BACKGROUND
proteins characterized by conserved motifs may belong to a gene family, which were represented by structural or functional similarity and evolutionary relationships.
wd <dig> proteins are a group of proteins that are highly conserved in evolution and are extremely abundant across a wide range of eukaryotic organisms  <cit> .
structurally, these proteins are characterized by the presence of approximately  <dig> amino acids core region, which contains a glycine-histidine  dipeptide at the n terminus and a tryptophan-aspartate  dipeptide at the c terminus separated by a region of variable lengths  <cit> .
usually, the wd <dig> protein contains several tandemly repeated units of such motif, which are required to form the secondary structure  <cit> .
the structure of several wd <dig> proteins has been determined, suggesting that the wd <dig> domain folds into a secondary structural of beta propeller despite large levels of sequence diversity.
for example, the mammalian gβ subunit of heterotrimeric gtpases involved in signal transduction forms a beta propeller structure containing seven wd <dig> repeats  <cit> .
wd <dig> gene family shows low level of sequence conservation with functional diversity in diverse pathways, and many wd <dig> proteins possess additional domains with other functional activities.
biochemical and structural studies have recognized wd <dig> proteins to be a broader spectrum of components in cytoplasm and nucleoplasm.
they participate in important cellular pathways, including signal transduction, rna processing, cytoskeleton dynamics, vesicular trafficking, nuclear export, regulation of cell division, and are especially prevalent in chromatin modification and transcriptional regulation  <cit> .
in the model plant arabidopsis thaliana, the wd  <dig> proteins have been identified and analyzed comprehensively  <cit> .
the results suggested that these proteins played key roles in plant-specific processes, with diversity in function conferred at least in part by divergence in upstream signaling pathways, downstream regulatory targets and/or structure outside of the wd  <dig> regions  <cit> .
a common characteristic feature of the wd <dig> proteins is that, the wd <dig> domain mediates diverse protein-protein or protein-dna interactions, thus interplays with multiple proteins to form dynamic complexes and functions as scaffolding protein  <cit> .
wd <dig> domain can mediate molecular recognitions with diverse partners through different sides of its surface.
the same wd <dig> proteins can either recruit different substrates in a similar mode or in distinct ways  <cit> .
considering the multiple interaction modes and the complex roles in cellular processes, it is difficult to identify the partners and pathways in relationship with wd <dig> proteins.
the availability of high-throughput interactomes from different species enables us to understand the networks of the wd <dig> proteins more comprehensively  <cit> .
for instance, wd <dig> domains were found to take part in more interaction pairs than any other domain in yeast, and being as the one of the most interacting domains in human interactome datasets  <cit> .
meanwhile, wd <dig> proteins can also act as a component of protein complexes involved in a variety of pathways  <cit> , or offer binding sites for other proteins  <cit> , demonstrating that they can interact with the appropriate partner in different processes.
the expression profiles in genome-wide scale provide essential data for building the co-expression network, thus allowed us to identify biological processes and gene functions  <cit> .
the comprehensive expression data in crep database encompassing the entire life cycle of rice  provided rich information for associating the wd <dig> genes in different pathways by co-expression analysis  <cit> .
and deeper understanding of their structures, expression profiles, interactions and functional diversity will be essential for our study in the detailed cellular processes mediated by wd <dig> proteins.
rice is one of the major staple foods for world population.
it is also an ideal model species for functional genomic analysis and represents an evolutionary lineage within the monocotyledons.
in this study, we discuss three important questions through the genome-wide scan and systematic characterization of oswd <dig> gene family during the whole life cycle in rice.
first, how many members belong to this family and what about their localizations, gene structures and other characteristics?
second, what are the expression patterns of the oswd <dig> genes, and what is the connection between the expression levels and their gene functions?
third, how does this gene family evolve or what are the evolutionary relationships between these oswd <dig> genes?
therefore, the answers would provide a solid base for future functional genomic studies of the oswd <dig> genes in rice.
RESULTS
collection and identification of the oswd <dig> genes in rice
in order to identify the oswd <dig> genes in the rice, the consensus protein sequence which is characteristic of wd <dig> genes in eukaryotes, geckxvlxghtstvtcvafspdgpllasgsrdgtikiwd, was generated by hmmemit from hmm profile .
we carried out blastp analysis using this sequence as a query in msu database http://rice.plantbiology.msu.edu/index.shtml, with a threshold e value of ≤  <dig>  a total of  <dig> sequences were identified as putative oswd <dig> genes.
by removal of different transcripts of the same gene, we identified  <dig> putative oswd <dig> genes.
these candidates were examined by smart and pfam searching for the presence of wd <dig> domain.
thus,  <dig> genes with the presence of wd <dig> domain were confirmed by smart, and additional  <dig> genes were identified as containing such domain in pfam.
therefore, there were a total of  <dig> oswd <dig> genes in the rice genome.
for convenience, the  <dig> oswd <dig> genes were named from oswd40- <dig> to oswd40- <dig> according to their positions on pseudomolecules.
as the table containing the accession numbers of each oswd <dig> gene is too large within one printed page, these data were exhibited as the additional file .
the detailed information of oswd <dig> genes were also listed in additional file 1: table s <dig>
except for the presence of a conserved wd <dig> domain, the oswd <dig> genes vary substantially in the size and sequences of their encoded proteins, and their physicochemical properties .
the position of the wd <dig> domain within the protein also varies.
the length of oswd <dig> proteins varied from  <dig> to  <dig> amino acids.
expasy analysis suggested that the oswd <dig> protein sequences had large variations in isoelectric point  values  and molecular weight  .
only  <dig> of the  <dig> oswd <dig> genes were predicted to be stable proteins, while the rest were unstable.
details on other parameters of protein sequences were shown in additional file 1: table s <dig>
classification and phylogenetic analysis of oswd <dig> proteins
the  <dig> oswd <dig> proteins were classified into  <dig> subfamilies according to their domain compositions .
one hundred and forty-five members merely with wd <dig> domain belonged to subfamily a. besides wd <dig> domain, oswd <dig> proteins containing several other known functional domains were classified into the following subfamilies.
