BACKGROUND
in plants, wrky proteins constitute a large family of transcription factors.
since their first identification in sweet potato and wild oats  <cit>  the wrky proteins have been described as transcriptional regulators in multiple plant species and in various processes such as pathogen defense  <cit> , trichome  <cit>  and seed development  <cit> , as well as leaf senescence  <cit> .
the common feature of wrky proteins is the existence of either one or two wrky domains.
the wrky domain is an approximately  <dig> amino acids long spanning dna binding domain, which contains a highly conserved amino acid motif, wrkygqk, at its n-terminus and a metal chelating zinc finger signature at the c-terminus.
the crystal structure of one wrky domain has been resolved recently  <cit>  and provides an insight into how the conserved residues of the wrky domain facilitate the binding to their cognate dna-element, the w-box.
while most of the functional studies of wrky proteins indicate a binding to the conserved nucleotide consensus sequence tgac of the w-box, the binding to another cis-element called sure has also been described  <cit> .
based on the number of wrky domains present and on the type of zinc finger wrky proteins have been classified into groups  <dig>  2a to 2e and  <dig>  <cit> .
wrky transcription factors seem to have their evolutionary origin in ancient eukaryotes with the most basal wrky genes identified in the unicellular eukaryote giardia lamblia and the slime mold dictyostelium discoideum  <cit> .
while the wrky-gcm <dig> superfamily has been found in all organism kingdoms, the conserved wrky-type signatures of these genes have not yet been detected in the lineage of true fungi and animals  <cit> .
in contrast, phylogenetic analysis revealed that the wrky transcription factor family has expanded to great extent in higher plants  <cit> .
for example, the wrky gene family consists of  <dig> members in arabidopsis  <cit>  and at least  <dig> members in rice  <cit> .
the cereal crop barley is of high importance for food, feed and brewing.
after maize, rice and wheat, barley ranked as number four in cereal production in  <dig>  <cit> .
due to its wide ecological potential and adaptability, it is cultivated worldwide and under diverse environmental conditions.
for example, barley is grown on soils and in altitudes that are unsuitable for wheat and oats  <cit> .
the genome of barley is estimated to be of approximately  <dig> mb in size and has not been fully sequenced yet.
however, the genbank est dataset for barley contains almost  <dig>  ests and provides a valuable resource for bioinformatics analyses  <cit> .
in addition, the affymetrix  <dig> k barley <dig> genechip microarray, representing probe sets for roughly  <dig>  genes  <cit>  was released in  <dig>  nowadays, a number of barley microarray experiments are accessible via different public sources such as barleybase  <cit>  or geo  <cit>  and allow for in silico expression analyses of barley genes.
several studies have shown an interest in barley wrky proteins  and their function.
hvwrky <dig>  also named hvwrky <dig>  has been reported to be involved in several regulatory processes, such as the response to cold and drought  <cit>  and the repression of the alpha-amylase gene amy32b during seed germination  <cit> .
recently, the same wrky protein and its paralog hvwrky <dig> have been described as repressors of the pamp-triggered basal defense upon infection with blumeria graminis  <cit> .
apart from hvwrky proteins functioning in response to biotic and abiotic stresses, the group  <dig> wrky protein susiba <dig> has been shown to mediate sucrose signaling in barley cells during regulation of starch synthesis  <cit> .
in this study we identified  <dig> members of the wrky gene family in barley based on publicly available sequence information.
we describe their phylogenetic relationship and try to assign putative orthologs from barley, arabidopsis and rice.
expression analysis using microarray data suggests the involvement of some of the hvwrky proteins in response to powdery mildew and in plant development.
moreover, correlated expression of orthologous barley and arabidopsis wrky genes imply that their function is retained in monocot and dicot species.
RESULTS
identification of  <dig> wrky protein coding sequences in barley
to estimate the minimum number of wrky genes in barley, publicly available sequence data was searched with the blast program.
the protein sequence of wrky domains from representative members for all subgroups from arabidopsis  were used as input.
the primary search resulted in  <dig> non-redundant hits, of which  <dig> were removed as they did not contain the conserved wrky domain signature.
the remaining  <dig> sequences were screened for partial overlaps by pairwise comparison; this analysis identified  <dig> wrky open reading frames  unique for barley.
a keyword search on ncbi returned eight previously annotated hvwrky protein sequences.
as indicated by corresponding est sequences, all eight orfs were expressed.
in order to create a uniform nomenclature with consecutive numbering, we renamed sequences annotated as putative wrky <dig>   <dig>   <dig>   <dig> and  <dig> to hvwrky <dig>   <dig>   <dig>   <dig> and  <dig>  respectively.
sequences of hvwrky <dig> and  <dig> could be extended based on available est information.
the sequence of putative wrky <dig> protein, cad <dig>  is identical with the sequence aas <dig> which has been published as hvwrky- <dig>  <cit> .
we designated both sequences as hvwrky <dig> and did not annotate any sequence as hvwrky <dig> to prevent confusion.
susiba <dig> received the formal name hvwrky <dig>  the remaining sequences were annotated accordingly as hvwrky <dig> to hvwrky <dig> and hvwrky <dig> to hvwrky <dig> .
seven of the hvwrky sequences included start and stop codons and, as judged by protein sequence alignments with wrky proteins from rice , constitute complete coding sequences.
the remaining  <dig> hvwrky sequences represented partial coding sequences.
to confirm that we were identifying clade members correctly, we extended sequence information by pcr hvwrky <dig> and hvwrky <dig>  a putative group  <dig> wrky and group 2d wrky, respectively.
this approach revealed a second wrky domain in the sequence of hvwrky <dig>  confirming that hvwrky <dig> is indeed a class  <dig> member.
in case of hvwrky <dig>  a characteristic domain with the highly conserved amino acid sequence harf  <cit>  could be detected at the beginning of the coding sequence confirming that it too, was correctly assigned by our analysis.
* ortholog could not be assigned with significance
while eulgem et al.
