BACKGROUND
designing expression cassettes allowing a precise control of where, when and at which level transcription should occur may ultimately be achieved through synthetic promoter engineering  <cit> .
the basic building blocks for such promoters are regions of cis-regulatory dna, which in eukaryotes often comprise clusters of cis-regulatory elements   bound by a combination of transcription factors .
the unraveling of eukaryotic transcriptional regulation is a challenging area of research driving the synergetic development of experimental and computational techniques  <cit> .
cis-regulatory motifs of plant promoters have commonly been delineated by the experimental manipulation of dna segments and reporter gene expression assays  <cit> .
plant cis-regulatory motifs are often reported as consensus sequences, a motif model of limited predictive power  <cit> .
collections of experimentally characterized plant cis-regulatory elements sequences such as the place database  <cit>  nevertheless remain an invaluable resource e.g.
for annotating motifs discovered in sequences that have not been characterized experimentally.
the majority of contemporary computational approaches for the discovery of cis-regulatory elements  <cit>  use the position weight matrix  motif model, based on the frequencies of nucleotides at each position in a collection of regulatory elements.
the seeder dna motif discovery algorithm, designed for fast and reliable prediction of cis-regulatory elements in eukaryotic promoters, uses a string-based approach to identify motifs that are statistically significant  in a set of positive sequences as compared to a background set of sequences and it was recently shown to outperform some popular motif discovery tools on biological benchmark data  <cit> .
the maturation of plant seeds, and more specifically protein storage in seeds, is regulated by a combination of hormonal, genetic and metabolic controls  <cit> .
in arabidopsis, four master regulators of seed maturation have been identified including three tfs of the b <dig> dna-binding domain family, namely abscisic acid insensitive <dig> , fusca <dig>  and leafy cotyledon <dig> , and a hap <dig> subunit of the ccaat-box binding transcription factor   <cit> .
known dicotyledonous seed maturation regulatory motifs include the ry motif and the acgt motif, which are targets of b <dig> and bzip transcription factors respectively  <cit> .
in rapeseed , a comprehensive analysis of the napa promoter revealed the presence of two regulatory element complexes, the b-box which contains the distb element  together with the proxb element , and the ry/g complex which contains two ry repeats  and one g-box   <cit> .
in bean , a comprehensive promoter analysis was performed on the phas promoter by chandrasekharan et al.
<cit> .
the site-directed substitution mutations analysis within the - <dig> region of the phas promoter revealed that the g-box, the ccaaat box, the e-box  and ry elements mediate levels of expression in embryos  <cit> .
several studies have shown that motifs conferring seed-specific expression reside in the proximal region of the promoter, often within  <dig> bp upstream of the transcriptional start .
the analysis of prolamin gene promoters from barley , wheat  and maize  uncovered a conserved ~ <dig> base pairs  conserved sequence containing two cres, the gcn4-like  element  , and the prolamin-box    <cit> .
an additional element called aaca  was further found to be involved in the seed-specific regulation of rice  glutelin genes  <cit> .
these three cres  are frequently found in monocotyledonous ssp gene promoters and are bound by tfs of the bzip, dof and myb families, respectively  <cit> .
in this work, we performed de novo motif discovery in  <dig> ssp gene promoters from brassicaceae, fabaceae and poaceae using discriminative seeding dna motif discovery, and uncovered the presence of family-specific conserved motifs, the validity of which was corroborated by matching to experimentally characterized plant seed-specific cres.
furthermore, we show that the discovered motifs constitute signatures of ssp gene promoters in the different species.
RESULTS
seed storage protein gene promoters contain conserved motifs
seed storage protein gene promoter sequences  from brassicaceae , fabaceae  and poaceae  were retrieved from public sequence databases.
discriminative seeding dna motif discovery  <cit>  was performed separately in each of the three plant families using a background model based on the complete set of promoters from a representative species, namely arabidopsis , soybean  and rice .
statistically significant conserved cis-regulatory motifs  were identified in ssp gene promoter sequences within each plant family.
discovered motifs were matched to consensus sequences of experimentally characterized plant cis-regulatory elements from the place database  <cit>  using the stamp suite of tools  <cit>  .
q-value, statistical significance of motif
place id, identifier of place consensus sequence matching motif
stamp alignment, alignment of motif consensus sequence  with place consensus sequence
e value, expectation value of the stamp alignment
five significant motifs were found in the fabaceae ssp gene promoters, including two ry-like motifs and one acgt-like motif .
motif f <dig> is a tata-box motif and is discussed below.
the fourth motif discovered  is possibly related to the e2fb motif  found in the tobacco  ribonucleotide reductase  <dig>  gene promoter  <cit> .
