BACKGROUND
although sessile plants lack a circulating, somatically adaptive immune system, they have developed specific mechanisms to respond to diverse environmental signals that may cause stress and restrict growth and development.
among these mechanisms, many genes that either directly protect plants against stress or further control the expression of other target genes are induced or repressed
<cit> .
therefore, plant defence responses are regulated by a network of signalling pathways that fine-tune the transcriptional activation of defence-related genes.
among these complex responses, it has been demonstrated that the transcriptional activation of defence-related genes requires the expression of particular transcription factors and/or alterations in their dna-binding activity
<cit> .
the wrky family is one of the largest families of transcription factors in plants.
since the first member was isolated in sweet potato, an increasing number of wrky members have been identified, including  <dig> members in arabidopsis thaliana and  <dig> members in oryza sativa <cit> .
the defining characteristic of wrky transcription factors is their dna-binding domain, known as the wrky domain, which is composed of a highly conserved peptide sequence  and a zinc-finger motif,
<cit> .
moreover, the conservation of the wrky domain is mirrored by the conservation of cognate cis-acting w-box elements tgac) in the target promoter region, which are essential for the function and binding of the wrky protein
<cit> .
the number of highly conserved wrky domains and the structure of the zinc-finger motif have resulted in the division of the wrky superfamily into three distinct groups , and group ii can be further split into five distinct subgroups  based on the presence of additional short conserved structural motifs
<cit> .
previous studies have examined the roles of plant wrky proteins in response to pathogens, and wrky proteins can function as either positive or negative regulators of the defence response.
constitutive overexpression of wrky <dig> results in increased resistance to pathogens and the constitutive expression of salicylic acid -induced pathogen-related  genes.
generally, the expression of atwrky <dig> is activated by sa and repressed by ja
<cit> .
furthermore, the enhanced resistance of cawrky1-silenced chilli pepper leaves to xanthomonas axonopodis pv.
vesicatoria suggests that cawrky <dig> acts as a negative regulator of the defence response
<cit> .
interestingly, a dual function in defence signalling has been suggested for atwrky <dig> and atwrky <dig> <cit> .
wrky41-overexpressing arabidopsis exhibited enhanced resistance to pseudomonas syringae but increased susceptibility to erwinia carotovora <cit> .
mechanistically, to increase the defence response, wrky transcription factors frequently upregulate the expression of several pathogen-related genes and nonexpressor of pr <dig>  by binding specifically to the w-box element in their promoter region
<cit> .
the expression of pr proteins, including antimicrobial chitinases and glucanases that directly destroy the cell walls of fungal pathogens, is temporarily and spatially activated following pathogen recognition
<cit> .
npr <dig> is a key regulator of the sa-dependent defence response and systemic acquired resistance , and plants under-expressing npr gene are more susceptible to infection with pathogens
<cit> .
these data suggest that the mechanism underlying the regulation of the defence response by wrky transcription factors is fairly complex and needs to be examined further.
importantly, gmwrky <dig> proteins in soybean  probably form homodimers and can efficiently activate reporter gene expression in a two-hybrid assay
<cit> .
interactions among wrky <dig>  wrky <dig> and wrky <dig> can result in both homocomplexes and heterocomplexes, thereby allowing for adaptation to different microbial pathogens
<cit> .
such protein-protein interactions may affect properties that are important for the regulatory functions of these proteins
<cit> .
few studies have examined the role of the wrky family of transcription factors in plant development.
the overexpression of gmwrky <dig> promotes lateral root development, and this promotion is probably the result of the activation of the downstream gene arf <dig>  a component of the auxin signalling pathway
<cit> .
some wrky transcription factors also function in seed development, dormancy and germination; leaf senescence; and trichome development, which are mostly dependent upon the abscisic acid  and gibberellin  signalling pathways
<cit> .
a recent study has demonstrated that the wrky <dig> a. thaliana mutants underwent secondary thickening in the walls of certain pith cells that were associated with the ectopic deposition of lignin, xylan and cellulose, leading to an approximately 50% increase in biomass density in stem tissues.
this finding suggests the possibility of significantly increasing the mass of fermentable cell wall components in bioenergy crops
<cit> .
together, the above findings suggest that the wrky transcription factors play a crucial role in plant development and disease resistance
<cit> .
whereas previous research has been largely restricted to arabidopsis, tobacco and rice, data regarding the wrky family of transcription factors in cotton , an important economic crop, are notably limited.
in the present study, a cdna clone, ghwrky <dig>  encoding a putative iid wrky gene, was isolated and characterised, and the gene expression patterns under various biotic and abiotic stresses were investigated.
we obtained information on the potential of overexpression of ghwrky <dig> to alter responses to fungal or viral pathogens and developmental processes in tobacco.
this study suggests the alteration of responses to disease attacks in ghwrky <dig> overexpressing tobacco may be related to multiple defence signalling pathways, and further via regulation of the reactive oxygen species  network.
RESULTS
cloning and characterisation of ghwrky15
due to the vital function of the wrky motif in the activity of the wrky family members, we designed a pair of degenerate primers targeting this region  in an attempt to isolate wrky family members from cotton.
using this primer pair, the internal conserved region of one iid wrky subfamily member was obtained.
next, rapid amplification of cdna ends pcr  was used to identify the full-length cdna, and a 419-bp 5' untranslated region  and a 546-bp 3' utr were amplified.
to verify the full-length cdna sequence, two specific primers were designed, and sequence analysis revealed that the full-length cdna contained a 942-bp open reading frame  encoding a 314-amino acid protein with a predicted molecular mass of  <dig>  kda and an isoelectric point of  <dig> .
because this putative cotton wrky family member shares a high degree of homology with wrky <dig> in a. thaliana, we designated this gene ghwrky <dig> .
a protein sequence comparison between ghwrky <dig> and the other plant wrky proteins demonstrated that the amino acid sequences of these proteins are highly similar, with a homology of  <dig> % to ntwrky <dig> ,  <dig> % to atwrky <dig> ,  <dig> % to stwrky <dig> , and  <dig> % to ptwrky <dig>  .
the predicted ghwrky <dig> protein contains an approximately 60-amino acid wrky domain that is composed of the conserved amino acid sequence  and a zinc-finger motif , and a short conserved structural motif , indicating that ghwrky <dig> belongs to group iid of the wrky family
<cit> .
additionally, a putative nuclear localisation signal , kkrk, was found at position 220– <dig> .
a phylogenetic analysis was performed to determine the evolutionary relationship between ghwrky <dig> and the other wrky proteins .
overall, wrky proteins can be classified into three groups, and group ii can be further divided into five subgroups .
the phylogenetic analysis demonstrated that ghwrky <dig> was closely related to group iid wrky family members, including ptwrky <dig>  stwrky <dig>  bnwrky <dig>  atwrky <dig> and atwrky <dig>  these results strongly suggest that ghwrky <dig> belongs to group iid of the wrky family, the members of which may share some similar functions in different species.
to examine the genomic organisation of the ghwrky <dig> locus, we amplified the ghwrky <dig> genomic sequence with a pair of specific primers that had been designed based on the full-length cdna sequence.
the full-length ghwrky <dig> genomic sequence was 1306 bp in length, and two short introns  that exhibited a high at content and had a typical 5'-gt splice donor and 3'-ag splice acceptor were detected.
the comparative analysis of the ghwrky <dig> , atwrky <dig> , atwrky <dig>  and vvwrky  genomic sequences revealed that the number and position of the introns were well-conserved .