seven members containing the lish domain were identified as subfamily b; five members containing the utp <dig>  utp <dig>  utp <dig>  and utp <dig> domain were identified as subfamily c; four members with the coatomer wd associated regionand/or coatomer  alpha subunit c-terminus were identified as d subfamily; e subfamily  had histone-binding protein rbbp <dig> or subunit c of caf <dig> complex domains before wd <dig> repeats; f subfamily  contained nle  domain n terminal to wd <dig> repeats; g subfamily  had protein kinase domain or heat repeat; h subfamily  contained the beige/beach domain; i subfamily  had zinc finger domain; j subfamily  contained breast carcinoma amplified sequence  <dig> ; k subfamily  contained other domains including f-box, u-box and domains with unknown function .
to explore the evolutionary relationships of the wd <dig> genes in rice and arabidopsis, an unrooted phylogenetic tree was generated from alignments of their full-length protein sequences.
the phylogenetic analysis revealed that all wd40s were clustered into five distinct groups , comprising  <dig>   <dig>   <dig>   <dig>  and  <dig> proteins, respectively .
wd <dig> proteins from rice and arabidopsis are present in all groups.
the oswd <dig> members were more closely to those in the same clade in arabidopsis than to other oswd <dig> proteins in the same species , which indicated synteny and conservation between rice and arabidopsis proteins.
most members in the same groups or subgroups shared one or more domains outside the wd <dig> domain, thus was consistent with the subfamily definition revealed above.
expression profiling of oswd <dig> genes during the whole life cycle of rice
to study the transcript accumulation of oswd <dig> genes in the entire life cycle of rice, the expression profiling covering  <dig> developmental stages  in minghui  <dig> were analyzed by affymetrix rice microarray data in crep database  <cit> .
probes for  <dig> of the  <dig> oswd <dig> genes could be identified in the affymetrix microarray.
thirty-six genes had two probe sets and the higher signal value of the probe sets was used for analysis.
two pairs of genes, oswd40- <dig> and oswd40- <dig>  as well as oswd40- <dig> and oswd40- <dig>  shared the same probe sets, respectively.
only  <dig> genes showed a "present" detection call at p value of  <dig>  in at least one of the investigated tissues, whereas two low expressing genes  were either "absent" or "marginal" under these developmental stages.
the transcripts of these two genes were at almost undetectable levels in all the stages analyzed, but it is possible that these genes might respond to specific stimuli or their expressions might be limited to specialized cell types that have not been analyzed in this investigation.
a hierarchical cluster displaying the logarithm of average signal values for the  <dig> oswd <dig> genes were generated.
based on which the expression patterns of oswd <dig> genes could be classified into two major groups .
one hundred and nineteen genes belonged to group i, most of which  showed high transcript accumulations  in all the tissues analyzed .
these oswd <dig> genes might play roles in housekeeping functions and gene oswd40- <dig> had the highest average expression level in the entire life cycle.
these  <dig> genes could be further divided into three subgroups.
subgroup ia consisted of  <dig> genes, with the average expression signal from  <dig>  to  <dig>  .
subgroup ib of  <dig> genes showed relatively high expression level in a serious of reproductive tissues or vegetative tissues .
notably, the expression levels of  <dig> genes in subgroup 1c were extremely high, with the average expression signal from  <dig> to  <dig>  .
group ii consisted of  <dig> genes, which showed relatively low expression signals or preferential expressions in some tissues .
this group can also divide into two subgroups.
subgroup iia contained  <dig> genes showing tissue-specific/preferential expressions, e.g., oswd40- <dig>   <dig>   <dig>   <dig> with predominant expression in stamen, and oswd40- <dig> and  <dig> as endosperm preferential expression genes.
interestingly, the expression of eight genes  which was high in panicles decreased in panicle  <dig> and then increased gradually as the panicles matured.
these genes might play essential roles in panicle developing.
subgroup iib comprised  <dig> genes, and genes in this subgroup showed almost negligible expressions .
in order to reveal more information in oswd <dig> expression pattern, genes that showed differential expression during various stages of development in comparison to seed were analyzed.
detailed p values and fold change values were given in additional file 5: table s <dig>  genes considered as preferential expression in a given stage showed tremendous differences .
up-regulated genes mainly accumulated in panicles and stamen, suggesting that oswd <dig> genes participate in various molecular pathways in flowering development.
surprisingly, although down-regulated genes accumulated in seedlings, they were activated in stamen, either .
this result suggested that oswd <dig> genes might control stamen regulation.
they might also play important roles in early developmental stages in seedlings.
responses of oswd <dig> genes under naa, kt, and ga <dig> treatments
phytohormones play a critical role in plant growth and development.
to investigate the oswd <dig> genes in response to phytohormone treatment, microarray analysis was performed.
we identified a total of  <dig> oswd <dig> genes that were differentially expressed with treatments of one or more of the phytohormone naa, kt, ga <dig> in seedlings.
the control indicated the expression level of corresponding oswd <dig> genes in rice seedlings in trefoil stage without treatment .
the fold change values with respect to control are given in additional file 5: table s <dig>
four oswd <dig> genes showed differential expression under all three phytohormone treatments, among which three genes  were up-regulated, whereas oswd40- <dig> was down-regulated.
the expression profile of the remaining genes in response to naa, kt, and ga <dig> was different.
for instance, three genes  were up-regulated under naa and ga <dig> treatments, two genes  were differentially expressed under naa and kt treatments, and six genes  were up-regulated to kt and ga <dig> treatment, respectively.
meanwhile, ten oswd <dig> genes showed differential expression specifically to one phytohormone treatment.
amongst the ten genes, oswd40- <dig>   <dig>   <dig>   <dig> and  <dig> were up-regulated specifically to ga <dig> treatment.
we also found that oswd40- <dig>   <dig>   <dig> and  <dig> were up-regulated, whereas oswd40- <dig> was down-regulated specifically to kt treatment.
the induction of oswd <dig> genes by phytohormones prompted us to check their promoter sequence  by searching against the place database http://www.dna.affrc.go.jp/place/signalscan.html.
the results suggested that all promoter regions of these  <dig> oswd <dig> genes contained various elements of auxin, gibberellin, and cytokinin .
chromosomal localization and gene duplication
the genomic distribution of oswd <dig> genes were determined by their chromosomal positions on rice chromosome pseudomolecules.