<cit>  defined wrky proteins by the conserved heptapeptide wrkygqk, slight variations of this sequence have been described for a number of rice wrky proteins  <cit> .
similarly, a number of hvwrky sequences have amino acid sequence substitutions in the conserved wrky signature.
in hvwrky <dig>   <dig> and  <dig> the wrky domain displays the sequence wrkygkk, in hvwrky <dig> and  <dig> wrkygek, in hvwrky <dig> wrkygqn and in the c-terminal domain of hvwrky <dig> wskygqm .
moreover, a unique zinc-finger motif  was described as a second characteristic feature of wrky proteins <cit> .
in group  <dig> wrky proteins, this motif is slightly altered to c-x7-c-x23-h-x-c. in barley, three of the so far characterized group  <dig> wrky proteins, namely hvwrky <dig> and  <dig>  showed an extension of the zinc finger motif  compared to the patterns described by eulgem et al.
<cit>  .
again, similar modifications of the zinc-finger patterns were detected for rice wrky proteins and have been discussed to be a monocot-specific feature  <cit> .
phylogenetic analysis of barley wrky proteins
while the phylogenetic analysis showed that wrky proteins are of monophyletic nature  <cit> , the assignment to defined groups is polyphyletic and either based on the number of wrky domains or on the type of zinc finger motif.
to study the phylogenetic relationship of the  <dig> hvwrky proteins we performed a multiple sequence alignment of the about  <dig> amino acids spanning the wrky domain of all hvwrkys.
to obtain a better separation of the different groups and subgroups, wrky domains from members of different arabidopsis and rice wrky proteins were included in the analysis.
for each of the groups  <dig>  2a to 2e and  <dig> one representative was chosen randomly.
these were: atwrky <dig>  - <dig>  - <dig>  - <dig>  - <dig>  - <dig>  - <dig> for arabidopsis and oswrky <dig>  - <dig>  - <dig>  - <dig>  - <dig>  - <dig>  - <dig> for rice .
both c-terminal and n-terminal wrky domains of group  <dig> wrky proteins have been treated as independent sequences, so that a total number of  <dig> barley,  <dig> arabidopsis and  <dig> rice wrky domains were analyzed.
as shown in figure  <dig> for all subgroups of wrky proteins, we could identify at least one representative in the barley genome.
for example, five hvwrky sequences code for proteins with two wrky domains and clearly group with the group  <dig> members atwrky <dig> and oswrky <dig>  the single wrky domain identified for hvwrky <dig> clusters within the c-terminal group  <dig> domains, indicating that a second n-terminal domain remains likely to be identified.
interestingly, the  <dig> identified group  <dig> wrky members of barley are distributed very unevenly among the five subgroups.
whereas  <dig> hvwrkys form a distinct subclade with the characteristic members of subgroup 2c, only a single barley wrky, hvwrky <dig>  belongs to group 2b.
group  <dig> is represented by  <dig> single-wrky domain barley proteins yet, both wrky domains of hvwrky <dig> cluster with these proteins.
again, this phenomenon has been observed for at least four rice wrky proteins: oswrky <dig>  - <dig>  - <dig> and - <dig>  <cit> .
the total number of barley wrky genes will remain unknown until the sequencing of the barley genome is completed.
however, we constructed an alignment and a phylogenetic tree of  <dig> arabidopsis,  <dig> barley and  <dig> rice wrky proteins to estimate this number .
as described earlier by zhang and wang  <cit> , group  <dig> and  <dig> domains were closer related to the c-terminal domains of group  <dig> whereas the n-terminal wrky domains of group  <dig> members form a separate monophyletic cluster.
on the other hand, separate subclades of the wrky domains of subgroup 2c underline the paraphyletic nature of these subgroups .
the grouping of hvwrky <dig>  hvwrky <dig> and oswrky <dig> based solely on the phylogenetic analysis of their wrky domain sequences can not be performed unambiguously.
however, the presence of two wrky domains in hvwrky <dig> and oswrky <dig> is indicative for group  <dig> members.
both c-terminal and n-terminal wrky domains of hvwrky <dig> and oswrky <dig> support basal monophyletic subclades of group  <dig> wrky proteins , which might be specific for grasses  <cit> .
the number of wrky proteins within each species assigned to the different paraphyletic subgroups  <dig> to  <dig> was calculated and is summarized in table  <dig>  a simple comparison of the total number of arabidopsis and rice wrky genes with the number of hvwrkys indicates that approximately 50% of the barley wrky genes have been identified.
also, the distribution of wrky proteins showed that hvwrkys are underestimated in several subgroups.
nevertheless, similar numbers of barley and rice wrky proteins in subgroups 2a and 2d may indicate that all hvwrky proteins of this subgroup have been identified.
* according to eulgem et al.
<cit>
**according to xie et al.
<cit>
monocot-specific radiation and identification of orthologous wrky genes
based on the wrky domains of all proteins from barley, arabidopsis and rice, we calculated a comprehensive phylogenetic tree to gain a closer insight into the evolutionary relationships between wrky proteins  and assign putative wrky orthologs between these sequences.
additionally, nine wrky domains of the moss physcomitrella patens, two each from giardia lamblia and dictyostelium discoideum, as well as additional zinc finger domains of other wrky-gcm1-like proteins were included as outgroups.
clades exist for almost all wrky subgroups which consist solely of rice wrky domains or a combination of rice and arabidopsis wrky domains; these clades might be comprised of orthologs of yet unidentified barley wrky proteins.
in contrast, rice and barley wrky domains form a number of monocot-specific clades inside group 2c and group  <dig>  these clades reveal clusters of orthologous wrky proteins for rice and barley.
for example in subgroup 2c, atwrky <dig> forms a basal accession to five barley and six rice wrky domains.
similarly, in a subgroup of group  <dig>  ten barley and nine rice wrky proteins constitute putative monocotyledonous orthologs that diverge from a group of three paralogous arabidopsis wrky proteins .
thus, the barley and rice wrky proteins that form paralogous or orthologous groups would indicate monocot-specific diversification of wrky genes.
this assumption was further supported by blast searches in dbest  <cit> , which did not return orthologous wrky domains of any other dicotyledonous species for the monocot-specific subclades .
thus, our results support the idea of radiation events in groups 2c and  <dig> wrky proteins that are specific for monocot species.
this diversification has most likely occurred after the ancestral divergence of dicotyledonous and monocotyledonous plants.
the assignment of orthologs is crucial for transferring the knowledge of wrky protein functions from the model plants rice and arabidopsis to barley.
therefore, based on the presented phylogenetic tree , we assigned orthologs for most of the wrky genes described .