the fabaceae e2fb-like motif  does not have similarity to any known plant seed-specific cis-regulatory elements; it is thus a novel putative ssp gene promoter cis-regulatory motif.
motifs enriched in the promoters of poaceae ssp genes  are distinct from those observed in the two other plant families.
the first motif discovered  is most similar to the gcn4-like motif .
the second motif  is similar to a variant of the prolamin-box motif  found in a rice glutelin promoter  <cit> .
this sequence has also been suggested to act as a prolamin-box variant in a wheat glutenin promoter  <cit> .
the third motif  is a strong match to the typical prolamin-box .
motif p <dig> is a tata-box motif and is discussed below.
the fifth motif  has some core similarity with a rice bell homeodomain transcription factor binding site  <cit> .
it is also similar to an skn-1-like motif identified in a rice glutelin gene promoter  <cit> .
motif p <dig> is related to the gcaa motif found in a maize zein promoter  <cit> .
motif p <dig> does not have similarity to any known monocotyledonous seed promoter motif but is weakly related to an opaque- <dig> recognition site  <cit> .
seed storage protein gene promoters contain tata-box motifs
the third motif discovered in fabaceae , and the fourth motif discovered in poaceae ssp gene promoters , are highly similar to a tata-box motif .
in fabaceae ssp gene promoters, the best matching subsequences to the tata-box motif  are localized between positions - <dig> to - <dig> upstream of the transcription start site .
no tata-box motif was initially discovered in brassicacea ssp gene promoters.
to investigate whether brassicaceae ssp gene promoters also contain a tata-box motif, we searched the brassicaceae promoter sequences with the tata-box motif found in fabaceae .
scoring promoter sequences with the f <dig> motif's pwm returned a highly similar tata-box motif .
in both brassicaceae and fabaceae, most best matching subsequences to the tata-box motif are also localized approximately - <dig> to - <dig> upstream of the transcriptional start .
some seed storage regulatory motifs are highly localized
the position of the best matching subsequences to discovered motifs  in promoter sequences, identified by the seeder algorithm  <cit> , is illustrated in figure  <dig>  the distribution of best matching subsequence positions  is represented in additional file  <dig>  several patterns emerge from this map:  the tata-box motif is highly localized to positions approx.
between - <dig> to - <dig> upstream of the transcriptional start in brassicaceae, fabaceae and poaceae ssp promoters;  brassicaceae and fabaceae ssp promoters have one ry motif localized in close proximity upstream of the tata-box, and one additional ry motif and one acgt motif at variable position upstream of the tata-box;  poaceae ssp promoters are characterized by one glm, two p-box, one skn- <dig> and one gcaa motifs scattered at variable positions upstream of the transcriptional start.
the combination of fabaceae seed storage motifs is a signature of seed storage protein gene promoters in the soybean genome
the recently sequenced soybean genome is predicted to contain over  <dig>  protein-coding genes .
this publicly available genome sequence set was used to retrieve  <dig>  promoter sequences.
we used the fabaceae pwms  to identify the best matching promoter sequences from the soybean genome by a pwm scoring and sequence matching strategy.
in order to assign a function to the genes whose promoters were enriched in these five motifs, we manually annotated the top-ten matching gene sequences from the genome.
the translated gene sequences corresponding to the top-ten scoring promoters were aligned with the swiss-prot database  using the smith-waterman algorithm.
all of the top-scoring promoters are associated with soybean genes coding for different subunits of glycinin, β-conglycinin or 7s globulin .
similar results were obtained in arabidopsis and rice , where eight out of the top-ten scoring arabidopsis promoters are associated with ssp genes and the top-ten scoring rice promoters are all associated with ssp genes.
gene id, gene identifier
pwm rank, total pwm matching score rank
c-b rank, total cluster-buster score rank
hit id, hit identifier
description, hit description
e value, hit alignment expectation value.
the promoters of soybean genes coding for different seed storage protein subunits vary in motif composition
although genes coding for different soybean ssp subunits have been shown to be expressed specifically in seeds during maturation, some subunits are differentially expressed   <cit> .
we investigated whether there were also differences in promoter motif composition.
soybean major ssp sequences from the swiss-prot database, namely glycinin subunits   <cit> , β-conglycinin subunits   <cit>  and basic 7s globulins  <cit> , were aligned against all soybean predicted peptides .
we identified  <dig> soybean peptide sequences with high similarity , and an additional two sequences with moderate similarity .
figure  <dig> shows the pwm scores for each fabaceae ssp promoter motif in soybean ssp gene promoters compared with a baseline .