specifically, the insert position of the second intron, called an r-type intron, was in the arginine codon of the wrky domain.
additionally, we also determined the copy number of the ghwrky <dig> locus in cotton using fluorescence real-time quantitative pcr  analysis with rna-dependent rna polymerase  <dig> , which is present in the cotton genome as single copy
<cit> , as an internal standard.
the standard curve of ghrdr <dig> and ghwrky <dig> is shown in additional file
<dig> , and the correlation coefficients between the standard curves of ghrdr <dig> and ghwrky <dig> were highly significant.
as shown in additional file
<dig> , our results demonstrate that ghwrky <dig> is probably a single-copy gene in cotton.
ghwrky <dig> is localised to the nucleus
to investigate the mechanism underlying the regulatory activity of ghwrky <dig> in development and during the stress response, the subcellular localisation of ghwrky <dig> was predicted using the psort programme.
an nls  was identified in ghwrky <dig>  suggesting that it is mostly located in the nucleus.
to confirm this prediction, a biolistic transformation system was used for a transient assay, and two constructs, 35s-ghwrky15::gfp and 35s-gfp , were individually introduced into onion epidermal cells.
the fluorescence was observed using a confocal laser scanning microscope with dapi staining to detect the nuclei and interference contrast images to detect the whole-cell structures.
as shown in figure
2b, the fluorescence was predominantly localised to the nucleus in the onion epidermal cells expressing 35s-ghwrky15::gfp, whereas the fluorescence was present throughout the cytoplasm and nucleus in the cells transfected with the control 35s-gfp construct.
these results indicate that the ghwrky <dig> protein is located in the nucleus, and this localisation may aid in the determination of both the function and the molecular mechanism underlying the function of this protein.
analysis of partial putative cis-acting elements in the ghwrky <dig> promoter
a 1012-bp fragment of the ghwrky <dig> promoter  was obtained using inverse pcr  and nested pcr.
an analysis of this region using the plantcare databases revealed a group of putative cis-acting elements that suggest that ghwrky <dig> may play a role in the response to environmental stress.
specifically, we identified pathogen/elicitor-related elements including rav1aat, are, w-box and tl1atsar, abiotic stress responsive element including acgtaterd <dig>  hse, ltr and mbs and tissue-specific and development-related elements including a skn- <dig> motif, circadian, ose2rootnodule, pollen1lelat <dig> and aacacoreosglub <dig>  all of the identified cis-elements are listed in table
<dig>
ghwrky <dig> expression in cotton following exposure to diverse biotic stresses, sa, methyl jasmonate  and et
to examine the role of ghwrky <dig> in response to biotic stress in cotton, cotton seedlings were infected with three different fungi  using the root dip method.
as shown in figure
3a, b, c and additional file
<dig> figure s3a, b, c, these pathogen treatments dramatically enhanced the accumulation of ghwrky <dig> transcripts.
the maximum accumulation of the ghwrky <dig> transcripts occurred  <dig>   <dig> and  <dig> d after treatment with these three fungi, respectively.
there was no obvious change in the expression of ghwrky <dig> under normal conditions without any pathogen infection within 7 days.
these data suggest that ghwrky <dig> may play important roles in the response to pathogens.
to elucidate the molecular mechanism underlying the responsiveness to various biotic stresses, we examined the expression of ghwrky <dig> following treatment with exogenously applied sa, meja and et, which are all phytohormones involved in different signalling pathways.
as shown in figure
3d, e, f, and additional file
<dig> , the expression of ghwrky <dig> was obviously enhanced by sa, meja  and et.
after the sa treatment, ghwrky <dig> expression was noticeably enhanced at 8 h. following the meja treatment, transcripts of ghwrky <dig> accumulated at 4 h and reached a peak from 8 h to 24 h. under et treatment, ghwrky <dig> accumulated at 2 h, and reached a peak at 4 h. but et treatment does not lead to a higher expression than sa or meja treatment, respectively.
therefore, ghwrky <dig> may be involved in the sa, et and ja signalling pathways that function in disease resistance.
tobacco plants overexpressing ghwrky <dig> exhibit enhanced viral and fungal resistance
to investigate the effects of constitutive overexpression of ghwrky <dig> on tobacco, full-length ghwrky <dig> was cloned into the binary vector pbi <dig> by replacing the gus gene, and tobacco lines overexpressing ghwrky <dig> were generated.
a total of  <dig> independent transgenic lines were obtained following selection with kanamycin, and these lines were confirmed using pcr.
additionally,  <dig> lines of transgenic t <dig> plants were randomly selected for an expression analysis.
the expression of the transformed ghwrky <dig> gene in the leaf tissue was detected using northern blots, whereas the control, wild-type  tobacco lines demonstrated no expression .
three independent lines, oe <dig> , oe <dig>  and oe <dig> , which exhibited different expression levels of the target gene were used for further functional analysis.
the disease resistance tests were performed on the t <dig> progeny of the three transgenic lines using two kinds of viruses and fungi and compared with the resistance of wt plants.
six-week-old transgenic tobacco plants were inoculated with tobacco mosaic virus  or cucumber mosaic virus .
minor disease symptoms were observed in the wt plants seven days after inoculation with tmv, with the leaf veins and adjacent tissue colour fading and appearing dark green, whereas no symptoms were seen in any of the three transgenic lines.
the leaves of the tmv-inoculated lines 10 days after inoculation are shown in figure
4b.
however, there were no significant differences in the disease symptoms between the transgenic and wt plants fourteen days after inoculation with tmv; both plants exhibited severely diseased, crinkled leaves, leaf distortion, plant dwarfism and slow growth.
for the cmv inoculation, no difference in the disease symptoms was observed between the wt and transgenic lines up to seven days after the inoculation, but the wt plants exhibited significantly more severe disease symptoms relative to the transgenic plants after fourteen days .
furthermore, the expression level of the coat proteins  gene was much lower in both the tmv- and cmv-inoculated transgenic plants than in the wt line .
additionally, as detected using elisas, the average expression of the tmv and cmv cps in the transgenic lines was lower than the expression in the wt line .
to investigate whether ghwrky15-overexpressing tobacco plants possess enhanced fungal resistance compared with wt plants, conidial suspensions of colletotrichum gossypii and phytophthora parasitica spores were sprayed on detached leaves.
visually, the detached leaves of the transgenic plants exhibited more resistance to both c. gossypii and p. parasitica infection than the wt plants .
to quantify the lesions, the number and diameter of the disease spots in the wild-type and transgenic plants were measured, and the data were consistent with what was observed visually .
overexpression of ghwrky <dig> affects the expression of pr genes and ethylene  biosynthesis-related genes
because many wrky transcription factors have been shown to be involved in activating the transcription of defence genes, particularly the transcription of pr genes
<cit> , the expression of pr genes was investigated using qpcr after the plants were infected with tmv or c. gossypii.
following infection with tmv, the expression of pr genes, including pr <dig>  pr <dig> , pr <dig> and pr <dig> , was greater in the three transgenic lines than the expression in the wt plants .
moreover, the expression of the sa signalling-related gene npr <dig> <cit>  was also slightly enhanced.
to further elucidate the possible mechanisms underlying the enhanced viral resistance of the transgenic plants, we examined the expression of two enzymes that are involved in et biosynthesis, 1-aminocyclopropane-1-carboxylic acid  synthase , which catalyses the conversion of s-adenosyl-l-met into acc, and acc oxidase , which converts acc into et
<cit> .