in a few cases orthologs could not be defined unambiguously.
however, as gene orthology implies similar gene function, the assignment presented provided a solid base for functional analyses.
powdery mildew infection affects expression of hvwrky genes
the analysis of temporal and spatial expression patterns is an initial step towards the functional characterization of genes.
the expression of numerous wrky genes from various plant species is affected by biotic and abiotic stresses  and implies the regulatory role of wrky proteins in stress responses.
in order to investigate if this is also true for hvwrky genes, we mined publicly available microarray data of barley leaves infected with powdery mildew .
a total of  <dig> probesets on the barley <dig> genechip could be assigned to  <dig> different hvwrky genes .
datasets of two experiments named bb <dig> and bb <dig> were retrieved from barleybase  <cit>  and analyzed.
figure  <dig> shows the expression profiles of  <dig> hvwrky genes are displayed for two genotypes  under infection and non-infection conditions  as well as during a time course experiment using the host genotype mla <dig> and the bgh isolate  <dig> .
even though the setup of the bb <dig> and bb <dig> experiments differ, some general trends can be seen in both experiments.
roughly 50% of all hvwrky genes show altered expression levels upon infection with blumeria graminis.
hvwrky <dig> and hvwrky <dig> have been previously shown to be involved in the regulation of defense mechanisms  <cit> .
hvwrky <dig> expression appears to be drastically increased under one experimental condition  and lower in the second , while hvwrky <dig> shows increased expression levels in both experiments.
in addition to these two wrky genes, a number of other hvwrky genes mediating pathogen responses could be assigned based on their expression profiles.
for example, the gene for hvwrky <dig>  which is closely related to the well-studied pathogen response regulators atwrky <dig>  - <dig> and - <dig>  shows an increase in expression after  <dig> h of pathogen treatment.
also, putative pathogen response regulators could be found in subgroups 2c and  <dig>  interestingly, these are wrky groups that have undergone monocot-specific radiation events, which might thus have been related to an increased biotic stress during evolution.
in addition to upregulated genes, there are hvwrky genes with decreased transcript levels, such as most of the hvwrky genes in group  <dig>  however, expression of several hvwrky genes was not affected upon powdery mildew infection and, therefore, might function in regulatory processes other than pathogen response such as abiotic stress or development.
orthologous barley and arabidopsis wrky genes exhibit correlative expression signatures during plant development
a number of wrky proteins have been reported to be involved in plant developmental processes  <cit> .
to investigate if hvwrky transcript levels vary during plant development, we used public microarray data of different developmental stages from barley  <cit>  and investigated whether phylogenetically related wrky genes of barley and arabidopsis exhibited similarities in their expression patterns.
as not all wrky genes of both barley and arabidopsis are represented on their respective microarrays, we were restricted to the expression patterns of  <dig> hvwrky genes and their putative arabidopsis orthologs.
we extracted expression data from the total microarray dataset for ten organs in barley at different developmental stages:coleoptile, mesocotyl, radicule, leaf, root, inflorescence, anthers, and caryopsis  <dig>   <dig> and  <dig> days post anthesis   <cit> .
the expression profiles reveal spatial and temporal variations in expression of hvwrky genes in different barley organs .
some genes, like hvwrky <dig>  - <dig> and - <dig>  have similar expression levels in all investigated tissues.
in contrast, genes such as hvwrky <dig>  - <dig>  - <dig>  - <dig> and - <dig> reveal more than 3-fold difference in expression levels in different organs.
furthermore, the transcript levels of barley wrky genes in the same organ show temporal variations.
for example, hvwrky <dig> is expressed in young caryopses at  <dig> d.p.a.
but is clearly reduced in later stages of caryopsis development .
similarly, the expression levels of hvwrky <dig> are low in radicules but significantly higher in older roots.
in addition, paralogous barley wrky genes such as hvwrky <dig> and - <dig> reveal similar expression patterns, implying redundant function.
next, we performed a comparative expression analysis for both barley and arabidopsis wrky genes on the extracted subsets of the microarray expression datasets of homologous organs by druka et al.
<cit>  and schmid et al.
<cit> .
the pearson correlation of the normalized signal intensities was calculated for the potentially orthologous wrky genes.
the resulting correlation coefficients ranged from - <dig>  to + <dig>  .
the average correlation of all the putative orthologous wrky genes was + <dig>  and differed significantly  from the average expression correlation of a control dataset composed of  <dig> randomly chosen gene pairs .
selection of only the best correlated pairs of hvwrky and atwrky orthologs even increased the average correlation coefficient to + <dig> .
this high correlation can be easily seen by the overall expression pattern of the compared wrky genes .
for example, the expression levels of the orthologous pair in group  <dig>  atwrky <dig> and hvwrky <dig>  hardly differ in any of the ten organs and are clearly correlated.
in contrast, the expression profiles of hvwrky <dig> and atwrky <dig> are observably anti-correlated.
genes for the group 2e wrky protein hvwrky <dig> and their closely related arabidopsis orthologs atwrky <dig> and - <dig> show a consistent increase in mrna level in radicules and roots with low expression levels in leaves.
according to our phylogenetic analysis, hvwrky <dig> has two potential orthologs in arabidopsis, namely atwrky <dig> and atwrky <dig>  the hvwrky <dig> expression profile resembles that of atwrky <dig>  in vegetative tissues but is more similar to the profile of atwrky <dig>  in reproductive organs such as the inflorescence and the anthers.
this might indicate that the arabidopsis wrky genes have undergone functional changes after a duplication event.
* for each hvwrky gene, the atwrky gene with the highest correlation value was chosen to calculate the average correlation of best pairs.