totally, all  <dig> oswd <dig> genes were dispersed on the  <dig> chromosomes, presenting unevenly in all regions of the chromosomes.
a diagrammatic representation of chromosomal distribution of oswd <dig> genes was depicted in figure  <dig> .
certain chromosomes had a relatively high density of oswd <dig> genes, e.g., a maximum of  <dig> genes were present on chromosome  <dig>  followed by  <dig> genes on chromosome  <dig>  on the other hand, only six oswd <dig> genes were present on chromosome  <dig>
segmental duplication and tandem duplication play important roles in generating the members of a gene family during the evolution  <cit> .
therefore, both segmental and tandem duplication events were investigated for elucidating the potential mechanism of evolution of oswd <dig> gene family.
analysis of the msu rgap rice segmental duplication database revealed only  <dig>  oswd <dig> genes could be assigned to msu rgap segmental duplication blocks at a maximal length distance permitted between collinear gene pairs of  <dig> kb.
the overall similarity of the cdna sequences of these genes ranged from  <dig> % to  <dig> % and all of them were found to have their counterparts on duplicated segments .
nineteen oswd <dig> genes  seemed to be produced from tandem duplications according to the criterion adopted in our analysis .
they were separated by a maximum of five intervening genes.
three group of the gene pairs were placed juxtaposed with no intervening gene.
the distance between these genes ranged from  <dig> kb to  <dig> kb .
interestingly, not all the tandemly duplicated genes in the same cluster had the same direction of transcription.
this might suggest the complex behavior of tandem duplications in this family.
all these results suggested that much of the diversity of the oswd <dig> gene family in rice is due to both tandem duplication and segmental genome duplication events.
the expression patterns of oswd <dig> genes for segmentally duplicated and tandemly duplicated genes were examined by affymetrix rice microarray data.
probe sets were available for  <dig> out of  <dig> segmental duplication genes and  <dig> out of  <dig> tandemly duplicated genes in microarray data.
a comparison of expression level revealed that two segmental duplicated genes in one pair always showed similar expression pattern, although one of the duplicated genes showed low expression level, or was not expressed at significant levels in most of the tissues .
we could therefore infer that immediately after segmental duplication, the two copies of genes might be functionally redundant.
however, it is possible that only one of the gene copies retains its function while the other one degenerates into a pseudogene  <cit> .
two tandemly duplicated genes in group  <dig> shared the same probe and oswd40- <dig> in group  <dig> did not have probe sets on affymetrix microarray.
therefore, we analyzed the rest  <dig> tandemly duplicated genes in  <dig> groups.
the expression pattern for tandem duplicated genes was more complicated .
the expression pattern was quite similar for four pairs of genes .
therefore, the gene copies might have maintained their functions during evolution, as evidenced by the similar expression pattern.
four pairs of genes  showed divergent expression profiles in most of the investigated tissues, as one of the genes was not expressed at significant levels in most of the tissues.
this indicated that one of the members changed its function during the course of evolution.
group  <dig> containing oswd40- <dig> and oswd40- <dig> have already been elucidated.
as reported by luo et al.
<cit> , osfie <dig>  and osfie <dig>  were likely to have duplicated in the ancestor of the grasses.
osfie <dig> was expressed only in endosperm with imprinted effect  <cit> , while osfie <dig> was not imprinted in endosperm and was expressed constitutively.
oswd40- <dig> in group  <dig> was not expressed at significant levels in all tissues, which might be induced by pseudo-functionalization after duplication  <cit> .
expression correlation and gene ontology  analyses
by using a permutation test, we set the threshold of the pearson's correlation coefficients  at  <dig>  and extracted  <dig> genes whose expression tightly correlates with  <dig> of the members in oswd <dig> family .
using the functional annotation and web-based go analysis, four networks were constructed regarding correlations between oswd <dig> genes and their co-expressed genes, reflecting functional bias and possible molecular pathways that they involved in .
the network in figure 7a contained  <dig> rice genes  and  <dig> co-expression links, including  <dig> oswd <dig> genes,  <dig> myb related genes and three basic helix-loop-helix  genes.
compared with the whole rice genome annotation, these oswd <dig> genes seem to affect the regulation of multiple biological processes, such as cellular biosynthetic/metabolic process, transcription, nucleobase, nucleoside, nucleotide and nucleic acid metabolic process, gene expression, biosynthetic process, nitrogen compound metabolic process, macromolecule biosynthetic process and so on.
besides, the molecular functions related to dna binding and nucleic acid binding were significantly enriched .
the network in figure 7b contained only  <dig> rice genes  and  <dig> co-expression links.
this network suggested the possibility that five oswd <dig> genes might play roles in different molecular pathways with the participation of four mads-box genes.
complex network has also been constructed in figure 7c, containing  <dig> rice genes  and  <dig> co-expression links.
the co-expression genes in this group were associated with histone-related proteins such as histone-lysine n-methyltransferase, histone deacetylase, single myb histone, jmjc domain containing proteins and so on.
analysis of their go terms identified functional modules enriched for chromosome organization, chromatin organization, organelle organization, and chromatin modification.
these genes also acted as important cellular components in nucleus and membrane-bounded organelle, and might take part in methyltransferase and transferase activities .
our analysis also indicated that the expression of some oswd <dig> genes was co-expressed with that of other oswd <dig> genes.
we found that  <dig> oswd <dig> genes might therefore correlate with or interact with each other, thus forming a more complex feedback network .
however, no significant go terms were identified.
discussion
oswd <dig> evolution and classification
gene duplications are one of the primary driving forces during the evolution, and the variations in family size and distribution of a gene family were related to either tandem or segmental duplications  <cit> .
as stated previously, oswd <dig> family expanded from both tandem and segmental duplications, and the number of oswd <dig> genes arranged in segmental duplications contributes to the 12% birth of new genes, while tandem duplication events contribute to the  <dig> % birth of new genes.
it was interesting that different subfamilies of oswd <dig> genes expanded in distinct manners: all segmental duplicated genes were favor for subfamily a, except oswd40- <dig> that belonged to subfamily i. however, tandem duplications were belonged to various subfamilies, including subfamily a, b, d, g, and k.