in all three transgenic lines, the expression levels of aco and acs were higher than those in wt plants infected with tmv .
similar results were obtained following c. gossypii infection, except that the transcription of pr <dig> was inhibited in the transgenic plants compared with the wt plants .
in addition, the expression of the disease-responsive gene sar <dig>  l was significantly upregulated .
overexpression of ghwrky <dig> decreases the accumulation of ros and activates the expression of oxidation-related genes
some virus-host plant interactions result in oxidative stress and the generation of ros
<cit> .
h2o <dig>  singlet oxygen, o2-, and hydroxyl radicals are all ros, and we monitored the accumulation of h2o <dig> in transgenic plants and wt plants following exposure to tmv, cmv or c. gossypii.
the accumulation of h2o <dig> was detected using histochemical analyses via dab staining.
as seen by the visible accumulation of a brown precipitate, the leaves of transgenic tobacco plants accumulated lower levels of h2o <dig> relative to the wt plants .
a microscopic analysis of the plants following viral infection also indicated less brown precipitate on the leaves of transgenic plants when compared with wt plants , which is consistent with the results defined visually.
a potential redox mediator, methyl viologen , was used to verify whether ghwrky <dig> is responsive to oxidative stress.
the expression of ghwrky <dig> was upregulated 2 h after treatment with  <dig>  mm mv, and it was significantly enhanced at 4 h .
to explore the underlying mechanism of the reduced ros levels in the transgenic plants during the disease resistance process, the expression of six genes was analysed in response to oxidative stress.
the enzyme sod acts as the first line of defence against ros by transforming o2- into h2o <dig>  and such enzymes as apx, cat and gpx play important roles in regulating intracellular h2o <dig> levels
<cit> .
to our surprise, with the exception of the upregulation of ntgpx, the expression of the other five antioxidant genes  was decreased in the transgenic plants relative to the wt plants under normal conditions .
however, when treated with mv, the expression of ntapx <dig> and ntgpx was enhanced  in the transgenic plants compared with the wild-type plants, whereas the ntcat <dig> and ntca expression levels were still decreased in the transgenic plants relative to the wild-type plants.
we observed no significant differences in the expression of ntsod and ntapx <dig> between the transgenic and wt lines.
these results suggest that the overexpression of ghwrky <dig> indeed affected the antioxidant system in tobacco, which led us to further investigate how ghwrky <dig> regulated the ros scavenging pathway.
thus, we examined the enzymatic activities of sod, pod, cat and apx in the oxidative response during treatment with tmv or cmv .
compared to the activities observed in wt plants, the activities of pod and apx were significantly increased in the oe lines, but there was a slight decrease in cat activity.
interestingly, the activity of sod was significantly increased following infection with tmv, whereas the activity of sod following infection with cmv was decreased.
these results indicate that the role of ghwrky <dig> in the ros-scavenging pathway is complicated.
expression of ghwrky <dig> affects plant growth and development
many wrky genes have been reported to affect plant development and growth throughout the entire development and growth period.
to investigate the effects of constitutive overexpression of ghwrky <dig> on plant growth and development in tobacco, the growth phenotype of both wt plants and transgenic lines was monitored.
except for the later germination of the transgenic plants relative to the wt plants , we observed no difference between the plants until approximately ten weeks.
the transgenic plants entered the shooting stages earlier than did the wild-type plants.
as shown in figure
10b, the stems of the oe plants began to elongate at approximately ten weeks, but the stems of the wt tobacco had no evident elongation.
interestingly, the stems of the transgenic plants were much thinner than the stems of the wt plants during the shooting stage .
microscopic analyses revealed that the cortical cells of the transgenic plants had smaller diameters relative to the cells of the wt plants , but no difference in the cell diameter of the pith was observed .
moreover, no difference was observed in the length of the cells in the wt and transgenic plants .
after the shooting stage of the transgenic plants began, overexpression of ghwrky <dig> resulted in higher transgenic plants relative to wild-type plants .
however, this difference in the stem length disappeared at the flower stage.
moreover, ghwrky15-overexpressing transgenic plants displayed premature flowering by two to three weeks relative to the wild-type plants under normal growth conditions .
additionally, the bottom leaves of the oe lines exhibited senescence earlier than the bottom leaves of the wt plants .
tissue-specific expression of ghwrky <dig> and the effects of abiotic stresses on ghwrky <dig> expression
to characterise the expression of ghwrky <dig> under abiotic stresses, including cold, high salinity, wounding and drought , northern blots were performed using total rna extracted from cotton seedlings.
whereas the expression of ghwrky <dig> exhibited negligible changes following nacl treatment , the other three treatments resulted in a dramatic increase in the expression of ghwrky <dig> within 8 h. as shown in figure
12b, d, e, the expression patterns were different.
ghwrky <dig> expression increased sharply within 2 h following wounding, and the high level due to the wounding was present until 8 h. following the cold treatment, increased ghwrky <dig> expression was detected at 2 h, and this expression continued gradually to increase.
the peak expression of ghwrky <dig> following drought treatment occurred at 8 h, after which the expression level decreased gradually.
these results indicate that ghwrky <dig> expression can be regulated by abiotic stresses.
intriguingly, there was a greater accumulation of ghwrky <dig> transcripts in the root and stem than in the leaves , and ga <dig> treatment led to a higher expression level from 4 h to 24 h , whereas aba did not increase expression until 24 h .
these results suggest that ghwrky <dig> may play a role not only in response to abiotic stresses but also in plant growth and development.
discussion
although five major families of plant transcription factors, including bzip, wrky, myb, erebf and homeodomain protein, have been shown to play roles in the regulation of the plant defence response
<cit> , little is known about the exact function or mechanism of individual transcription factors.
generally, it is believed that the wrky family of transcription factors plays major roles in plant responses to biotic and abiotic stresses and during development
<cit> .
most functional analyses of wrky transcription factors have been restricted to a. thaliana and o. sativa, and few wrky family members from cotton have been reported previously.
to explore the function of the wrky transcription factors in cotton, we isolated a wrky gene from g. hirsutum, which is an important economic crop cultivated worldwide.
due to the high similarity between the sequence of the protein encoded by the wrky gene obtained from cotton and those of several wrkys obtained from n. tabacum, a. thaliana, solanum tuberosum and populus tomentosa, we verified that the gene we isolated from cotton is a wrky transcription factor gene.
the phylogenetic tree and the short conserved harf structural motif further indicated that the ghwrky gene belongs to subgroup iid, with atwrky <dig>  atwrky <dig> and atwrky <dig> as its closest homologues in a. thaliana <cit> ; as a result, we designated the isolated gene ghwrky <dig>  consistent with the putative role of wrky proteins as transcription factors, a nuclear targeting sequence  was identified in the c-terminal region of the protein, and the targeting experiment suggested that gfp-tagged ghwrky <dig> was located in nucleus.
these results suggest that, similar to ghwrky <dig> <cit> , ghwrky <dig> might function in the nucleus.
to investigate the effects of constitutive overexpression of ghwrky <dig> on the response to pathogen resistance, ghwrky <dig> overexpressing-tobacco plants were inoculated with two types of viruses and two pathogenic fungi.
the plants constitutively overexpressing ghwrky <dig> were more resistant to tmv and cmv infection compared with the wt tobacco plants.