** correlation of  <dig> random genes of barley and arabidopsis.
probesets on the barley <dig> genechip and ath <dig> genechip were randomly selected and the average of the pearson correlation of their expression profiles was calculated.
next, we performed rt-pcr experiments on cdna from three different organs  and to extend our studies we also included samples from rice, oryza sativa japonica, for the analysis.
the signal intensities of the pcr products were quantified and normalized to the actin controls to calculate normalized fold intensities for each organ and organism .
first of all, the expression trajectories of all tested barley and arabidopsis genes generally reproduced those observed in the microarrays experiments .
transcripts of the three orthologs hvwrky <dig>  oswrky <dig> and atwrky <dig> were the most abundant and expressed to the same amount in all organs.
although hvwrky <dig> and oswrky <dig> constitute close relatives, their normalized intensities differ especially in leaves where no hvwrky <dig> transcript could be detected .
intriguingly, the expression of the closely related group 2c wrkys hvwrky <dig>  oswrky <dig>  atwrky <dig> and atwrky <dig> was highly correlated and strongly increased in seed stages compared to leaves .
from this we can conclude that these four wrky genes are of high general importance in all of the three plants for seed development as the comparative organs, siliques and caryopses or infructescences, differ greatly in their structure and composition.
discussion
in this study we present the annotation of  <dig> members of the wrky gene family in barley and classify them according to the wrky groups  <dig> to  <dig> .
comparison to the total number of wrkys in arabidopsis and rice  allowed us to identify about 50% of all barley wrky genes.
this is in accordance with a study by ryu and colleagues  <cit>  who found est sequences for 44% of all oswrky genes.
in order to transfer the knowledge of wrky function in arabidopsis and rice to barley, we created a phylogenetic tree including the wrky protein sequences of all three species.
for example, atwrky <dig>  has been shown to bind to calmodulin in a ca2+- dependent manner by a calmodulin-binding domain , which has been found in all other group 2d wrky proteins of arabidopsis  <cit> .
following our phyogenetic analysis, hvwrky <dig> is the ortholog of atwrky <dig>  and might therefore have similar calmodulin-binding capacity.
even though the current sequence of hvwrky <dig> does not span the cambd, we could identify such a cambd for hvwrky <dig> and hvwrky <dig> which are members of the same subclade.
also, a sequence alignment of all group 2d oswrky proteins revealed the cambd in four of six oswrky proteins , arguing for a conserved function of this domain in combination with the wrky binding domain in angiosperms.
interestingly, although most identified wrky clades have at least one member involved in pathogen stress, none of the group 2d hvwrky genes displayed significant changes in expression levels upon powdery mildew infection .
this underlines the uniformity of group 2d hvwrky proteins, and also leads to the speculation whether these hvwrky proteins are impaired in the regulation of pathogen response in a calmodulin-binding dependent manner.
for rice and arabidopsis members of wrky group 2a an involvement in the response to fungal pathogens has been demonstrated  <cit> .
recently, hvwrky <dig> and hvwrky <dig> have been shown to play an important role in the regulation of the response to the fungal pathogen blumeria graminis  <cit> .
our alignment tree indicates the existence of two further group 2a wrky proteins in barley, hvwrky <dig> and hvwrky <dig>  which therefore are also potential regulators of pathogen response.
in fact, the expression levels of all group 2a hvwrky genes alter upon powdery mildew infection .
interestingly, three out of six rice members of the monocot-specific subclade of group 2c having atwrky <dig> as a basal member show increased expression levels upon infection with the fungus magnaporthe grisea  <cit> .
the same is true for at least two other 2c barley members, namely hvwrky <dig> and hvwrky <dig>  which show changes in expression levels in blumeria graminis challenged leaves .
this could indicate that the diversification of this particular subclade of wrky transcription factors might have occurred in response to pathogen stress.
as all identified pathogen-responsive oswrkys  so far have corresponding orthologs in barley, these proteins are also potential regulators of pathogen response.
xie and co-workers  <cit>  showed synergistic interactions of oswrky <dig>  and oswrky <dig>  in regulation of amy32b gene expression in rice aleurone cells and parts of their work was used for a comparative analysis in barley, where hvwrky <dig>  was shown to be involved in regulating amy32b expression  <cit> .
in fact, based on our alignment, oswrky <dig> and hvwrky <dig> were shown to be putative orthologs.
however, an interaction partner of hvwrky <dig> with functional similarities to oswrky <dig> has not been described so far  <cit> , although one may postulate that the ortholog to oswrky <dig>  hvwrky <dig>  constitutes a good candidate as an interaction partner to hvwrky <dig>  in summary, the sequence comparison of wrky proteins of different species aids in assigning functions to different wrky proteins and allows for target-oriented hypothesis testing.
microarray-based studies have become a highly valuable source for functional genomics  <cit>  and huge publicly available datasets allow scientists to monitor the expression of their genes of interest.
in this study, we performed a cross-species expression analysis using the microarray data of developmental studies in barley and arabidopsis  <cit> .
we considered similarities in expression profiles as an indicator for conserved functional similarities of orthologous genes.
our study was limited by the fact that only  <dig> of the hvwrky genes and  <dig> of the atwrky genes are represented by probesets on the respective microarrays.
also, there was an obvious risk of not comparing true orthologs as only about 50% of all hvwrky genes could be identified .
although cultivation of barley and arabidopsis plants differed, the homologous organs we considered are known to harbor plant lineage-specific functions ; we observed an average positive pearson correlation of + <dig>  in the gene expression of putative wrky orthologs between these two species .
this is consistent with the results of genome-wide expression comparisons of five developmental stages of arabidopsis and rice, which returned correlation coefficients in the same range  <cit> .
similarly, a comparative study of the expression of members of the basic helix-loop-helix transcription factors in arabidopsis and rice indicates conserved expression patterns of orthologs in four corresponding plant organs  <cit> , further indicating that protein function is likely conserved between homologous organs.
when only considering the best pairing wrky orthologs, the average pearson correlation for gene expression increased to + <dig> .
this is mostly due to a reduction in the number of putatively orthologous gene-pairs exhibiting no correlation .
several reasons can be suggested which might explain why some of these putative orthologs of barley and arabidopsis show random or negatively correlated expression profiles.
first, the function of the orthologous wrky genes might have changed during evolution, mirrored by changes in expression patterns.
moreover, the expression of these wrky genes might not be related to any developmental processes and instead are only activated upon abiotic and biotic stresses.
since the limitation of identifying orthologs between barley and arabidopsis was based on the available barley est data, it is possible that a better hvwrky ortholog may exist for each atwrky in our current assignment.
it is possible, therefore, that poor correlations observed in this study could be improved once more est or genomic sequence is available.
nevertheless, the current phylogenetic assignments cover and establish a solid orthology for future studies.
in this study we have shown the potential value of comparative expression analysis between sequenced model species and non-sequenced species of high economic impact.
nowadays, microarrays for several non-sequenced crop species are commercially available.
thus, the reported combination of phylogenetic and expressional analysis may offer a valuable tool for future studies and may contribute to the understanding of gene functions in these crops.
