of ghmpk16 to isolate a genomic ghmpk clone, two pairs of specific primers, fl1/z and fl2/z were designed based on the ghmpk cdna sequence, and the cotton genomic dna was used as the template to generate two pcr fragments. these two fragments were further linked together through their overlapping region and the full-length ghmpk genomic sequence of bp was deduced. a comparison between the ghmpk genomic and cdna sequences indicated that ten introns were present in the gene. interestingly, exon was only bp, intron did not contain a 3'-splice acceptor ag signal, and intron did not contain the conserved 5'-splice donor gt signal. a comparative analysis of the homologous genes from arabidopsis thaliana, oryza sativa and vitis vinifera was performed. this analysis indicated that the size of the exons was conserved, but the length of the introns for each member was different. to date, the most noticeable difference in ghmpk was the number of exons. ghmpk has exons, and the others have southern blots were used to investigate the genomic organization of the ghmpk gene. genomic dna was completely digested with ecor v, xba i, hind iii and ecor i and hybridised to the 3' partial sequence of ghmpk which contains no restriction sites of ecor v, hind iii and ecor i and only one xba i site presents in the probe region. as shown in figure only one band was observed for the ecor v, hind iii and ecor i digestions, and two bands were observed for the xba i digestion. these results imply that there is a single ghmpk gene in the cotton genome. subcellular localization of ghmpk16 to reveal the cellular localization of ghmpk a reporter gene encoding gfp was fused to ghmpk and placed under the control of the camv35s promoter, and immunoblot analysis indicated that ghmpk16::gfp was an integrated fusion protein. the biolistic transformation system was used for a transient assay in onion epidermal cells. the nuclear localization of gfp-conjugated ghmpk was confirmed in individual transgenic cells by gfp fluorescence, using dapi staining to detect the nuclei and interference contrast images to detect whole-cell structures. as shown in figure 3b, the 35s-ghmpk16::gfp construct localized to the nucleus, and the 35s-gfp control construct showed gfp signals in both the cytoplasm and the nucleus. in addition, a program that predicts the subcellular localization of proteins predicted that ghmpk is localized in the nucleus with an expected accuracy of nearly 74%. these results indicate that the ghmpk protein is likely localized in the nucleus. expression pattern of ghmpk under diverse abiotic and biotic stresses in cotton to determine whether ghmpk expression is triggered by abiotic stresses, cotton seedlings were exposed to low and high temperatures, mannitol, nacl and wounding treatments. ghmpk showed a slightly response to low and high temperatures. ghmpk transcripts accumulated within h following mannitol treatment, and the induction was still present h after treatment. as shown in figure 4e, ghmpk expression increased and reached the maximum at h after the nacl treatment. wounding rapidly and transiently enhanced the expression level of ghmpk. taken together, the expression profiles indicate that ghmpk is induced by various abiotic stresses. to explore the roles of ghmpk in plant biotic stresses, cotton seedlings were inoculated with pathogens, including xanthomonas campestris pv. malvacearum, colletotrichum gossypii and fusarium oxysporum f. sp. vasinfectum. as shown in figure, infection by all of these pathogens elevated the transcription level of ghmpk although the induction kinetic was variable. there was no significant change in ghmpk transcript level without pathogen treatment during days. these results indicate that ghmpk may be intimately involved in the plant pathogen defense response. additionally, we examined the response of ghmpk to exogenously applied salicylic acid, methyl jasmonate and aba, which are plant signaling molecules involved in plant defense signaling pathways. the expression of ghmpk was induced by all of these signal molecules. furthermore, the solvent control did not significantly induce the expression of ghmpk ghmpk promoter analysis inverse-pcr was used to obtain a bp fragment of the 5' flanking region upstream of the transcriptional start site, as determined by the ghmpk cdna sequence. in order to find cis-acting elements, the plantcare databases were analyzed. sequence analysis revealed that the ghmpk promoter contains tata and caat motifs located at nucleotides - and - relative to the transcriptional start site, respectively, which is characteristic of eukaryotic gene promoters. as shown in figure the bp promoter region contains several motifs probably related to pathogen and drought signals, such as a box-w element, an ere element, two tca-element and a mbs element. this suggests that these putative cis-acting elements are responsible for enhanced expression of ghmpk during stress conditions. enhanced resistance of ghmpk transgenic plants to pathogenic infections in order to further explore the function of ghmpk in plant defense, the coding sequence of ghmpk was cloned into the plant binary vector pbi and transformed into arabidopsis. a total of independent transgenic lines were obtained by kanamycin-resistance selection and confirmed by pcr. rna blot analysis was performed to determine the transgenic expression levels in randomly selected lines. two representative lines exhibiting different expression levels were selected and their t transgenic plants used to evaluate disease resistance. to analyze bacterial resistance responses in wild-type and ghmpk16-overexpressing plants, 3-week-old plants were inoculated with p. solanacearum and monitored daily for the appearance of typical disease symptoms. p. solanacearum caused more severe chlorotic symptoms in the