this observation diverges from previous research demonstrating that the virus-induced gene silencing  of nbwrky <dig>  nbwrky <dig>  or nbwrky <dig> suppressed resistance to tmv and that the size of the hypersensitive response  lesions was also reduced in nbwrky1-silenced plants
<cit> .
the ghwrky <dig> transgenic plants also exhibited increased resistance to fungal pathogens  compared with wild-type plants.
previously, overexpression studies examining a pair of allelic genes oswrky45- <dig> and oswrky45- <dig> demonstrated that these genes positively regulated resistance to the rice fungal pathogen magnaporthe grisea <cit> .
our data presented above indicate that ghwrky <dig> overexpression increases the tolerance of the plant to pathogens; therefore, the upregulation of ghwrky <dig> in cotton seedlings in response to pathogens, including r. solani, c. gossypii and f. oxysporum, suggests that ghwrky <dig> may participate in the regulation of plant responses to disease defence.
the increased expression of pr <dig>  pr <dig> and pr <dig> contributed to the increased resistance of transgenic ghwrky15-overexpressing plants to tmv and c. gossypii.
furthermore, previous studies have demonstrated that wrky dna binding-proteins can regulate the transcription of a number of defence-related genes, including the well-studied pr genes, via binding to the w-box in the promoter of these genes
<cit> .
it is noted that the expression of the pr <dig> gene in transgenic plants during c. gossypii infection was down-regulated compared with the infected wt plants, suggesting that the molecular mechanism underlying the activity of ghwrky <dig> is fairly complex under different biotic stresses or that ghwrky <dig> may be an integrator of several signalling pathways.
sa and ja are two well-studied key mediators of plant resistance.
in arabidopsis, more than  <dig> wrky genes can be differentially regulated after infection with p. syringae or treatment with sa
<cit> .
infection of ghwrky <dig> transgenic plants with tmv and c. gossypii increased the expression of npr <dig>  which is a key regulator of the sa-dependent defence pathway and sar.
furthermore, the analysis of the expression of ghwrky <dig> in cotton seedlings showed that ghwrky <dig> expression can be affected by sa and meja, supporting our hypothesis that ghwrky <dig> plays a role in resistance to viral and fungal pathogens via sa- and ja-dependent defence pathways.
interestingly, reports of wrky proteins that function in both sa- and ja-dependent pathways are relatively limited
<cit> .
moreover, the expression of two key enzymes that are involved in et biosynthesis  is increased in ghwrky <dig> transgenic plants following infection with fungi and viruses.
and there was significant increase of ghwrky <dig> expression in cotton seedlings following et treatment.
this suggests that the increased disease resistance of ghwrky <dig> overexpressing tobacco compared to the wide type may partially depend on the expression of et synthesis related genes.
additionally, ghwrky <dig> expression could be increased under various abiotic stress conditions, including low temperature, wounding and drought, and a group of cis-elements related to biotic and abiotic stresses were identified in the ghwrky <dig> promoter.
altogether, these data support our hypothesis that the mechanism of response to various environmental stresses, especially viral and fungal infection, in ghwrky <dig> overexpressing plants may be related with multiple signalling pathways, including sa, ja or et signalling pathways.
mounting evidence suggests that the ros network is essential for the induction of disease resistance
<cit> .
for instance, mitogen-activated protein kinase  cascades members ghmapk <dig> and ghmapk <dig> are both involved in the response to infection and oxidative stress
<cit> .
following challenge with a virus or a fungus, a lower accumulation of h2o <dig> and a higher activity of antioxidant enzymes, including pod and apx, were detected in the ghwrky15-overexpressing plants relative to the wt plants.
we reasoned that ghwrky <dig> upregulates the activity of pod and apx to increase the oxidative stress tolerance and contributes to the resistance towards biotic stresses in transgenic plants.
however, except for ntgpx, the expression of the other five antioxidant enzymes examined was reduced in unstressed transgenic seedlings compared with the wild-type plants.
these results contrast previous research examining some genes, which appear to play positive roles in the stress response and enhance the expression of most antioxidant enzymes
<cit> .
to explain this difference, the expression of the six antioxidant enzymes was examined during treatment with mv, which is a potential redox mediator.
although there was no significant difference in the expression of ntsod and ntapx <dig> in the transgenic plants compared with the wt plants following the treatment with mv, the expression of these genes was increased compared with the expression in plants receiving no treatment.
additionally, the upregulation of ntgpx and ntapx <dig> expression during mv treatment suggests that ghwrky <dig> overexpression can help plants maintain ros homoeostasis under oxidative stresses to alleviate injury to the organism.
most recently, it has been reported that there was a substantial increase in the musawrky <dig> level when banana plantlets were subjected to h2o <dig>  moreover, a putative thioredoxin-dependent peroxidase gene belonging to the ros detoxification system was induced in musawrky71-overexpressing cultures
<cit> .
furthermore, in arabidopsis, the maximum expression of wrky <dig> was detected in seven-week-old plants overexpressing wrky <dig>  at which point the h2o <dig> level increased significantly
<cit> .
in wild-type cotton seedlings, a substantial increase in the level of ghwrky <dig> was also observed following exposure to mv.
intriguingly, some of the growth and development phenomena observed in plants overexpressing ghwrky <dig> are similar to those observed in atwrky <dig> over-expressing plants.
similar to ghwrky15-overexpressing plants, atwrky53-overexpressing plants demonstrated early flowering after sowing, and the senescence of the whole plant with partly necrotic leaves was visible earlier when compared with wild-type plants
<cit> .
interestingly, overexpression of ghwrky <dig> in n. tabacum cv.
nc <dig> promoted stem elongation and inhibited the transversal growth of stems.
atwrky <dig> controls cell fate in thickened pith cells by acting as a negative regulator of nst <dig> and c3h zinc finger tfs, which, in turn, regulate secondary cell wall synthesis
<cit> .
although no difference in the diameter and length of pith cells was observed in our transgenic plants, overexpression of ghwrky <dig> may affect the fate of cells in the cortex, which had a smaller diameter relative to wt plants.
the phytohormone ga is an essential regulator of stem elongation and other developmental processes throughout the life cycle of a plant
<cit> .
the induction of ghwrky <dig> expression during ga <dig> treatment combined with the higher expression of ghwrky <dig> in the roots and stems compared with the cotyledon of cotton may clarify the underlying mechanism of the influence of ghwrky <dig> overexpression on plant growth and development.
CONCLUSIONS
in conclusion, we identified and characterised a group iid wrky member in cotton.
our results strongly suggest that overexpression of ghwrky <dig> confers tobacco resistance to both viral and fungal pathogens, and indicate that overexpression of ghwrky <dig> regulates the stem growth or development in tobacco.
the elucidation of the regulatory mechanism of overexpression of ghwrky <dig> in tobacco may provide a converging node of regulatory pathways in response to pathogens and oxidative stress and in the regulation of plant development.
although the influence of overexpression of ghwrky <dig> in tobacco was explored, the mechanism underlying the function of ghwrky <dig> should be elucidated in transgenic cotton and loss-of function data in cotton are particularly needed.
moreover, other research focusing ghwrky <dig> is still needed, for example, the study of its interactions with other wrky proteins or mapk cascades members.