the promoters of genes coding respectively for glycinins gy <dig> , gy <dig> , gy <dig> , gy <dig>  and gy <dig>  scored relatively high  for the presence of fabaceae ssp gene promoter motifs.
the promoters of all genes coding for the β-conglycinin subunits, namely α' , α  and β  were among the top- <dig> scoring promoters  out of the  <dig>  soybean promoters.
the promoters of the gene coding for the basic 7s globulin  <dig>  was also among the top-ten promoters , while that of the gene coding for the basic 7s globulin  <dig>  scored lower .
the products of two genes flanking gene glyma <dig> g <dig>  on chromosome  <dig>  are equivalently good matches to the three β-conglycinin subunits  , making a precise annotation difficult for those two genes.
interestingly, the promoter of glyma <dig> g <dig>  scored very low  while that of glyma <dig> g <dig>  was among the top- <dig> scoring promoters .
discussion
we have applied the seeder discriminative dna motif discovery algorithm to an in-depth analysis of ssp gene promoters from brassicaceae, fabaceae and poaceae.
most discovered motifs match experimentally characterized cis-regulatory element consensus sequences, which strongly supports the validity of the discovered motifs.
the analysis of brassicaceae ssp gene promoters highlighted the presence of three significant motifs corresponding to two ry motifs and one acgt motif.
it is interesting to contrast this result with that obtained from the analysis of promoters of arabidopsis seed-specific marker genes where one ry motif and one acgt motif were significantly enriched  <cit> .
the three motifs match components of the ry/g complex experimentally characterized in the rapeseed napa promoter  <cit> .
the analysis of brassicaceae ssp gene promoter sequences using the seeder algorithm did not initially reveal enrichment in a tata-box motif.
this could be explained by the proportion of promoters containing a tata-box in the background set of sequences, or by the relatively low complexity of tata-box motifs which makes them hard to discriminate from background, particularly if we take into account the fact that promoter sequences are generally a/t rich  <cit> .
we used a pwm corresponding to a putative fabaceae tata-box motif to retrieve, in brassicaceae ssp gene promoter sequences, a motif highly localized around position - <dig> to - <dig> relative to the transcriptional start site.
the localization, the information content of the motif and the fact that it is very similar to tata-box motifs found in fabaceae and poaceae ssp gene promoters suggest that this motif indeed corresponds to a brassicaceae ssp gene promoter tata-box motif, in accordance with reported occurrences of tata-box motifs in the promoters of e.g.
napa and napb  <cit> .
fabaceae ssp gene promoters have also revealed enrichment in two ry motifs.
the ry motif has long been known to be conserved in legume seed-protein gene promoters  <cit>  and ry cres have been proven to be functional e.g.
in soybean  <cit>  and broad bean   <cit> .
a novel, e2fb-like motif was discovered in fabaceae ssp gene promoters.
e2f transcription factors are involved in the control of cell cycle  <cit> .
the role of this e2fb-like motif in seed-specific gene expression will require further experimental verification.
position weight matrices corresponding to motifs discovered in fabaceae, arabidopsis and rice ssp gene promoters were used to score the respective whole genome sets of promoter sequences.
the top-ten scoring promoters are associated with ssp-coding genes in soybean and rice, as are eight out of the top-ten scoring promoters in arabidopsis.
this combination of a few motifs is thus sufficient to constitute a signature of ssp gene promoters.
the fact that the promoter of some soybean genes coding for ssp protein subunits did score relatively low to the combination of fabaceae ssp gene promoter motifs may indicate alternative regulatory mechanisms for those genes.
furthermore, the promoters of other soybean ssp protein genes such as those coding for albumin- <dig>  and 2s albumin  did also score relatively low  and could be regulated by a different set of tfs.
in soybean, experimental ssp gene promoter analyses have focused on the α' and β subunits of β-conglycinin  <cit> .
experimental analyses have revealed the importance of the proximal region  and the presence of several factors binding the promoters  and the presence of a ry cis-regulatory element.
the study by fujiwara and beachy  <cit>  disproved a cis-regulatory role for the binding sites of sef <dig> and sef <dig> located within the proximal promoter and confirmed the role of the ry element in seed-specific gene regulation.
the work by yoshino et al.