we also noticed that the expression level of three tandemly duplicated genes, oswd40- <dig>   <dig>  and  <dig> were lower than that of their corresponding copies, suggesting they might lose their functions during the evolution.
however, these three genes were differentially expressed under phytohormone treatments.
whereas another copy of these tandemly duplicated genes did not show differential expression under phytohormone treatments.
therefore, one would tentatively suppose that while oswd40- <dig>   <dig>  and  <dig> slowly lost their ancestral functions, they might evolve new functions in phytohormone pathways during the evolution.
oswd <dig> genes may initiate their diverse functions by performing protein complex with myb and bhlh transcription factors
the multiple metabolism pathways in plants must be regulated by coordinated expression of different genes.
the co-expression networks reflect the correlation of the expression pattern of different genes, and are suggestive in tracing the genes in the same pathway.
here, our co-expression analysis has revealed that the function of oswd <dig> proteins might require the participation of various members of myb and bhlh transcription factors .
oswd40- <dig> was found to be co-expressed with five myb factors, os12g <dig>  os06g <dig>  os01g <dig>  os02g <dig> and os06g <dig>  as well as two bhlh factors os06g <dig> and os08g <dig>  oswd40- <dig> was homologous to plant smu genes, which appeared to be involved in splicing of specific pre-mrnas that affected multiple aspects of development  <cit> .
thus, whether transcription factors could serve as a link to mrna process was a suggestive direction in further study.
we also noticed that oswd40- <dig> was found to be co-expressed with these transcription factors.
compared with the co-expression genes of oswd40- <dig>  four myb factors os02g <dig>  os08g <dig>  os08g <dig>  and os08g <dig> might be the potential interaction factors of oswd40- <dig> in addition.
oswd40- <dig> might be a homolog of arabidopsis lis, which restricted gametic cell fate in female gametophyte  <cit> .
oswd40- <dig> was found to be co-expressed with three myb factors, os02g <dig>  os01g <dig> and os06g <dig>  as well as a bhlh factor os06g <dig>  these three oswd <dig> genes were co-expressed with the same group of myb and bhlh transcription factors, suggesting that they may take part in correlated molecular pathways by interaction with these partners.
previous genetic analyses found that ectopic expression of maize wd <dig> protein pac <dig> in an arabidopsis ttg <dig> mutant was able to complement the mutant phenotypes  <cit> , suggesting that wd <dig> proteins can interact with similar partners.
we might speculate that some oswd <dig> genes might be in relation with each other by controlling the expression of these transcription factors.
another oswd <dig> gene that attracted our attention was oswd40- <dig>  which was co-expressed with another group of myb and bhlh transcription factors that were different from the co-expression genes mentioned above.
we also found that oswd40- <dig> might be related with other partners such as genes in set family, hdac family, and hac .
this result suggested that oswd40- <dig> might participate in the regulation of another pathway.
a general wd/myb/bhlh complex for regulation of the anthocyanin biosynthetic pathway was also found in antirrhinum majus, petunia hybrida and arabidopsis thaliana  <cit> .
meanwhile, a bhlh protein lc in maize was found to interact with myb transcription factors to activate anthocyanin expression  <cit> .
another lc-like bhlh protein was also found to require a myb protein to perform its function  <cit> .
results suggested that many mybs interacted directly with lc-like bhlh proteins and the wd <dig> repeat protein  <cit> .
therefore, it seems that wd <dig> proteins allow protein-protein interactions between the bhlh and myb proteins, and wd <dig> proteins in rice might also require mybs and bhlhs to form a transcription complex to participate a range of pathways.
oswd <dig> genes may be involved in histone-related functions with members in set family
histone expression and histone post-translational modifications play pivotal roles in chromatin remodeling and epigenetic regulation in plant development  <cit> .
our co-expression analysis has revealed that oswd <dig> genes may function with histone-related proteins.
although the exact pathways mediate by these genes are still unclear, one might speculate that these oswd <dig> genes play important roles in histone modification.
an important group of enzymes involved in histone modification is the histone-lysine n-methyltransferases.
these proteins participate in the establishment and/or maintenance of euchromatic or heterochromatic states of active or transcriptionally repressed sequences  <cit> .
here, a total of  <dig> histone-lysine n-methyltransferases were identified to be co-expressed with the oswd <dig> proteins, both of which contain the set domain that is responsible for the catalytic activity of the enzymes, suggesting possible interactions between the oswd <dig> and set genes in a family level.
one might also tentatively speculate that the wd <dig> and set domain must be the key functional structure for interaction by a conserved mechanism.
functional studies of several arabidopsis genes encoding wd <dig> proteins also suggest that they might be implicated in histone modification in different pathways.
a wd <dig> domain cyclophilin, cyclophilin <dig> , which functions in gene repression and organogenesis in arabidopsis, serves as a highly conserved histone remodeling factor involved in chromatin-based gene silencing  <cit> .
another wd <dig> protein msi <dig> in arabidopsis has also been proposed to exhibit pleiotropic phenotypes by epigenetic regulation  <cit> .
therefore, oswd <dig> genes in rice might also involved in similar pathways by histone modulation.
in a word, characterization of oswd <dig> proteins function in histone modification could therefore open new perspectives for understanding the molecular mechanism of epigenetic regulation.
oswd <dig> genes may take part in reproductive pathways with mads-box transcription factors
wd <dig> genes identified in different plant species are involved in various developmental processes  <cit> .
in our study, the expression patterns of oswd <dig> genes and the co-expression analysis provide useful information for establishing their putative functions.
the available evidence suggests that oswd <dig> genes may take part in reproductive pathways with mads-box transcription factors in rice.
mads-box transcription factors are essential for various aspects of pathways in flower development both in dicotyledon and monocotyledon  <cit> .
our result suggested that oswd40- <dig> was co-expressed with three mads-box transcription factors, os03g <dig>  os06g <dig>  and os01g <dig> .
oswd40- <dig> was homologous to arabidopsis fve/msi <dig>  a key regulator that interacted with cul4-ddb <dig> and a prc2-like complex to control epigenetic regulation of flowering time  <cit> .
it was also reported that os03g <dig>  was required for rice inflorescence and spikelet development  <cit> .
therefore, it would be interesting to investigate whether oswd40- <dig> play roles in rice flower development with mads-box transcription factors.
we also found that three oswd <dig> genes, oswd40- <dig>   <dig>  and  <dig>  were co-expressed with a mads gene os04g <dig>  these four genes were expressed preferentially in stamen , suggesting that they may function in stamen development.
all these studies support the results that mads-box transcription factors are essential for flower developmental processes in relationship with oswd <dig> genes.