 although sessile plants lack a circulating, somatically adaptive immune system, they have developed specific mechanisms to respond to diverse environmental signals that may cause stress and restrict growth and development. among these mechanisms, many genes that either directly protect plants against stress or further control the expression of other target genes are induced or repressed. therefore, plant defence responses are regulated by a network of signalling pathways that fine-tune the transcriptional activation of defence-related genes. among these complex responses, it has been demonstrated that the transcriptional activation of defence-related genes requires the expression of particular transcription factors and/or alterations in their dna-binding activity. the wrky family is one of the largest families of transcription factors in plants. since the first member was isolated in sweet potato, an increasing number of wrky members have been identified, including members in arabidopsis thaliana and members in oryza sativa. the defining characteristic of wrky transcription factors is their dna-binding domain, known as the wrky domain, which is composed of a highly conserved peptide sequence and a zinc-finger motif,. moreover, the conservation of the wrky domain is mirrored by the conservation of cognate cis-acting w-box elements tgac) in the target promoter region, which are essential for the function and binding of the wrky protein. the number of highly conserved wrky domains and the structure of the zinc-finger motif have resulted in the division of the wrky superfamily into three distinct groups, and group ii can be further split into five distinct subgroups based on the presence of additional short conserved structural motifs. previous studies have examined the roles of plant wrky proteins in response to pathogens, and wrky proteins can function as either positive or negative regulators of the defence response. constitutive overexpression of wrky results in increased resistance to pathogens and the constitutive expression of salicylic acid -induced pathogen-related genes. generally, the expression of atwrky is activated by sa and repressed by ja. furthermore, the enhanced resistance of cawrky1-silenced chilli pepper leaves to xanthomonas axonopodis pv. vesicatoria suggests that cawrky acts as a negative regulator of the defence response. interestingly, a dual function in defence signalling has been suggested for atwrky and atwrky. wrky41-overexpressing arabidopsis exhibited enhanced resistance to pseudomonas syringae but increased susceptibility to erwinia carotovora. mechanistically, to increase the defence response, wrky transcription factors frequently upregulate the expression of several pathogen-related genes and nonexpressor of pr by binding specifically to the w-box element in their promoter region. the expression of pr proteins, including antimicrobial chitinases and glucanases that directly destroy the cell walls of fungal pathogens, is temporarily and spatially activated following pathogen recognition. npr is a key regulator of the sa-dependent defence response and systemic acquired resistance, and plants under-expressing npr gene are more susceptible to infection with pathogens. these data suggest that the mechanism underlying the regulation of the defence response by wrky transcription factors is fairly complex and needs to be examined further. importantly, gmwrky proteins in soybean probably form homodimers and can efficiently activate reporter gene expression in a two-hybrid assay. interactions among wrky wrky and wrky can result in both homocomplexes and heterocomplexes, thereby allowing for adaptation to different microbial pathogens. such protein-protein interactions may affect properties that are important for the regulatory functions of these proteins. few studies have examined the role of the wrky family of transcription factors in plant development. the overexpression of gmwrky promotes lateral root development, and this promotion is probably the result of the activation of the downstream gene arf a component of the auxin signalling pathway. some wrky transcription factors also function in seed development, dormancy and germination; leaf senescence; and trichome development, which are mostly dependent upon the abscisic acid and gibberellin signalling pathways. a recent study has demonstrated that the wrky a. thaliana mutants underwent secondary thickening in the walls of certain pith cells that were associated with the ectopic deposition of lignin, xylan and cellulose, leading to an approximately 50% increase in biomass density in stem tissues. this finding suggests the possibility of significantly increasing the mass of fermentable cell wall components in bioenergy crops. together, the above findings suggest that the wrky transcription factors play a crucial role in plant development and disease resistance. whereas previous research has been largely restricted to arabidopsis, tobacco and rice, data regarding the wrky family of transcription factors in cotton, an important economic crop, are notably limited. in the present study, a cdna clone, ghwrky encoding a putative iid wrky gene, was isolated and characterised, and the gene expression patterns under various biotic and abiotic stresses were investigated. we obtained information on the potential of overexpression of ghwrky to alter responses to fungal or viral pathogens and developmental processes in tobacco. this study suggests the alteration of responses to disease attacks in ghwrky overexpressing tobacco may be related to multiple defence signalling pathways, and further via regulation of the reactive oxygen species network.  cloning and characterisation of ghwrky15 due to the vital function of the wrky motif in the activity of the wrky family members, we designed a pair of degenerate primers targeting this region in an attempt to isolate wrky family members from cotton. using this primer pair, the internal conserved region of one iid wrky subfamily member was obtained. next, rapid amplification of cdna ends pcr was used to identify the full-length cdna, and a 419-bp 5' untranslated region and a 546-bp 3' utr were amplified. to verify the full-length cdna sequence, two specific primers were designed, and sequence analysis revealed that the full-length cdna contained a 942-bp open reading frame encoding a 314-amino acid protein with a predicted molecular mass of  kda and an isoelectric point of. because this putative cotton wrky family member shares a high degree of homology with wrky in a. thaliana, we designated this gene ghwrky. a protein sequence comparison between ghwrky and the other plant wrky proteins demonstrated that the amino acid sequences of these proteins are highly similar, with a homology of % to ntwrky, % to atwrky, % to stwrky, and % to ptwrky. the predicted ghwrky protein contains an approximately 60-amino acid wrky domain that is composed of the conserved amino acid sequence and a zinc-finger motif, and a short conserved structural motif, indicating that ghwrky belongs to group iid of the wrky family. additionally, a putative nuclear localisation signal, kkrk, was found at position 220–. a phylogenetic analysis was performed to determine the evolutionary relationship between ghwrky and the other wrky proteins. overall, wrky proteins can be classified into three groups, and group ii can be further divided into five subgroups. the phylogenetic analysis demonstrated that ghwrky was closely related to group iid wrky family members, including ptwrky stwrky bnwrky atwrky and atwrky these results strongly suggest that ghwrky belongs to group iid of the wrky family, the members of which may share some similar functions in different species. to examine the genomic organisation of the ghwrky locus, we amplified the ghwrky genomic sequence with a pair of specific primers that had been designed based on the full-length cdna sequence. the full-length ghwrky genomic sequence was 1306 bp in length, and two short introns that exhibited a high at content and had a typical 5'-gt splice donor and 3'-ag splice acceptor were detected. the comparative analysis of the ghwrky, atwrky, atwrky and vvwrky genomic sequences revealed that the number and position of the introns were well-conserved. specifically, the insert position of the second intron, called an r-type intron, was in the arginine codon of the wrky domain. additionally, we also determined the copy number of the ghwrky locus in cotton using fluorescence real-time quantitative pcr analysis with rna-dependent rna polymerase, which is present in the cotton genome as single copy, as an internal standard. the standard curve of ghrdr and ghwrky is shown in additional file, and the correlation coefficients between the standard curves of ghrdr and ghwrky were highly significant. as shown in additional file, our results demonstrate that ghwrky is probably a single-copy gene in cotton. ghwrky is localised to the nucleus to investigate the mechanism underlying the regulatory activity of ghwrky in development and during the stress response, the subcellular localisation of ghwrky was predicted using the psort programme. an nls was identified in ghwrky suggesting that it is mostly located in the nucleus. to confirm this