<cit>  on the promoter of the α subunit of β-conglycinin also suggests a role for ry elements in seed-specific gene regulation.
the promoters of genes coding for glycinin subunits gy <dig> and gy <dig> have also been analyzed experimentally  <cit>  yet although an a/t-rich sef-binding sequence has been identified, the only clearly confirmed cis-regulatory element therein is a ry element.
our results suggest that soybean ssp promoters may be characterized by four cis-regulatory motifs, in addition to a tata-box motif.
motifs enriched in the promoters of poaceae ssp genes were all good matches to experimentally characterized plant seed-specific cis-regulatory elements including a glm motif, two prolamin-box-like motifs, a skn-1-like motif and a tata-box motif.
a recent study  <cit>  has identified a barley protein homologous to the arabidopsis fusca <dig> that regulates ssp genes and binds ry boxes; this was the first report of a possible implication of the ry motif in seed-specific gene regulation in a monocotyledonous plant species.
our computational analysis did not reveal significant enrichment in ry motifs among poaceae ssp gene promoters.
this however does not necessarily refute a possible role for b3-type transcription factors and ry-like elements in the transcriptional regulation of some poaceae ssp genes, which could be an attribute of a limited number of genes only, and not a general feature of poaceae ssp gene promoters.
on the other hand, motifs containing the aaag core of dof transcription factor binding sites  <cit>  were found only in poaceae ssp gene promoters.
soybean dof-type transcription factor have been reported to be involved in the regulation of the lipid content in soybean seeds  <cit> , and a prolamin-box motif has been reported in pea   <cit> .
however, prolamin-box motifs have been reported mostly in poaceae promoters .
indeed, our results suggest that prolamin-box-like motifs are conserved in poaceae ssp gene promoters, but are not featured in brassicaceae or fabaceae ssp gene promoters.
CONCLUSIONS
presented results highlight motifs that are conserved in ssp gene promoters within three plant families.
promoter/motif combinations generated in this analysis can be further validated experimentally, e.g.
in a framework such as that used by  <cit> .
most motifs conserved in ssp gene promoters have a high degree of similarity with experimentally characterized cis-regulatory elements; this is an indicator that they are indeed functional in seed-specific gene regulation.
the same methodology can be applied to analyze various data sets and decipher transcriptional regulation mechanisms in plants and other eukaryotes.

 designing expression cassettes allowing a precise control of where, when and at which level transcription should occur may ultimately be achieved through synthetic promoter engineering. the basic building blocks for such promoters are regions of cis-regulatory dna, which in eukaryotes often comprise clusters of cis-regulatory elements bound by a combination of transcription factors. the unraveling of eukaryotic transcriptional regulation is a challenging area of research driving the synergetic development of experimental and computational techniques. cis-regulatory motifs of plant promoters have commonly been delineated by the experimental manipulation of dna segments and reporter gene expression assays. plant cis-regulatory motifs are often reported as consensus sequences, a motif model of limited predictive power. collections of experimentally characterized plant cis-regulatory elements sequences such as the place database nevertheless remain an invaluable resource e.g. for annotating motifs discovered in sequences that have not been characterized experimentally. the majority of contemporary computational approaches for the discovery of cis-regulatory elements use the position weight matrix motif model, based on the frequencies of nucleotides at each position in a collection of regulatory elements. the seeder dna motif discovery algorithm, designed for fast and reliable prediction of cis-regulatory elements in eukaryotic promoters, uses a string-based approach to identify motifs that are statistically significant in a set of positive sequences as compared to a background set of sequences and it was recently shown to outperform some popular motif discovery tools on biological benchmark data. the maturation of plant seeds, and more specifically protein storage in seeds, is regulated by a combination of hormonal, genetic and metabolic controls. in arabidopsis, four master regulators of seed maturation have been identified including three tfs of the b dna-binding domain family, namely abscisic acid insensitive, fusca and leafy cotyledon, and a hap subunit of the ccaat-box binding transcription factor. known dicotyledonous seed maturation regulatory motifs include the ry motif and the acgt motif, which are targets of b and bzip transcription factors respectively. in rapeseed, a comprehensive analysis of the napa promoter revealed the presence of two regulatory element complexes, the b-box which contains the distb element together with the proxb element, and the ry/g complex which contains two ry repeats and one g-box. in bean, a comprehensive promoter analysis was performed on the phas promoter by chandrasekharan et al.. the site-directed substitution mutations analysis within the - region of the phas promoter revealed that the g-box, the ccaaat box, the e-box and ry elements mediate levels of expression in embryos. several studies have shown that motifs conferring seed-specific expression reside in the proximal region of the promoter, often within bp upstream of the transcriptional start. the analysis of prolamin gene promoters from barley, wheat and maize uncovered a conserved ~ base pairs conserved sequence containing two cres, the gcn4-like element, and the prolamin-box. an additional element called aaca was further found to be involved in the seed-specific regulation of rice glutelin genes. these three cres are frequently found in monocotyledonous ssp gene promoters and are bound by tfs of the bzip, dof and myb families, respectively. in this work, we performed de novo motif discovery in ssp gene promoters from brassicaceae, fabaceae and poaceae using discriminative seeding dna motif discovery, and uncovered the presence of family-specific conserved motifs, the validity of which was corroborated by matching to experimentally characterized plant seed-specific cres. furthermore, we show that the discovered motifs constitute signatures of ssp gene promoters in the different species.  