CONCLUSIONS
in conclusion, using an in silico approach, a total of  <dig> oswd <dig> genes were found to be present in rice genome.
genomic framework revealed the potential mechanisms responsible for the evolution of oswd <dig> genes in rice.
the expression profiling of oswd <dig> gene family covering rice life cycle could provide deep insights into their potential functions during rice growth and development.
some genes appear to be differentially expressed in different tissues/organs, vegetative and reproductive development stages, and expression of some genes is influenced under phytohormones.
these data will provide the basis for understanding the evolutionary history of oswd <dig> members and their roles in rice growth and development.
the findings in our work would be useful in selecting candidate genes for functional studies of oswd <dig> members in rice.
however, future research by adopting transformation strategies or insertion mutagenesis is required to elucidate the precise functions of these oswd <dig> genes.

 proteins characterized by conserved motifs may belong to a gene family, which were represented by structural or functional similarity and evolutionary relationships. wd proteins are a group of proteins that are highly conserved in evolution and are extremely abundant across a wide range of eukaryotic organisms. structurally, these proteins are characterized by the presence of approximately amino acids core region, which contains a glycine-histidine dipeptide at the n terminus and a tryptophan-aspartate dipeptide at the c terminus separated by a region of variable lengths. usually, the wd protein contains several tandemly repeated units of such motif, which are required to form the secondary structure. the structure of several wd proteins has been determined, suggesting that the wd domain folds into a secondary structural of beta propeller despite large levels of sequence diversity. for example, the mammalian gβ subunit of heterotrimeric gtpases involved in signal transduction forms a beta propeller structure containing seven wd repeats. wd gene family shows low level of sequence conservation with functional diversity in diverse pathways, and many wd proteins possess additional domains with other functional activities. biochemical and structural studies have recognized wd proteins to be a broader spectrum of components in cytoplasm and nucleoplasm. they participate in important cellular pathways, including signal transduction, rna processing, cytoskeleton dynamics, vesicular trafficking, nuclear export, regulation of cell division, and are especially prevalent in chromatin modification and transcriptional regulation. in the model plant arabidopsis thaliana, the wd proteins have been identified and analyzed comprehensively. the results suggested that these proteins played key roles in plant-specific processes, with diversity in function conferred at least in part by divergence in upstream signaling pathways, downstream regulatory targets and/or structure outside of the wd regions. a common characteristic feature of the wd proteins is that, the wd domain mediates diverse protein-protein or protein-dna interactions, thus interplays with multiple proteins to form dynamic complexes and functions as scaffolding protein. wd domain can mediate molecular recognitions with diverse partners through different sides of its surface. the same wd proteins can either recruit different substrates in a similar mode or in distinct ways. considering the multiple interaction modes and the complex roles in cellular processes, it is difficult to identify the partners and pathways in relationship with wd proteins. the availability of high-throughput interactomes from different species enables us to understand the networks of the wd proteins more comprehensively. for instance, wd domains were found to take part in more interaction pairs than any other domain in yeast, and being as the one of the most interacting domains in human interactome datasets. meanwhile, wd proteins can also act as a component of protein complexes involved in a variety of pathways, or offer binding sites for other proteins, demonstrating that they can interact with the appropriate partner in different processes. the expression profiles in genome-wide scale provide essential data for building the co-expression network, thus allowed us to identify biological processes and gene functions. the comprehensive expression data in crep database encompassing the entire life cycle of rice provided rich information for associating the wd genes in different pathways by co-expression analysis. and deeper understanding of their structures, expression profiles, interactions and functional diversity will be essential for our study in the detailed cellular processes mediated by wd proteins. rice is one of the major staple foods for world population. it is also an ideal model species for functional genomic analysis and represents an evolutionary lineage within the monocotyledons. in this study, we discuss three important questions through the genome-wide scan and systematic characterization of oswd gene family during the whole life cycle in rice. first, how many members belong to this family and what about their localizations, gene structures and other characteristics? second, what are the expression patterns of the oswd genes, and what is the connection between the expression levels and their gene functions? third, how does this gene family evolve or what are the evolutionary relationships between these oswd genes? therefore, the answers would provide a solid base for future functional genomic studies of the oswd genes in rice.  collection and identification of the oswd genes in rice in order to identify the oswd genes in the rice, the consensus protein sequence which is characteristic of wd genes in eukaryotes, geckxvlxghtstvtcvafspdgpllasgsrdgtikiwd, was generated by hmmemit from hmm profile. we carried out blastp analysis using this sequence as a query in msu database http://rice.plantbiology.msu.edu/index.shtml, with a threshold e value of ≤ a total of sequences were identified as putative oswd genes. by removal of different transcripts of the same gene, we identified putative oswd genes. these candidates were examined by smart and pfam searching for the presence of wd domain. thus, genes with the presence of wd domain were confirmed by smart, and additional genes were identified as containing such domain in pfam. therefore, there were a total of oswd genes in the rice genome. for convenience, the oswd genes were named from oswd40- to oswd40- according to their positions on pseudomolecules. as the table containing the accession numbers of each oswd gene is too large within one printed page, these data were exhibited as the additional file. the detailed information of oswd genes were also listed in additional file 1: table s except for the presence of a conserved wd domain, the oswd genes vary substantially in the size and sequences of their encoded proteins, and their physicochemical properties. the position of the wd domain within the protein also varies. the length of oswd proteins varied from to amino acids. expasy analysis suggested that the oswd protein sequences had large variations in isoelectric point values and molecular weight. only of the oswd genes were predicted to be stable proteins, while the rest were unstable. details on other parameters of protein sequences were shown in additional file 1: table s classification and phylogenetic analysis of oswd proteins the oswd proteins were classified into subfamilies according to their domain compositions. one hundred and forty-five members merely with wd domain belonged to subfamily a. besides wd domain, oswd proteins containing several other known functional domains were classified into the following subfamilies. seven members containing the lish domain were identified as subfamily b; five members containing the utp utp utp and utp domain were identified as subfamily c; four members with the coatomer wd associated regionand/or coatomer alpha subunit c-terminus were identified as d subfamily; e