CONCLUSIONS
the basis for deriving orthology between related proteins of different plant species is their degree in similarity.
here, we could provide that this orthology is not restricted to sequence similarities alone but reaches out to orthology in expression patterns.
we speculate that this observation is not restricted to the wrky proteins based on observations made in studies of other gene families.
nonetheless, the combination of phylogenetic characterization of hvwrky genes and the subsequent comparative expression analysis of orthologous genes in arabidopsis provides a solid basis for further comparative functional genomics studies of this gene family in the cereal crop barley.

 in plants, wrky proteins constitute a large family of transcription factors. since their first identification in sweet potato and wild oats the wrky proteins have been described as transcriptional regulators in multiple plant species and in various processes such as pathogen defense, trichome and seed development, as well as leaf senescence. the common feature of wrky proteins is the existence of either one or two wrky domains. the wrky domain is an approximately amino acids long spanning dna binding domain, which contains a highly conserved amino acid motif, wrkygqk, at its n-terminus and a metal chelating zinc finger signature at the c-terminus. the crystal structure of one wrky domain has been resolved recently and provides an insight into how the conserved residues of the wrky domain facilitate the binding to their cognate dna-element, the w-box. while most of the functional studies of wrky proteins indicate a binding to the conserved nucleotide consensus sequence tgac of the w-box, the binding to another cis-element called sure has also been described. based on the number of wrky domains present and on the type of zinc finger wrky proteins have been classified into groups 2a to 2e and. wrky transcription factors seem to have their evolutionary origin in ancient eukaryotes with the most basal wrky genes identified in the unicellular eukaryote giardia lamblia and the slime mold dictyostelium discoideum. while the wrky-gcm superfamily has been found in all organism kingdoms, the conserved wrky-type signatures of these genes have not yet been detected in the lineage of true fungi and animals. in contrast, phylogenetic analysis revealed that the wrky transcription factor family has expanded to great extent in higher plants. for example, the wrky gene family consists of members in arabidopsis and at least members in rice. the cereal crop barley is of high importance for food, feed and brewing. after maize, rice and wheat, barley ranked as number four in cereal production in. due to its wide ecological potential and adaptability, it is cultivated worldwide and under diverse environmental conditions. for example, barley is grown on soils and in altitudes that are unsuitable for wheat and oats. the genome of barley is estimated to be of approximately mb in size and has not been fully sequenced yet. however, the genbank est dataset for barley contains almost ests and provides a valuable resource for bioinformatics analyses. in addition, the affymetrix k barley genechip microarray, representing probe sets for roughly genes was released in nowadays, a number of barley microarray experiments are accessible via different public sources such as barleybase or geo and allow for in silico expression analyses of barley genes. several studies have shown an interest in barley wrky proteins and their function. hvwrky also named hvwrky has been reported to be involved in several regulatory processes, such as the response to cold and drought and the repression of the alpha-amylase gene amy32b during seed germination. recently, the same wrky protein and its paralog hvwrky have been described as repressors of the pamp-triggered basal defense upon infection with blumeria graminis. apart from hvwrky proteins functioning in response to biotic and abiotic stresses, the group wrky protein susiba has been shown to mediate sucrose signaling in barley cells during regulation of starch synthesis. in this study we identified members of the wrky gene family in barley based on publicly available sequence information. we describe their phylogenetic relationship and try to assign putative orthologs from barley, arabidopsis and rice. expression analysis using microarray data suggests the involvement of some of the hvwrky proteins in response to powdery mildew and in plant development. moreover, correlated expression of orthologous barley and arabidopsis wrky genes imply that their function is retained in monocot and dicot species.  identification of wrky protein coding sequences in barley to estimate the minimum number of wrky genes in barley, publicly available sequence data was searched with the blast program. the protein sequence of wrky domains from representative members for all subgroups from arabidopsis were used as input. the primary search resulted in non-redundant hits, of which were removed as they did not contain the conserved wrky domain signature. the remaining sequences were screened for partial overlaps by pairwise comparison; this analysis identified wrky open reading frames unique for barley. a keyword search on ncbi returned eight previously annotated hvwrky protein sequences. as indicated by corresponding est sequences, all eight orfs were expressed. in order to create a uniform nomenclature with consecutive numbering, we renamed sequences annotated as putative wrky and to hvwrky and respectively. sequences of hvwrky and could be extended based on available est information. the sequence of putative wrky protein, cad is identical with the sequence aas which has been published as hvwrky-. we designated both sequences as hvwrky and did not annotate any sequence as hvwrky to prevent confusion. susiba received the formal name hvwrky the remaining sequences were annotated accordingly as hvwrky to hvwrky and hvwrky to hvwrky. seven of the hvwrky sequences included start and stop codons and, as judged by protein sequence alignments with wrky proteins from rice, constitute complete coding sequences. the remaining hvwrky sequences represented partial coding sequences. to confirm that we were identifying clade members correctly, we extended sequence information by pcr hvwrky and hvwrky a putative group wrky and group 2d wrky, respectively. this approach revealed a second wrky domain in the sequence of hvwrky confirming that hvwrky is indeed a class member. in case of hvwrky a characteristic domain with the highly conserved amino acid sequence harf could be detected at the beginning of the coding sequence confirming that it too, was correctly assigned by our analysis. * ortholog could not be assigned with significance while eulgem et al. defined wrky proteins by the conserved heptapeptide wrkygqk, slight variations of this sequence have been described for a number of rice wrky proteins. similarly, a number of hvwrky sequences have amino acid sequence substitutions in the conserved wrky signature. in hvwrky and the wrky domain displays the sequence wrkygkk, in hvwrky and wrkygek, in hvwrky wrkygqn and in the c-terminal domain of hvwrky wskygqm. moreover, a unique zinc-finger motif was described as a second characteristic feature of wrky proteins. in group wrky proteins, this motif is slightly altered to c-x7-c-x23-h-x-c. in barley, three of the so far characterized group wrky proteins, namely hvwrky and showed an extension of the zinc finger motif compared to the patterns described by eulgem et al.. again, similar modifications of the zinc-finger patterns were detected for rice wrky proteins and have been discussed to be a monocot-specific feature. phylogenetic analysis of barley wrky proteins while the phylogenetic analysis showed that wrky proteins are of monophyletic nature, the assignment to defined groups is polyphyletic and either based on the number of wrky domains or on the