prediction, a biolistic transformation system was used for a transient assay, and two constructs, 35s-ghwrky15::gfp and 35s-gfp, were individually introduced into onion epidermal cells. the fluorescence was observed using a confocal laser scanning microscope with dapi staining to detect the nuclei and interference contrast images to detect the whole-cell structures. as shown in figure 2b, the fluorescence was predominantly localised to the nucleus in the onion epidermal cells expressing 35s-ghwrky15::gfp, whereas the fluorescence was present throughout the cytoplasm and nucleus in the cells transfected with the control 35s-gfp construct. these results indicate that the ghwrky protein is located in the nucleus, and this localisation may aid in the determination of both the function and the molecular mechanism underlying the function of this protein. analysis of partial putative cis-acting elements in the ghwrky promoter a 1012-bp fragment of the ghwrky promoter was obtained using inverse pcr and nested pcr. an analysis of this region using the plantcare databases revealed a group of putative cis-acting elements that suggest that ghwrky may play a role in the response to environmental stress. specifically, we identified pathogen/elicitor-related elements including rav1aat, are, w-box and tl1atsar, abiotic stress responsive element including acgtaterd hse, ltr and mbs and tissue-specific and development-related elements including a skn- motif, circadian, ose2rootnodule, pollen1lelat and aacacoreosglub all of the identified cis-elements are listed in table  ghwrky expression in cotton following exposure to diverse biotic stresses, sa, methyl jasmonate and et to examine the role of ghwrky in response to biotic stress in cotton, cotton seedlings were infected with three different fungi using the root dip method. as shown in figure 3a, b, c and additional file figure s3a, b, c, these pathogen treatments dramatically enhanced the accumulation of ghwrky transcripts. the maximum accumulation of the ghwrky transcripts occurred and d after treatment with these three fungi, respectively. there was no obvious change in the expression of ghwrky under normal conditions without any pathogen infection within 7 days. these data suggest that ghwrky may play important roles in the response to pathogens. to elucidate the molecular mechanism underlying the responsiveness to various biotic stresses, we examined the expression of ghwrky following treatment with exogenously applied sa, meja and et, which are all phytohormones involved in different signalling pathways. as shown in figure 3d, e, f, and additional file, the expression of ghwrky was obviously enhanced by sa, meja and et. after the sa treatment, ghwrky expression was noticeably enhanced at 8 h. following the meja treatment, transcripts of ghwrky accumulated at 4 h and reached a peak from 8 h to 24 h. under et treatment, ghwrky accumulated at 2 h, and reached a peak at 4 h. but et treatment does not lead to a higher expression than sa or meja treatment, respectively. therefore, ghwrky may be involved in the sa, et and ja signalling pathways that function in disease resistance. tobacco plants overexpressing ghwrky exhibit enhanced viral and fungal resistance to investigate the effects of constitutive overexpression of ghwrky on tobacco, full-length ghwrky was cloned into the binary vector pbi by replacing the gus gene, and tobacco lines overexpressing ghwrky were generated. a total of independent transgenic lines were obtained following selection with kanamycin, and these lines were confirmed using pcr. additionally, lines of transgenic t plants were randomly selected for an expression analysis. the expression of the transformed ghwrky gene in the leaf tissue was detected using northern blots, whereas the control, wild-type tobacco lines demonstrated no expression. three independent lines, oe, oe and oe, which exhibited different expression levels of the target gene were used for further functional analysis. the disease resistance tests were performed on the t progeny of the three transgenic lines using two kinds of viruses and fungi and compared with the resistance of wt plants. six-week-old transgenic tobacco plants were inoculated with tobacco mosaic virus or cucumber mosaic virus. minor disease symptoms were observed in the wt plants seven days after inoculation with tmv, with the leaf veins and adjacent tissue colour fading and appearing dark green, whereas no symptoms were seen in any of the three transgenic lines. the leaves of the tmv-inoculated lines 10 days after inoculation are shown in figure 4b. however, there were no significant differences in the disease symptoms between the transgenic and wt plants fourteen days after inoculation with tmv; both plants exhibited severely diseased, crinkled leaves, leaf distortion, plant dwarfism and slow growth. for the cmv inoculation, no difference in the disease symptoms was observed between the wt and transgenic lines up to seven days after the inoculation, but the wt plants exhibited significantly more severe disease symptoms relative to the transgenic plants after fourteen days. furthermore, the expression level of the coat proteins gene was much lower in both the tmv- and cmv-inoculated transgenic plants than in the wt line. additionally, as detected using elisas, the average expression of the tmv and cmv cps in the transgenic lines was lower than the expression in the wt line. to investigate whether ghwrky15-overexpressing tobacco plants possess enhanced fungal resistance compared with wt plants, conidial suspensions of colletotrichum gossypii and phytophthora parasitica spores were sprayed on detached leaves. visually, the detached leaves of the transgenic plants exhibited more resistance to both c. gossypii and p. parasitica infection than the wt plants. to quantify the lesions, the number and diameter of the disease spots in the wild-type and transgenic plants were measured, and the data were consistent with what was observed visually. overexpression of ghwrky affects the expression of pr genes and ethylene biosynthesis-related genes because many wrky transcription factors have been shown to be involved in activating the transcription of defence genes, particularly the transcription of pr genes, the expression of pr genes was investigated using qpcr after the plants were infected with tmv or c. gossypii. following infection with tmv, the expression of pr genes, including pr pr, pr and pr, was greater in the three transgenic lines than the expression in the wt plants. moreover, the expression of the sa signalling-related gene npr was also slightly enhanced. to further elucidate the possible mechanisms underlying the enhanced viral resistance of the transgenic plants, we examined the expression of two enzymes that are involved in et biosynthesis, 1-aminocyclopropane-1-carboxylic acid synthase, which catalyses the conversion of s-adenosyl-l-met into acc, and acc oxidase, which converts acc into et. in all three transgenic lines, the expression levels of aco and acs were higher than those in wt plants infected with tmv. similar results were obtained following c. gossypii infection, except that the transcription of pr was inhibited in the transgenic plants compared with the wt plants. in addition, the expression of the disease-responsive gene sar  l was significantly upregulated. overexpression of ghwrky decreases the accumulation of ros and activates the expression of oxidation-related genes some virus-host plant interactions result in oxidative stress and the generation of ros. h2o singlet oxygen, o2-, and hydroxyl radicals are all ros, and we monitored the accumulation of h2o in transgenic plants and wt plants following exposure to tmv, cmv or c. gossypii. the accumulation of h2o was detected using histochemical analyses via dab staining. as seen by the visible accumulation of a brown precipitate, the leaves of transgenic tobacco plants accumulated lower levels of h2o relative to the wt plants. a microscopic analysis of the plants following viral infection also indicated less brown precipitate on the leaves of transgenic plants when compared with wt plants, which is consistent with the results defined visually. a potential redox mediator, methyl viologen, was used to verify whether ghwrky is responsive to oxidative stress. the expression of ghwrky was upregulated 2 h after treatment with  mm mv, and it was significantly enhanced at 4 h. to explore the underlying mechanism of the reduced ros levels in the transgenic plants during the disease resistance process, the expression of six genes was analysed in response to oxidative stress. the enzyme sod acts as the first line of defence against ros by transforming o2- into h2o and such enzymes as apx, cat and gpx play important roles in regulating intracellular h2o levels. to our surprise, with the exception of the upregulation of ntgpx, the expression of the other five antioxidant genes was decreased in the transgenic plants relative to the wt plants under normal conditions. however, when treated with mv, the expression of ntapx and ntgpx was enhanced in the transgenic plants compared with the wild-type plants, whereas the ntcat and ntca expression levels were still decreased in the transgenic plants relative