subfamily had histone-binding protein rbbp or subunit c of caf complex domains before wd repeats; f subfamily contained nle domain n terminal to wd repeats; g subfamily had protein kinase domain or heat repeat; h subfamily contained the beige/beach domain; i subfamily had zinc finger domain; j subfamily contained breast carcinoma amplified sequence; k subfamily contained other domains including f-box, u-box and domains with unknown function. to explore the evolutionary relationships of the wd genes in rice and arabidopsis, an unrooted phylogenetic tree was generated from alignments of their full-length protein sequences. the phylogenetic analysis revealed that all wd40s were clustered into five distinct groups, comprising and proteins, respectively. wd proteins from rice and arabidopsis are present in all groups. the oswd members were more closely to those in the same clade in arabidopsis than to other oswd proteins in the same species, which indicated synteny and conservation between rice and arabidopsis proteins. most members in the same groups or subgroups shared one or more domains outside the wd domain, thus was consistent with the subfamily definition revealed above. expression profiling of oswd genes during the whole life cycle of rice to study the transcript accumulation of oswd genes in the entire life cycle of rice, the expression profiling covering developmental stages in minghui were analyzed by affymetrix rice microarray data in crep database. probes for of the oswd genes could be identified in the affymetrix microarray. thirty-six genes had two probe sets and the higher signal value of the probe sets was used for analysis. two pairs of genes, oswd40- and oswd40- as well as oswd40- and oswd40- shared the same probe sets, respectively. only genes showed a "present" detection call at p value of in at least one of the investigated tissues, whereas two low expressing genes were either "absent" or "marginal" under these developmental stages. the transcripts of these two genes were at almost undetectable levels in all the stages analyzed, but it is possible that these genes might respond to specific stimuli or their expressions might be limited to specialized cell types that have not been analyzed in this investigation. a hierarchical cluster displaying the logarithm of average signal values for the oswd genes were generated. based on which the expression patterns of oswd genes could be classified into two major groups. one hundred and nineteen genes belonged to group i, most of which showed high transcript accumulations in all the tissues analyzed. these oswd genes might play roles in housekeeping functions and gene oswd40- had the highest average expression level in the entire life cycle. these genes could be further divided into three subgroups. subgroup ia consisted of genes, with the average expression signal from to. subgroup ib of genes showed relatively high expression level in a serious of reproductive tissues or vegetative tissues. notably, the expression levels of genes in subgroup 1c were extremely high, with the average expression signal from to. group ii consisted of genes, which showed relatively low expression signals or preferential expressions in some tissues. this group can also divide into two subgroups. subgroup iia contained genes showing tissue-specific/preferential expressions, e.g., oswd40- with predominant expression in stamen, and oswd40- and as endosperm preferential expression genes. interestingly, the expression of eight genes which was high in panicles decreased in panicle and then increased gradually as the panicles matured. these genes might play essential roles in panicle developing. subgroup iib comprised genes, and genes in this subgroup showed almost negligible expressions. in order to reveal more information in oswd expression pattern, genes that showed differential expression during various stages of development in comparison to seed were analyzed. detailed p values and fold change values were given in additional file 5: table s genes considered as preferential expression in a given stage showed tremendous differences. up-regulated genes mainly accumulated in panicles and stamen, suggesting that oswd genes participate in various molecular pathways in flowering development. surprisingly, although down-regulated genes accumulated in seedlings, they were activated in stamen, either. this result suggested that oswd genes might control stamen regulation. they might also play important roles in early developmental stages in seedlings. responses of oswd genes under naa, kt, and ga treatments phytohormones play a critical role in plant growth and development. to investigate the oswd genes in response to phytohormone treatment, microarray analysis was performed. we identified a total of oswd genes that were differentially expressed with treatments of one or more of the phytohormone naa, kt, ga in seedlings. the control indicated the expression level of corresponding oswd genes in rice seedlings in trefoil stage without treatment. the fold change values with respect to control are given in additional file 5: table s four oswd genes showed differential expression under all three phytohormone treatments, among which three genes were up-regulated, whereas oswd40- was down-regulated. the expression profile of the remaining genes in response to naa, kt, and ga was different. for instance, three genes were up-regulated under naa and ga treatments, two genes were differentially expressed under naa and kt treatments, and six genes were up-regulated to kt and ga treatment, respectively. meanwhile, ten oswd genes showed differential expression specifically to one phytohormone treatment. amongst the ten genes, oswd40- and were up-regulated specifically to ga treatment. we also found that oswd40- and were up-regulated, whereas oswd40- was down-regulated specifically to kt treatment. the induction of oswd genes by phytohormones prompted us to check their promoter sequence by searching against the place database http://www.dna.affrc.go.jp/place/signalscan.html. the results suggested that all promoter regions of these oswd genes contained various elements of auxin, gibberellin, and cytokinin. chromosomal localization and gene duplication the genomic distribution of oswd genes were determined by their chromosomal positions on rice chromosome pseudomolecules. totally, all oswd genes were dispersed on the chromosomes, presenting unevenly in all regions of the chromosomes. a diagrammatic representation of chromosomal distribution of oswd genes was depicted in figure. certain chromosomes had a relatively high density of oswd genes, e.g., a maximum of genes were present on chromosome followed by genes on chromosome on the other hand, only six oswd genes were present on chromosome segmental duplication and tandem duplication play important roles in generating the members of a gene family during the evolution. therefore, both segmental and tandem duplication events were investigated for elucidating the potential mechanism of evolution of oswd gene family. analysis of the msu rgap rice segmental duplication database revealed only oswd genes could be assigned to msu rgap segmental duplication blocks at a maximal length distance permitted between collinear gene pairs of kb. the overall similarity of the cdna sequences of these genes ranged from % to % and all of them were found to have their counterparts on duplicated segments. nineteen oswd genes seemed to be produced from tandem duplications according to the criterion adopted in our analysis. they were separated by a maximum of five intervening genes. three group of the gene pairs were placed juxtaposed with no intervening gene. the distance between these genes ranged from kb to kb. interestingly, not all the tandemly duplicated genes in the same cluster had the same direction of transcription. this might suggest the complex behavior of tandem duplications in this family. all these results suggested that much of the diversity of the oswd gene family in rice is due to both tandem duplication and segmental genome duplication events. the