type of zinc finger motif. to study the phylogenetic relationship of the hvwrky proteins we performed a multiple sequence alignment of the about amino acids spanning the wrky domain of all hvwrkys. to obtain a better separation of the different groups and subgroups, wrky domains from members of different arabidopsis and rice wrky proteins were included in the analysis. for each of the groups 2a to 2e and one representative was chosen randomly. these were: atwrky - - - - - - for arabidopsis and oswrky - - - - - - for rice. both c-terminal and n-terminal wrky domains of group wrky proteins have been treated as independent sequences, so that a total number of barley, arabidopsis and rice wrky domains were analyzed. as shown in figure for all subgroups of wrky proteins, we could identify at least one representative in the barley genome. for example, five hvwrky sequences code for proteins with two wrky domains and clearly group with the group members atwrky and oswrky the single wrky domain identified for hvwrky clusters within the c-terminal group domains, indicating that a second n-terminal domain remains likely to be identified. interestingly, the identified group wrky members of barley are distributed very unevenly among the five subgroups. whereas hvwrkys form a distinct subclade with the characteristic members of subgroup 2c, only a single barley wrky, hvwrky belongs to group 2b. group is represented by single-wrky domain barley proteins yet, both wrky domains of hvwrky cluster with these proteins. again, this phenomenon has been observed for at least four rice wrky proteins: oswrky - - and -. the total number of barley wrky genes will remain unknown until the sequencing of the barley genome is completed. however, we constructed an alignment and a phylogenetic tree of arabidopsis, barley and rice wrky proteins to estimate this number. as described earlier by zhang and wang, group and domains were closer related to the c-terminal domains of group whereas the n-terminal wrky domains of group members form a separate monophyletic cluster. on the other hand, separate subclades of the wrky domains of subgroup 2c underline the paraphyletic nature of these subgroups. the grouping of hvwrky hvwrky and oswrky based solely on the phylogenetic analysis of their wrky domain sequences can not be performed unambiguously. however, the presence of two wrky domains in hvwrky and oswrky is indicative for group members. both c-terminal and n-terminal wrky domains of hvwrky and oswrky support basal monophyletic subclades of group wrky proteins, which might be specific for grasses. the number of wrky proteins within each species assigned to the different paraphyletic subgroups to was calculated and is summarized in table a simple comparison of the total number of arabidopsis and rice wrky genes with the number of hvwrkys indicates that approximately 50% of the barley wrky genes have been identified. also, the distribution of wrky proteins showed that hvwrkys are underestimated in several subgroups. nevertheless, similar numbers of barley and rice wrky proteins in subgroups 2a and 2d may indicate that all hvwrky proteins of this subgroup have been identified. * according to eulgem et al. **according to xie et al. monocot-specific radiation and identification of orthologous wrky genes based on the wrky domains of all proteins from barley, arabidopsis and rice, we calculated a comprehensive phylogenetic tree to gain a closer insight into the evolutionary relationships between wrky proteins and assign putative wrky orthologs between these sequences. additionally, nine wrky domains of the moss physcomitrella patens, two each from giardia lamblia and dictyostelium discoideum, as well as additional zinc finger domains of other wrky-gcm1-like proteins were included as outgroups. clades exist for almost all wrky subgroups which consist solely of rice wrky domains or a combination of rice and arabidopsis wrky domains; these clades might be comprised of orthologs of yet unidentified barley wrky proteins. in contrast, rice and barley wrky domains form a number of monocot-specific clades inside group 2c and group these clades reveal clusters of orthologous wrky proteins for rice and barley. for example in subgroup 2c, atwrky forms a basal accession to five barley and six rice wrky domains. similarly, in a subgroup of group ten barley and nine rice wrky proteins constitute putative monocotyledonous orthologs that diverge from a group of three paralogous arabidopsis wrky proteins. thus, the barley and rice wrky proteins that form paralogous or orthologous groups would indicate monocot-specific diversification of wrky genes. this assumption was further supported by blast searches in dbest, which did not return orthologous wrky domains of any other dicotyledonous species for the monocot-specific subclades. thus, our results support the idea of radiation events in groups 2c and wrky proteins that are specific for monocot species. this diversification has most likely occurred after the ancestral divergence of dicotyledonous and monocotyledonous plants. the assignment of orthologs is crucial for transferring the knowledge of wrky protein functions from the model plants rice and arabidopsis to barley. therefore, based on the presented phylogenetic tree, we assigned orthologs for most of the wrky genes described. in a few cases orthologs could not be defined unambiguously. however, as gene orthology implies similar gene function, the assignment presented provided a solid base for functional analyses. powdery mildew infection affects expression of hvwrky genes the analysis of temporal and spatial expression patterns is an initial step towards the functional characterization of genes. the expression of numerous wrky genes from various plant species is affected by biotic and abiotic stresses and implies the regulatory role of wrky proteins in stress responses. in order to investigate if this is also true for hvwrky genes, we mined publicly available microarray data of barley leaves infected with powdery mildew. a total of probesets on the barley genechip could be assigned to different hvwrky genes. datasets of two experiments named bb and bb were retrieved from barleybase and analyzed. figure shows the expression profiles of hvwrky genes are displayed for two genotypes under infection and non-infection conditions as well as during a time course experiment using the host genotype mla and the bgh isolate. even though the setup of the bb and bb experiments differ, some general trends can be seen in both experiments. roughly 50% of all hvwrky genes show altered expression levels upon infection with blumeria graminis. hvwrky and hvwrky have been previously shown to be involved in the regulation of defense mechanisms. hvwrky expression appears to be drastically increased under one experimental condition and lower in the second, while hvwrky shows increased expression levels in both experiments. in addition to these two wrky genes, a number of other hvwrky genes mediating pathogen responses could be assigned based on their expression profiles. for example, the gene for hvwrky which is closely related to the well-studied pathogen response regulators atwrky - and - shows an increase in expression after h of pathogen treatment. also, putative pathogen response regulators could be found in subgroups 2c and interestingly, these are wrky groups that have undergone monocot-specific radiation events, which might thus have been related to an increased biotic stress during evolution. in addition to upregulated genes, there are hvwrky genes with decreased transcript levels, such as most of the hvwrky genes in group however, expression of several hvwrky genes was not affected upon powdery mildew infection and, therefore, might function in regulatory processes other than pathogen response such as abiotic stress or development. orthologous barley and arabidopsis wrky genes exhibit correlative expression