wild-type plants compared to the transgenic plants d after inoculation. as shown in figure 6c, the bacterial titres in the inoculated leaves of the transgenic plants were significantly reduced as compared to those in wild-type plants at 3- and 6-day post-inoculation, which is consistent with the observed symptoms. the rate of bacterial growth in the inoculated leaves was lower for transgenic line # than for transgenic line # these results indicate that ghmpk can enhance resistance to p. solanacearum in a dose-dependent manner in transgenic plants. in order to better understand the role of ghmpk in disease resistance in transgenic plants, three marker genes associated with pathogen responses were selected, and the transcript levels of those genes were analyzed by reverse transcription-pcr. the expression of the three marker genes, especially atpr and atpdf, was more rapidly and strongly induced by the p. solanacearum infection in transgenic plants than in wild-type plants. these results demonstrate that the overexpression of ghmpk activates defense-related gene expression when plants are challenged with p. solanacearum. as shown in figure ghmpk16-overexpressing plants were evaluated for their resistance to fungal pathogens. detached leaves were inoculation with c. nicotianae and a. alternate, and after days, the lesions in the wild-type leaves were larger than those in the transgenic leaves, and line # which displayed the highest level of ghmpk expression, exhibited the least severe disease symptoms. to quantify the lesions, the diameters of the disease spots were measured, and the data were consistent with the visual observations. these results indicate that ghmpk can enhance resistance to infections by pathogenic fungi in transgenic plants. after inoculating whole plants with c. nicotianae and a. alternate, the expression of atpr atpr and atpdf was observed at 3- and 6-day post-infection in both the wild-type and transgenic plants, and the expression patterns of the three marker genes were similar to those observed with the p. solanacearum challenge. reduced drought tolerance and rapid h2o accumulation in ghmpk16-overexpressing plants compared with wild-type plants, the ghmpk16-overexpressing plants exhibited obvious drought sensitivity. as shown in figure 8a, the leaves began to turn yellow and curl in both the 3-week-old wild-type and transgenic plants after a 15-day drought treatment. additionally, drought treatment led to more serious damage in the transgenic plants than in the wild-type plants. under normal conditions, no overt morphological differences were observed between wild-type and transgenic plants. we tested the seed germination capacity on 1/ ms agar medium with mm mannitol to mimic drought conditions and found that the germination percentage was higher for wild-type plants than for transgenic plants. the root lengths of both wild-type and transgenic plants were used as indicators of drought stress tolerance. root growth was inhibited by mannitol treatment to a greater extent in transgenic seedlings than in wild-type seedlings. these results suggest that the overexpression of ghmpk in arabidopsis results in reduced drought tolerance. one of the important responses of plants under osmotic stress is the accumulation of reactive oxygen species. among the different ros, only h2o can cross plant membranes and thus directly function in cell-to-cell signaling. therefore, we examined endogenous h2o accumulation in wild-type and transgenic plants in response to mannitol treatment. mannitol was used to treat one-week-old seedlings, and the leaves were collected after and h. staining with'-diaminobenzidine revealed different levels of h2o production in the leaves of wild-type and transgenic plants after osmotic stress treatment. specifically, h2o accumulated in transgenic plants at a significantly faster speed than that in wild-type plants. these results suggest that the overexpression of ghmpk leads to the rapid production of ros or ineffective scavenge of excess ros after osmotic stress. discussion and  in this study, we isolated and characterised ghmpk a gene whose product belongs to the family of group d mapks. ghmpk is the first characterized mapk gene from cotton belonging to the group d mapks. the corresponding protein is characterized by the presence of a tdy activation motif in its t-loop, the lack of a cd domain and an extended c-terminal region compared to the tey subtypes of mapks. these structural differences suggest that group d mapks may function differently from other mapks. the subcellular localization of group d mapks has not been established. osbwmk a group d mapk from rice, is found in the nucleus and mediates pathogenesis-related gene expression by activating the oserebp transcription factor. intriguingly, the alternative splicing of osbwmk generates three different transcriptional variants that produce proteins with different subcellular localizations. in arabidopsis, atmpk is located in the cytoplasm. analysis of the subcellular localization of a gfp-tagged ghmpk protein in transiently transformed onion epidermal cells revealed that ghmpk is likely localized in the nucleus. therefore, we speculated that ghmpk may act as a transcriptional activator by activating the expression of a set of target genes in the nucleus. gene expression patterns are usually an indicator of gene function. notably, ghmpk can be induced by various pathogens, including x. campestris pv. malvacearum, r.solani and c. gossypii. rna blot analyzes revealed that the expression of ghmpk could be induced by signal molecules such as sa, meja, and aba. these results imply that ghmpk may play a role in plant defense responses and in the regulation of certain components of multiple stress-signaling pathways. consistent with this hypothesis, sequence analysis of the ghmpk promoter