expression patterns of oswd genes for segmentally duplicated and tandemly duplicated genes were examined by affymetrix rice microarray data. probe sets were available for out of segmental duplication genes and out of tandemly duplicated genes in microarray data. a comparison of expression level revealed that two segmental duplicated genes in one pair always showed similar expression pattern, although one of the duplicated genes showed low expression level, or was not expressed at significant levels in most of the tissues. we could therefore infer that immediately after segmental duplication, the two copies of genes might be functionally redundant. however, it is possible that only one of the gene copies retains its function while the other one degenerates into a pseudogene. two tandemly duplicated genes in group shared the same probe and oswd40- in group did not have probe sets on affymetrix microarray. therefore, we analyzed the rest tandemly duplicated genes in groups. the expression pattern for tandem duplicated genes was more complicated. the expression pattern was quite similar for four pairs of genes. therefore, the gene copies might have maintained their functions during evolution, as evidenced by the similar expression pattern. four pairs of genes showed divergent expression profiles in most of the investigated tissues, as one of the genes was not expressed at significant levels in most of the tissues. this indicated that one of the members changed its function during the course of evolution. group containing oswd40- and oswd40- have already been elucidated. as reported by luo et al., osfie and osfie were likely to have duplicated in the ancestor of the grasses. osfie was expressed only in endosperm with imprinted effect, while osfie was not imprinted in endosperm and was expressed constitutively. oswd40- in group was not expressed at significant levels in all tissues, which might be induced by pseudo-functionalization after duplication. expression correlation and gene ontology analyses by using a permutation test, we set the threshold of the pearson's correlation coefficients at and extracted genes whose expression tightly correlates with of the members in oswd family. using the functional annotation and web-based go analysis, four networks were constructed regarding correlations between oswd genes and their co-expressed genes, reflecting functional bias and possible molecular pathways that they involved in. the network in figure 7a contained rice genes and co-expression links, including oswd genes, myb related genes and three basic helix-loop-helix genes. compared with the whole rice genome annotation, these oswd genes seem to affect the regulation of multiple biological processes, such as cellular biosynthetic/metabolic process, transcription, nucleobase, nucleoside, nucleotide and nucleic acid metabolic process, gene expression, biosynthetic process, nitrogen compound metabolic process, macromolecule biosynthetic process and so on. besides, the molecular functions related to dna binding and nucleic acid binding were significantly enriched. the network in figure 7b contained only rice genes and co-expression links. this network suggested the possibility that five oswd genes might play roles in different molecular pathways with the participation of four mads-box genes. complex network has also been constructed in figure 7c, containing rice genes and co-expression links. the co-expression genes in this group were associated with histone-related proteins such as histone-lysine n-methyltransferase, histone deacetylase, single myb histone, jmjc domain containing proteins and so on. analysis of their go terms identified functional modules enriched for chromosome organization, chromatin organization, organelle organization, and chromatin modification. these genes also acted as important cellular components in nucleus and membrane-bounded organelle, and might take part in methyltransferase and transferase activities. our analysis also indicated that the expression of some oswd genes was co-expressed with that of other oswd genes. we found that oswd genes might therefore correlate with or interact with each other, thus forming a more complex feedback network. however, no significant go terms were identified. discussion oswd evolution and classification gene duplications are one of the primary driving forces during the evolution, and the variations in family size and distribution of a gene family were related to either tandem or segmental duplications. as stated previously, oswd family expanded from both tandem and segmental duplications, and the number of oswd genes arranged in segmental duplications contributes to the 12% birth of new genes, while tandem duplication events contribute to the % birth of new genes. it was interesting that different subfamilies of oswd genes expanded in distinct manners: all segmental duplicated genes were favor for subfamily a, except oswd40- that belonged to subfamily i. however, tandem duplications were belonged to various subfamilies, including subfamily a, b, d, g, and k. we also noticed that the expression level of three tandemly duplicated genes, oswd40- and were lower than that of their corresponding copies, suggesting they might lose their functions during the evolution. however, these three genes were differentially expressed under phytohormone treatments. whereas another copy of these tandemly duplicated genes did not show differential expression under phytohormone treatments. therefore, one would tentatively suppose that while oswd40- and slowly lost their ancestral functions, they might evolve new functions in phytohormone pathways during the evolution. oswd genes may initiate their diverse functions by performing protein complex with myb and bhlh transcription factors the multiple metabolism pathways in plants must be regulated by coordinated expression of different genes. the co-expression networks reflect the correlation of the expression pattern of different genes, and are suggestive in tracing the genes in the same pathway. here, our co-expression analysis has revealed that the function of oswd proteins might require the participation of various members of myb and bhlh transcription factors. oswd40- was found to be co-expressed with five myb factors, os12g os06g os01g os02g and os06g as well as two bhlh factors os06g and os08g oswd40- was homologous to plant smu genes, which appeared to be involved in splicing of specific pre-mrnas that affected multiple aspects of development. thus, whether transcription factors could serve as a link to mrna process was a suggestive direction in further study. we also noticed that oswd40- was found to be co-expressed with these transcription factors. compared with the co-expression genes of oswd40- four myb factors os02g os08g os08g and os08g might be the potential interaction factors of oswd40- in addition. oswd40- might be a homolog of arabidopsis lis, which restricted gametic cell fate in female gametophyte. oswd40- was found to be co-expressed with three myb factors, os02g os01g and os06g as well as a bhlh factor os06g these three oswd genes were co-expressed with the same group of myb and bhlh transcription factors, suggesting that they may take part in correlated molecular pathways by interaction with these partners. previous genetic analyses found that ectopic expression of maize wd protein pac in an arabidopsis ttg mutant was able to complement the mutant phenotypes, suggesting that wd proteins can interact with similar partners. we might speculate that some oswd genes might be in relation with each other by controlling the expression of these transcription factors. another oswd gene that attracted our attention was oswd40- which was co-expressed with another group of myb and bhlh transcription factors that were different from the co-expression genes mentioned above. we also found that oswd40- might be related with other partners such as genes in set family, hdac family, and hac. this result suggested that oswd40- might participate in the regulation of another pathway. a general wd/myb/bhlh complex