signatures during plant development a number of wrky proteins have been reported to be involved in plant developmental processes. to investigate if hvwrky transcript levels vary during plant development, we used public microarray data of different developmental stages from barley and investigated whether phylogenetically related wrky genes of barley and arabidopsis exhibited similarities in their expression patterns. as not all wrky genes of both barley and arabidopsis are represented on their respective microarrays, we were restricted to the expression patterns of hvwrky genes and their putative arabidopsis orthologs. we extracted expression data from the total microarray dataset for ten organs in barley at different developmental stages:coleoptile, mesocotyl, radicule, leaf, root, inflorescence, anthers, and caryopsis and days post anthesis. the expression profiles reveal spatial and temporal variations in expression of hvwrky genes in different barley organs. some genes, like hvwrky - and - have similar expression levels in all investigated tissues. in contrast, genes such as hvwrky - - - and - reveal more than 3-fold difference in expression levels in different organs. furthermore, the transcript levels of barley wrky genes in the same organ show temporal variations. for example, hvwrky is expressed in young caryopses at d.p.a. but is clearly reduced in later stages of caryopsis development. similarly, the expression levels of hvwrky are low in radicules but significantly higher in older roots. in addition, paralogous barley wrky genes such as hvwrky and - reveal similar expression patterns, implying redundant function. next, we performed a comparative expression analysis for both barley and arabidopsis wrky genes on the extracted subsets of the microarray expression datasets of homologous organs by druka et al. and schmid et al.. the pearson correlation of the normalized signal intensities was calculated for the potentially orthologous wrky genes. the resulting correlation coefficients ranged from - to +. the average correlation of all the putative orthologous wrky genes was + and differed significantly from the average expression correlation of a control dataset composed of randomly chosen gene pairs. selection of only the best correlated pairs of hvwrky and atwrky orthologs even increased the average correlation coefficient to +. this high correlation can be easily seen by the overall expression pattern of the compared wrky genes. for example, the expression levels of the orthologous pair in group atwrky and hvwrky hardly differ in any of the ten organs and are clearly correlated. in contrast, the expression profiles of hvwrky and atwrky are observably anti-correlated. genes for the group 2e wrky protein hvwrky and their closely related arabidopsis orthologs atwrky and - show a consistent increase in mrna level in radicules and roots with low expression levels in leaves. according to our phylogenetic analysis, hvwrky has two potential orthologs in arabidopsis, namely atwrky and atwrky the hvwrky expression profile resembles that of atwrky in vegetative tissues but is more similar to the profile of atwrky in reproductive organs such as the inflorescence and the anthers. this might indicate that the arabidopsis wrky genes have undergone functional changes after a duplication event. * for each hvwrky gene, the atwrky gene with the highest correlation value was chosen to calculate the average correlation of best pairs. ** correlation of random genes of barley and arabidopsis. probesets on the barley genechip and ath genechip were randomly selected and the average of the pearson correlation of their expression profiles was calculated. next, we performed rt-pcr experiments on cdna from three different organs and to extend our studies we also included samples from rice, oryza sativa japonica, for the analysis. the signal intensities of the pcr products were quantified and normalized to the actin controls to calculate normalized fold intensities for each organ and organism. first of all, the expression trajectories of all tested barley and arabidopsis genes generally reproduced those observed in the microarrays experiments. transcripts of the three orthologs hvwrky oswrky and atwrky were the most abundant and expressed to the same amount in all organs. although hvwrky and oswrky constitute close relatives, their normalized intensities differ especially in leaves where no hvwrky transcript could be detected. intriguingly, the expression of the closely related group 2c wrkys hvwrky oswrky atwrky and atwrky was highly correlated and strongly increased in seed stages compared to leaves. from this we can conclude that these four wrky genes are of high general importance in all of the three plants for seed development as the comparative organs, siliques and caryopses or infructescences, differ greatly in their structure and composition. discussion in this study we present the annotation of members of the wrky gene family in barley and classify them according to the wrky groups to. comparison to the total number of wrkys in arabidopsis and rice allowed us to identify about 50% of all barley wrky genes. this is in accordance with a study by ryu and colleagues who found est sequences for 44% of all oswrky genes. in order to transfer the knowledge of wrky function in arabidopsis and rice to barley, we created a phylogenetic tree including the wrky protein sequences of all three species. for example, atwrky has been shown to bind to calmodulin in a ca2+- dependent manner by a calmodulin-binding domain, which has been found in all other group 2d wrky proteins of arabidopsis. following our phyogenetic analysis, hvwrky is the ortholog of atwrky and might therefore have similar calmodulin-binding capacity. even though the current sequence of hvwrky does not span the cambd, we could identify such a cambd for hvwrky and hvwrky which are members of the same subclade. also, a sequence alignment of all group 2d oswrky proteins revealed the cambd in four of six oswrky proteins, arguing for a conserved function of this domain in combination with the wrky binding domain in angiosperms. interestingly, although most identified wrky clades have at least one member involved in pathogen stress, none of the group 2d hvwrky genes displayed significant changes in expression levels upon powdery mildew infection. this underlines the uniformity of group 2d hvwrky proteins, and also leads to the speculation whether these hvwrky proteins are impaired in the regulation of pathogen response in a calmodulin-binding dependent manner. for rice and arabidopsis members of wrky group 2a an involvement in the response to fungal pathogens has been demonstrated. recently, hvwrky and hvwrky have been shown to play an important role in the regulation of the response to the fungal pathogen blumeria graminis. our alignment tree indicates the existence of two further group 2a wrky proteins in barley, hvwrky and hvwrky which therefore are also potential regulators of pathogen response. in fact, the expression levels of all group 2a hvwrky genes alter upon powdery mildew infection. interestingly, three out of six rice members of the monocot-specific subclade of group 2c having atwrky as a basal member show increased expression levels upon infection with the fungus magnaporthe grisea. the same is true for at least two other 2c barley members, namely hvwrky and hvwrky which show changes in expression levels in blumeria graminis challenged leaves. this could indicate that the diversification of this particular subclade of wrky transcription factors might have occurred in response to pathogen stress. as all identified pathogen-responsive oswrkys so far have corresponding orthologs in barley, these proteins are also potential regulators of pathogen response. xie and co-workers showed synergistic interactions of oswrky and oswrky in regulation of amy32b gene expression in rice aleurone cells and parts of