to the wild-type plants. we observed no significant differences in the expression of ntsod and ntapx between the transgenic and wt lines. these results suggest that the overexpression of ghwrky indeed affected the antioxidant system in tobacco, which led us to further investigate how ghwrky regulated the ros scavenging pathway. thus, we examined the enzymatic activities of sod, pod, cat and apx in the oxidative response during treatment with tmv or cmv. compared to the activities observed in wt plants, the activities of pod and apx were significantly increased in the oe lines, but there was a slight decrease in cat activity. interestingly, the activity of sod was significantly increased following infection with tmv, whereas the activity of sod following infection with cmv was decreased. these results indicate that the role of ghwrky in the ros-scavenging pathway is complicated. expression of ghwrky affects plant growth and development many wrky genes have been reported to affect plant development and growth throughout the entire development and growth period. to investigate the effects of constitutive overexpression of ghwrky on plant growth and development in tobacco, the growth phenotype of both wt plants and transgenic lines was monitored. except for the later germination of the transgenic plants relative to the wt plants, we observed no difference between the plants until approximately ten weeks. the transgenic plants entered the shooting stages earlier than did the wild-type plants. as shown in figure 10b, the stems of the oe plants began to elongate at approximately ten weeks, but the stems of the wt tobacco had no evident elongation. interestingly, the stems of the transgenic plants were much thinner than the stems of the wt plants during the shooting stage. microscopic analyses revealed that the cortical cells of the transgenic plants had smaller diameters relative to the cells of the wt plants, but no difference in the cell diameter of the pith was observed. moreover, no difference was observed in the length of the cells in the wt and transgenic plants. after the shooting stage of the transgenic plants began, overexpression of ghwrky resulted in higher transgenic plants relative to wild-type plants. however, this difference in the stem length disappeared at the flower stage. moreover, ghwrky15-overexpressing transgenic plants displayed premature flowering by two to three weeks relative to the wild-type plants under normal growth conditions. additionally, the bottom leaves of the oe lines exhibited senescence earlier than the bottom leaves of the wt plants. tissue-specific expression of ghwrky and the effects of abiotic stresses on ghwrky expression to characterise the expression of ghwrky under abiotic stresses, including cold, high salinity, wounding and drought, northern blots were performed using total rna extracted from cotton seedlings. whereas the expression of ghwrky exhibited negligible changes following nacl treatment, the other three treatments resulted in a dramatic increase in the expression of ghwrky within 8 h. as shown in figure 12b, d, e, the expression patterns were different. ghwrky expression increased sharply within 2 h following wounding, and the high level due to the wounding was present until 8 h. following the cold treatment, increased ghwrky expression was detected at 2 h, and this expression continued gradually to increase. the peak expression of ghwrky following drought treatment occurred at 8 h, after which the expression level decreased gradually. these results indicate that ghwrky expression can be regulated by abiotic stresses. intriguingly, there was a greater accumulation of ghwrky transcripts in the root and stem than in the leaves, and ga treatment led to a higher expression level from 4 h to 24 h, whereas aba did not increase expression until 24 h. these results suggest that ghwrky may play a role not only in response to abiotic stresses but also in plant growth and development. discussion although five major families of plant transcription factors, including bzip, wrky, myb, erebf and homeodomain protein, have been shown to play roles in the regulation of the plant defence response, little is known about the exact function or mechanism of individual transcription factors. generally, it is believed that the wrky family of transcription factors plays major roles in plant responses to biotic and abiotic stresses and during development. most functional analyses of wrky transcription factors have been restricted to a. thaliana and o. sativa, and few wrky family members from cotton have been reported previously. to explore the function of the wrky transcription factors in cotton, we isolated a wrky gene from g. hirsutum, which is an important economic crop cultivated worldwide. due to the high similarity between the sequence of the protein encoded by the wrky gene obtained from cotton and those of several wrkys obtained from n. tabacum, a. thaliana, solanum tuberosum and populus tomentosa, we verified that the gene we isolated from cotton is a wrky transcription factor gene. the phylogenetic tree and the short conserved harf structural motif further indicated that the ghwrky gene belongs to subgroup iid, with atwrky atwrky and atwrky as its closest homologues in a. thaliana; as a result, we designated the isolated gene ghwrky consistent with the putative role of wrky proteins as transcription factors, a nuclear targeting sequence was identified in the c-terminal region of the protein, and the targeting experiment suggested that gfp-tagged ghwrky was located in nucleus. these results suggest that, similar to ghwrky, ghwrky might function in the nucleus. to investigate the effects of constitutive overexpression of ghwrky on the response to pathogen resistance, ghwrky overexpressing-tobacco plants were inoculated with two types of viruses and two pathogenic fungi. the plants constitutively overexpressing ghwrky were more resistant to tmv and cmv infection compared with the wt tobacco plants. this observation diverges from previous research demonstrating that the virus-induced gene silencing of nbwrky nbwrky or nbwrky suppressed resistance to tmv and that the size of the hypersensitive response lesions was also reduced in nbwrky1-silenced plants. the ghwrky transgenic plants also exhibited increased resistance to fungal pathogens compared with wild-type plants. previously, overexpression studies examining a pair of allelic genes oswrky45- and oswrky45- demonstrated that these genes positively regulated resistance to the rice fungal pathogen magnaporthe grisea. our data presented above indicate that ghwrky overexpression increases the tolerance of the plant to pathogens; therefore, the upregulation of ghwrky in cotton seedlings in response to pathogens, including r. solani, c. gossypii and f. oxysporum, suggests that ghwrky may participate in the regulation of plant responses to disease defence. the increased expression of pr pr and pr contributed to the increased resistance of transgenic ghwrky15-overexpressing plants to tmv and c. gossypii. furthermore, previous studies have demonstrated that wrky dna binding-proteins can regulate the transcription of a number of defence-related genes, including the well-studied pr genes, via binding to the w-box in the promoter of these genes. it is noted that the expression of the pr gene in transgenic plants during c. gossypii infection was down-regulated compared with the infected wt plants, suggesting that the molecular mechanism underlying the activity of ghwrky is fairly complex under different biotic stresses or that ghwrky may be an integrator of several signalling pathways. sa and ja are two well-studied key mediators of plant resistance. in arabidopsis, more than wrky genes can be differentially regulated after infection with p. syringae or treatment with sa. infection of ghwrky transgenic plants with tmv and c. gossypii increased the expression of npr which is a key regulator of the sa-dependent defence pathway and sar. furthermore, the analysis of the expression of ghwrky in cotton seedlings showed that ghwrky expression can be affected by sa and meja, supporting our hypothesis that ghwrky plays a role in resistance to viral and fungal pathogens via sa- and ja-dependent defence pathways. interestingly, reports of wrky proteins that function in both sa- and ja-dependent pathways are relatively limited. moreover, the expression of two key enzymes that are involved in et biosynthesis is increased in ghwrky transgenic plants following infection with fungi and viruses. and there was significant increase of ghwrky expression in cotton seedlings following et treatment. this suggests that the increased disease resistance of ghwrky overexpressing tobacco compared to the wide type may partially depend on the expression of et synthesis related genes. additionally, ghwrky expression could be increased under various abiotic stress conditions, including low temperature, wounding and drought, and a group of cis-elements related to biotic and abiotic stresses were identified in the ghwrky promoter. altogether, these data support our hypothesis that the mechanism of response to various environmental stresses, especially viral and fungal infection, in ghwrky overexpressing plants