for regulation of the anthocyanin biosynthetic pathway was also found in antirrhinum majus, petunia hybrida and arabidopsis thaliana. meanwhile, a bhlh protein lc in maize was found to interact with myb transcription factors to activate anthocyanin expression. another lc-like bhlh protein was also found to require a myb protein to perform its function. results suggested that many mybs interacted directly with lc-like bhlh proteins and the wd repeat protein. therefore, it seems that wd proteins allow protein-protein interactions between the bhlh and myb proteins, and wd proteins in rice might also require mybs and bhlhs to form a transcription complex to participate a range of pathways. oswd genes may be involved in histone-related functions with members in set family histone expression and histone post-translational modifications play pivotal roles in chromatin remodeling and epigenetic regulation in plant development. our co-expression analysis has revealed that oswd genes may function with histone-related proteins. although the exact pathways mediate by these genes are still unclear, one might speculate that these oswd genes play important roles in histone modification. an important group of enzymes involved in histone modification is the histone-lysine n-methyltransferases. these proteins participate in the establishment and/or maintenance of euchromatic or heterochromatic states of active or transcriptionally repressed sequences. here, a total of histone-lysine n-methyltransferases were identified to be co-expressed with the oswd proteins, both of which contain the set domain that is responsible for the catalytic activity of the enzymes, suggesting possible interactions between the oswd and set genes in a family level. one might also tentatively speculate that the wd and set domain must be the key functional structure for interaction by a conserved mechanism. functional studies of several arabidopsis genes encoding wd proteins also suggest that they might be implicated in histone modification in different pathways. a wd domain cyclophilin, cyclophilin, which functions in gene repression and organogenesis in arabidopsis, serves as a highly conserved histone remodeling factor involved in chromatin-based gene silencing. another wd protein msi in arabidopsis has also been proposed to exhibit pleiotropic phenotypes by epigenetic regulation. therefore, oswd genes in rice might also involved in similar pathways by histone modulation. in a word, characterization of oswd proteins function in histone modification could therefore open new perspectives for understanding the molecular mechanism of epigenetic regulation. oswd genes may take part in reproductive pathways with mads-box transcription factors wd genes identified in different plant species are involved in various developmental processes. in our study, the expression patterns of oswd genes and the co-expression analysis provide useful information for establishing their putative functions. the available evidence suggests that oswd genes may take part in reproductive pathways with mads-box transcription factors in rice. mads-box transcription factors are essential for various aspects of pathways in flower development both in dicotyledon and monocotyledon. our result suggested that oswd40- was co-expressed with three mads-box transcription factors, os03g os06g and os01g. oswd40- was homologous to arabidopsis fve/msi a key regulator that interacted with cul4-ddb and a prc2-like complex to control epigenetic regulation of flowering time. it was also reported that os03g was required for rice inflorescence and spikelet development. therefore, it would be interesting to investigate whether oswd40- play roles in rice flower development with mads-box transcription factors. we also found that three oswd genes, oswd40- and were co-expressed with a mads gene os04g these four genes were expressed preferentially in stamen, suggesting that they may function in stamen development. all these studies support the results that mads-box transcription factors are essential for flower developmental processes in relationship with oswd genes.  in conclusion, using an in silico approach, a total of oswd genes were found to be present in rice genome. genomic framework revealed the potential mechanisms responsible for the evolution of oswd genes in rice. the expression profiling of oswd gene family covering rice life cycle could provide deep insights into their potential functions during rice growth and development. some genes appear to be differentially expressed in different tissues/organs, vegetative and reproductive development stages, and expression of some genes is influenced under phytohormones. these data will provide the basis for understanding the evolutionary history of oswd members and their roles in rice growth and development. the findings in our work would be useful in selecting candidate genes for functional studies of oswd members in rice. however, future research by adopting transformation strategies or insertion mutagenesis is required to elucidate the precise functions of these oswd genes. 
the results suggested that oswd genes may perform their diverse functions by complex network, thus were predictive for understanding their biological pathways. the analysis also revealed that oswd genes might interact with each other to take part in metabolic pathways, suggesting a more complex feedback network. all of these analyses suggest that the functions of oswd genes are diversified, which provide useful references for selecting candidate genes for further functional studies. systematic characterization and co-expression analysis of oswd genes enable us to understand the networks of the wd proteins and their biological processes and gene functions in rice. we also used a combined analysis of expression correlation and gene ontology annotation to infer the biological role of the oswd genes in rice. under phytohormone treatments, oswd genes were differentially expressed with treatments of one or more of the phytohormone naa, kt, or ga in rice seedlings. in this study, we identify and analyze potential oswd genes in rice, describing their gene structures, genome localizations, and evolutionary relationship of each member. expression profiles covering the whole life cycle in rice has revealed that transcripts of oswd were accumulated differentially during vegetative and reproductive development and preferentially up or down-regulated in different tissues. oryza sativaexpression profilesmicroarraywd geneco-expression network
 wd proteins represent a large family in eukaryotes, which have been involved in a broad spectrum of crucial functions. systematic characterization and co-expression analysis of oswd genes enable us to understand the networks of the wd proteins and their biological processes and gene functions in rice.  in this study, we identify and analyze potential oswd genes in rice, describing their gene structures, genome localizations, and evolutionary relationship of each member. expression profiles covering the whole life cycle in rice has revealed that transcripts of oswd were accumulated differentially during vegetative and reproductive development and preferentially up or down-regulated in different tissues. under phytohormone treatments, oswd genes were differentially expressed with treatments of one or more of the phytohormone naa, kt, or ga in rice seedlings. we also used a combined analysis of expression correlation and gene ontology annotation to infer the biological role of the oswd genes in rice. the results suggested that oswd genes may perform their diverse functions by complex network, thus were predictive for understanding their biological pathways. the analysis also revealed that oswd genes might interact with each other to take part in metabolic pathways, suggesting a more complex feedback network.  all of these analyses suggest that the functions of oswd genes are diversified, which provide useful references for selecting candidate genes for further functional studies. oryza sativaexpression profilesmicroarraywd geneco-expression network 