revealed several motifs, such as box-w element, ere element and tca-element, related to motifs with important roles in defense signaling. recently, a variety of mapk genes have been identified and subsequently explored by both genetic and biochemical approaches. increasing evidence has shown that the mapk cascade is an important pathway in pathogen-triggered signal transduction. signal molecules such as sa, jasmonic acid and ethylene, regulate distinct sets of pathogenesis-related genes in different pathogen defense pathways. sa and meja are involved in two major defense signaling pathways, the sa-dependent and ja/et-dependent defense mechanisms, which act against different types of pathogens. in arabidopsis, atmpk responds to the balance between sa and meja through the eds1/pad module and regulates the sa and ja/et-related defense responses. when ghmpk is ectopically expressed in arabidopsis, the transgenic arabidopsis show enhanced disease resistance against bacteria and fungi. pr is known as a marker gene for the sa signaling pathway, and atpr and atpdf are markers for the ja/et signaling pathway. along with the enhanced disease resistance in ghmpk16-overexpressing plants, the expression of the examined defense-related genes atpr atpr and atpdf was elevated. thus, it is reasonable to speculate that ghmpk may be involved in the crosstalk between the sa- and ja/et- mediated pathogen defense pathways. ghmpk showed a response to various abiotic stresses, such as low or high temperatures, mannitol and nacl. interestingly, transgenic plants overexpressing ghmpk exhibited obvious drought sensitivity. ghmpk overexpression resulted in a significant reduction in both germination percentage and root growth after mannitol treatment, and seedling growth was more severely inhibited by drought stress in ghmpk16-overexpressing plants than in wild-type plants. aba responds to the environmental signals to protect plants from abiotic stresses, such as cold, drought and salt stresses. the expression of ghmpk was strongly induced by exogenous aba and the ghmpk16-overexpressing plants were sensitive to drought tolerance comparing with wild-type arabidopsis, suggesting that ghmpk may be involved in aba signaling and caused negative function for drought tolerance. dab staining revealed that h2o rapidly accumulation in ghmpk16-overexpressing transgenic plants after drought treatment. excessive ros can injure plants. ghmpk accelerates the accumulation of h2o and this may be the reason that drought causes more serious damage in the transgenic plants. in arabidopsis, mpk and mpk function downstream of ros to regulate guard cell aba signaling positively. guard cell-specific inhibition of arabidopsis mpk expression causes abnormal stomatal responses to aba and h2o. more physiological, biochemical and molecular experiments are needed to elucidate the mechanism of ghmpk in response to drought stress. on the basis of these observations, we propose that ghmpk functions in at least two signaling pathways, one that responds to pathogens and another that is involved in drought stress. ghmpk may serve as a point for crosstalk between biotic and abiotic stress response signaling. further studies on the function and regulation of ghmpk are required. 
members of group a and b have been extensively characterized, but little information on the group d mapks has been reported. mitogen-activated protein kinase cascades play pivotal roles in mediating biotic and abiotic stress responses. the analysis of the promoter region of ghmpk revealed a group of putative cis-acting elements related to stress responses. southern blot analysis suggests ghmpk is single copy in the cotton genome, and rna blot analysis indicates that ghmpk transcripts accumulate following pathogen infection and treatment with multiple defense-related signal molecules. in plants, mapks are classified into four major groups according to their sequence homology and conserved phosphorylation motifs. transgenic arabidopsis overexpressing ghmpk displayed significant resistance to fungi and bacteria pathogen, and the transcripts of pathogen-related genes were more rapidly and strongly induced in the transgenic plants. these results suggest that ghmpk might be involved in multiple signal transduction pathways, including biotic and abiotic stress signaling pathways. furthermore, transgenic arabidopsis showed reduced drought tolerance and rapid h2o accumulation.
 mitogen-activated protein kinase cascades play pivotal roles in mediating biotic and abiotic stress responses. in plants, mapks are classified into four major groups according to their sequence homology and conserved phosphorylation motifs. members of group a and b have been extensively characterized, but little information on the group d mapks has been reported.  in this study, we isolated and characterised ghmpk the first group d mapk gene found in cotton. southern blot analysis suggests ghmpk is single copy in the cotton genome, and rna blot analysis indicates that ghmpk transcripts accumulate following pathogen infection and treatment with multiple defense-related signal molecules. the analysis of the promoter region of ghmpk revealed a group of putative cis-acting elements related to stress responses. subcellular localization analysis suggests that ghmpk acts in the nucleus. transgenic arabidopsis overexpressing ghmpk displayed significant resistance to fungi and bacteria pathogen, and the transcripts of pathogen-related genes were more rapidly and strongly induced in the transgenic plants. furthermore, transgenic arabidopsis showed reduced drought tolerance and rapid h2o accumulation.  these results suggest that ghmpk might be involved in multiple signal transduction pathways, including biotic and abiotic stress signaling pathways. cotton ghmpk16pathogen resistancedrought sensitivity 