may be related with multiple signalling pathways, including sa, ja or et signalling pathways. mounting evidence suggests that the ros network is essential for the induction of disease resistance. for instance, mitogen-activated protein kinase cascades members ghmapk and ghmapk are both involved in the response to infection and oxidative stress. following challenge with a virus or a fungus, a lower accumulation of h2o and a higher activity of antioxidant enzymes, including pod and apx, were detected in the ghwrky15-overexpressing plants relative to the wt plants. we reasoned that ghwrky upregulates the activity of pod and apx to increase the oxidative stress tolerance and contributes to the resistance towards biotic stresses in transgenic plants. however, except for ntgpx, the expression of the other five antioxidant enzymes examined was reduced in unstressed transgenic seedlings compared with the wild-type plants. these results contrast previous research examining some genes, which appear to play positive roles in the stress response and enhance the expression of most antioxidant enzymes. to explain this difference, the expression of the six antioxidant enzymes was examined during treatment with mv, which is a potential redox mediator. although there was no significant difference in the expression of ntsod and ntapx in the transgenic plants compared with the wt plants following the treatment with mv, the expression of these genes was increased compared with the expression in plants receiving no treatment. additionally, the upregulation of ntgpx and ntapx expression during mv treatment suggests that ghwrky overexpression can help plants maintain ros homoeostasis under oxidative stresses to alleviate injury to the organism. most recently, it has been reported that there was a substantial increase in the musawrky level when banana plantlets were subjected to h2o moreover, a putative thioredoxin-dependent peroxidase gene belonging to the ros detoxification system was induced in musawrky71-overexpressing cultures. furthermore, in arabidopsis, the maximum expression of wrky was detected in seven-week-old plants overexpressing wrky at which point the h2o level increased significantly. in wild-type cotton seedlings, a substantial increase in the level of ghwrky was also observed following exposure to mv. intriguingly, some of the growth and development phenomena observed in plants overexpressing ghwrky are similar to those observed in atwrky over-expressing plants. similar to ghwrky15-overexpressing plants, atwrky53-overexpressing plants demonstrated early flowering after sowing, and the senescence of the whole plant with partly necrotic leaves was visible earlier when compared with wild-type plants. interestingly, overexpression of ghwrky in n. tabacum cv. nc promoted stem elongation and inhibited the transversal growth of stems. atwrky controls cell fate in thickened pith cells by acting as a negative regulator of nst and c3h zinc finger tfs, which, in turn, regulate secondary cell wall synthesis. although no difference in the diameter and length of pith cells was observed in our transgenic plants, overexpression of ghwrky may affect the fate of cells in the cortex, which had a smaller diameter relative to wt plants. the phytohormone ga is an essential regulator of stem elongation and other developmental processes throughout the life cycle of a plant. the induction of ghwrky expression during ga treatment combined with the higher expression of ghwrky in the roots and stems compared with the cotyledon of cotton may clarify the underlying mechanism of the influence of ghwrky overexpression on plant growth and development.  in conclusion, we identified and characterised a group iid wrky member in cotton. our results strongly suggest that overexpression of ghwrky confers tobacco resistance to both viral and fungal pathogens, and indicate that overexpression of ghwrky regulates the stem growth or development in tobacco. the elucidation of the regulatory mechanism of overexpression of ghwrky in tobacco may provide a converging node of regulatory pathways in response to pathogens and oxidative stress and in the regulation of plant development. although the influence of overexpression of ghwrky in tobacco was explored, the mechanism underlying the function of ghwrky should be elucidated in transgenic cotton and loss-of function data in cotton are particularly needed. moreover, other research focusing ghwrky is still needed, for example, the study of its interactions with other wrky proteins or mapk cascades members. 
additionally, exposure to abiotic stresses, including cold, wounding and drought, resulted in the accumulation of ghwrky transcripts. as a large family of regulatory proteins, wrky transcription factors play essential roles in the processes of adaptation to diverse environmental stresses and plant growth and development. although several studies have investigated the role of wrky transcription factors during these processes, the mechanisms underlying the function of wrky members need to be further explored, and research focusing on the wrky family in cotton crops is extremely limited. consistently, northern blot analysis showed that ghwrky expression was significantly induced in cotton seedlings following fungal infection or treatment with salicylic acid, methyl jasmonate or methyl viologen. additionally, a group of cis-acting elements associated with the response to environmental stress and plant growth and development were detected in the promoter. it is intriguing that ghwrky overexpression in tobacco affects plant growth and development, especially stem elongation. this finding suggests that the role of the wrky proteins in disease resistance may be closely related to their function in regulating plant growth and development. in the present study, a gene encoding a putative wrky family member, ghwrky was isolated from cotton. importantly, increased activity of the antioxidant enzymes pod and apx during infection and enhanced expression of ntapx and ntgpx in transgenic tobacco following methyl viologen treatment were observed. furthermore, ghwrky15-overexpressing tobacco exhibited more resistance to viral and fungal infections compared with wild-type tobacco. overall, our data suggest that overexpression of ghwrky may contribute to the alteration of defence resistance to both viral and fungal infections, probably through regulating the ros system via multiple signalling pathways in tobacco. moreover, ghwrky transcription was greater in the roots and stems compared with the expression in the cotyledon of cotton, and the stems of transgenic plants displayed faster elongation at the earlier shooting stages compared with wide type tobacco.
 as a large family of regulatory proteins, wrky transcription factors play essential roles in the processes of adaptation to diverse environmental stresses and plant growth and development. although several studies have investigated the role of wrky transcription factors during these processes, the mechanisms underlying the function of wrky members need to be further explored, and research focusing on the wrky family in cotton crops is extremely limited.  in the present study, a gene encoding a putative wrky family member, ghwrky was isolated from cotton. ghwrky is present as a single copy gene, and a transient expression analysis indicated that ghwrky was localised to the nucleus. additionally, a group of cis-acting elements associated with the response to environmental stress and plant growth and development were detected in the promoter. consistently, northern blot analysis showed that ghwrky expression was significantly induced in cotton seedlings following fungal infection or treatment with salicylic acid, methyl jasmonate or methyl viologen. furthermore, ghwrky15-overexpressing tobacco exhibited more resistance to viral and fungal infections compared with wild-type tobacco. the ghwrky15-overexpressing tobacco also exhibited increased rna expression of several pathogen-related genes, nonexpressor of pr and two genes that encode enzymes involved in et biosynthesis. importantly, increased activity of the antioxidant enzymes pod and apx during infection and enhanced expression of ntapx and ntgpx in transgenic tobacco following methyl viologen treatment were observed. moreover, ghwrky transcription was greater in the roots and stems compared with the expression in the cotyledon of cotton, and the stems of transgenic plants displayed faster elongation at the earlier shooting stages compared with wide type tobacco. additionally, exposure to abiotic stresses, including cold, wounding and drought, resulted in the accumulation of ghwrky transcripts.  overall, our data suggest that overexpression of ghwrky may contribute to the alteration of defence resistance to both viral and fungal infections, probably through regulating the ros system via multiple signalling pathways in tobacco. it is intriguing that ghwrky overexpression in tobacco affects plant growth and development, especially stem elongation. this finding suggests that the role of the wrky proteins in disease resistance may be closely related to their function in regulating plant growth and development. ghwrky15cottondisease resistancesarosplant development 