their work was used for a comparative analysis in barley, where hvwrky was shown to be involved in regulating amy32b expression. in fact, based on our alignment, oswrky and hvwrky were shown to be putative orthologs. however, an interaction partner of hvwrky with functional similarities to oswrky has not been described so far, although one may postulate that the ortholog to oswrky hvwrky constitutes a good candidate as an interaction partner to hvwrky in summary, the sequence comparison of wrky proteins of different species aids in assigning functions to different wrky proteins and allows for target-oriented hypothesis testing. microarray-based studies have become a highly valuable source for functional genomics and huge publicly available datasets allow scientists to monitor the expression of their genes of interest. in this study, we performed a cross-species expression analysis using the microarray data of developmental studies in barley and arabidopsis. we considered similarities in expression profiles as an indicator for conserved functional similarities of orthologous genes. our study was limited by the fact that only of the hvwrky genes and of the atwrky genes are represented by probesets on the respective microarrays. also, there was an obvious risk of not comparing true orthologs as only about 50% of all hvwrky genes could be identified. although cultivation of barley and arabidopsis plants differed, the homologous organs we considered are known to harbor plant lineage-specific functions; we observed an average positive pearson correlation of + in the gene expression of putative wrky orthologs between these two species. this is consistent with the results of genome-wide expression comparisons of five developmental stages of arabidopsis and rice, which returned correlation coefficients in the same range. similarly, a comparative study of the expression of members of the basic helix-loop-helix transcription factors in arabidopsis and rice indicates conserved expression patterns of orthologs in four corresponding plant organs, further indicating that protein function is likely conserved between homologous organs. when only considering the best pairing wrky orthologs, the average pearson correlation for gene expression increased to +. this is mostly due to a reduction in the number of putatively orthologous gene-pairs exhibiting no correlation. several reasons can be suggested which might explain why some of these putative orthologs of barley and arabidopsis show random or negatively correlated expression profiles. first, the function of the orthologous wrky genes might have changed during evolution, mirrored by changes in expression patterns. moreover, the expression of these wrky genes might not be related to any developmental processes and instead are only activated upon abiotic and biotic stresses. since the limitation of identifying orthologs between barley and arabidopsis was based on the available barley est data, it is possible that a better hvwrky ortholog may exist for each atwrky in our current assignment. it is possible, therefore, that poor correlations observed in this study could be improved once more est or genomic sequence is available. nevertheless, the current phylogenetic assignments cover and establish a solid orthology for future studies. in this study we have shown the potential value of comparative expression analysis between sequenced model species and non-sequenced species of high economic impact. nowadays, microarrays for several non-sequenced crop species are commercially available. thus, the reported combination of phylogenetic and expressional analysis may offer a valuable tool for future studies and may contribute to the understanding of gene functions in these crops.  the basis for deriving orthology between related proteins of different plant species is their degree in similarity. here, we could provide that this orthology is not restricted to sequence similarities alone but reaches out to orthology in expression patterns. we speculate that this observation is not restricted to the wrky proteins based on observations made in studies of other gene families. nonetheless, the combination of phylogenetic characterization of hvwrky genes and the subsequent comparative expression analysis of orthologous genes in arabidopsis provides a solid basis for further comparative functional genomics studies of this gene family in the cereal crop barley. 
wrky proteins belong to the wrky-gcm superfamily of zinc finger transcription factors that have been subject to a large plant-specific diversification. for the cereal crop barley, three different wrky proteins have been characterized so far as regulators in sucrose signaling, pathogen defense, and in response to cold and drought. according to their structural features, the hvwrky factors were classified into the previously defined polyphyletic wrky subgroups to furthermore, we could assign putative orthologs of the hvwrky proteins in arabidopsis and rice. hvwrky proteins exhibited not only sequence similarities between orthologs with arabidopsis, but also relatedness in their expression patterns. this correlative expression is indicative for a putative conserved function of related wrky proteins in monocot and dicot species. to gain insight into their putative functions, we examined expression profiles of wrky genes from publicly available microarray data resources and found group specific expression patterns. hvwrky genes have not only undergone radiation in monocot or dicot species, but exhibit evolutionary traits specific to grasses. in this study, we used available sequence information to identify a minimum number of barley wrky transcription factor genes. while putative orthologs of the hvwrky transcription factors have been inferred from phylogenetic sequence analysis, we performed a comparative expression analysis of wrky genes in arabidopsis and barley. indeed, highly correlative expression profiles were found between some of the putative orthologs. however, their phylogenetic relationship remained unresolved.
 wrky proteins belong to the wrky-gcm superfamily of zinc finger transcription factors that have been subject to a large plant-specific diversification. for the cereal crop barley, three different wrky proteins have been characterized so far as regulators in sucrose signaling, pathogen defense, and in response to cold and drought. however, their phylogenetic relationship remained unresolved.  in this study, we used available sequence information to identify a minimum number of barley wrky transcription factor genes. according to their structural features, the hvwrky factors were classified into the previously defined polyphyletic wrky subgroups to furthermore, we could assign putative orthologs of the hvwrky proteins in arabidopsis and rice. while in most cases clades of orthologous proteins were formed within each group or subgroup, other clades were composed of paralogous proteins for the grasses and arabidopsis only, which is indicative of specific gene radiation events. to gain insight into their putative functions, we examined expression profiles of wrky genes from publicly available microarray data resources and found group specific expression patterns. while putative orthologs of the hvwrky transcription factors have been inferred from phylogenetic sequence analysis, we performed a comparative expression analysis of wrky genes in arabidopsis and barley. indeed, highly correlative expression profiles were found between some of the putative orthologs.  hvwrky genes have not only undergone radiation in monocot or dicot species, but exhibit evolutionary traits specific to grasses. hvwrky proteins exhibited not only sequence similarities between orthologs with arabidopsis, but also relatedness in their expression patterns. this correlative expression is indicative for a putative conserved function of related wrky proteins in monocot and dicot species. 