seed storage protein gene promoters contain conserved motifs seed storage protein gene promoter sequences from brassicaceae, fabaceae and poaceae were retrieved from public sequence databases. discriminative seeding dna motif discovery was performed separately in each of the three plant families using a background model based on the complete set of promoters from a representative species, namely arabidopsis, soybean and rice. statistically significant conserved cis-regulatory motifs were identified in ssp gene promoter sequences within each plant family. discovered motifs were matched to consensus sequences of experimentally characterized plant cis-regulatory elements from the place database using the stamp suite of tools. q-value, statistical significance of motif place id, identifier of place consensus sequence matching motif stamp alignment, alignment of motif consensus sequence with place consensus sequence e value, expectation value of the stamp alignment five significant motifs were found in the fabaceae ssp gene promoters, including two ry-like motifs and one acgt-like motif. motif f is a tata-box motif and is discussed below. the fourth motif discovered is possibly related to the e2fb motif found in the tobacco ribonucleotide reductase gene promoter. the fabaceae e2fb-like motif does not have similarity to any known plant seed-specific cis-regulatory elements; it is thus a novel putative ssp gene promoter cis-regulatory motif. motifs enriched in the promoters of poaceae ssp genes are distinct from those observed in the two other plant families. the first motif discovered is most similar to the gcn4-like motif. the second motif is similar to a variant of the prolamin-box motif found in a rice glutelin promoter. this sequence has also been suggested to act as a prolamin-box variant in a wheat glutenin promoter. the third motif is a strong match to the typical prolamin-box. motif p is a tata-box motif and is discussed below. the fifth motif has some core similarity with a rice bell homeodomain transcription factor binding site. it is also similar to an skn-1-like motif identified in a rice glutelin gene promoter. motif p is related to the gcaa motif found in a maize zein promoter. motif p does not have similarity to any known monocotyledonous seed promoter motif but is weakly related to an opaque- recognition site. seed storage protein gene promoters contain tata-box motifs the third motif discovered in fabaceae, and the fourth motif discovered in poaceae ssp gene promoters, are highly similar to a tata-box motif. in fabaceae ssp gene promoters, the best matching subsequences to the tata-box motif are localized between positions - to - upstream of the transcription start site. no tata-box motif was initially discovered in brassicacea ssp gene promoters. to investigate whether brassicaceae ssp gene promoters also contain a tata-box motif, we searched the brassicaceae promoter sequences with the tata-box motif found in fabaceae. scoring promoter sequences with the f motif's pwm returned a highly similar tata-box motif. in both brassicaceae and fabaceae, most best matching subsequences to the tata-box motif are also localized approximately - to - upstream of the transcriptional start. some seed storage regulatory motifs are highly localized the position of the best matching subsequences to discovered motifs in promoter sequences, identified by the seeder algorithm, is illustrated in figure the distribution of best matching subsequence positions is represented in additional file several patterns emerge from this map: the tata-box motif is highly localized to positions approx. between - to - upstream of the transcriptional start in brassicaceae, fabaceae and poaceae ssp promoters; brassicaceae and fabaceae ssp promoters have one ry motif localized in close proximity upstream of the tata-box, and one additional ry motif and one acgt motif at variable position upstream of the tata-box; poaceae ssp promoters are characterized by one glm, two p-box, one skn- and one gcaa motifs scattered at variable positions upstream of the transcriptional start. the combination of fabaceae seed storage motifs is a signature of seed storage protein gene promoters in the soybean genome the recently sequenced soybean genome is predicted to contain over protein-coding genes. this publicly available genome sequence set was used to retrieve promoter sequences. we used the fabaceae pwms to identify the best matching promoter sequences from the soybean genome by a pwm scoring and sequence matching strategy. in order to assign a function to the genes whose promoters were enriched in these five motifs, we manually annotated the top-ten matching gene sequences from the genome. the translated gene sequences corresponding to the top-ten scoring promoters were aligned with the swiss-prot database using the smith-waterman algorithm. all of the top-scoring promoters are associated with soybean genes coding for different subunits of glycinin, β-conglycinin or 7s globulin. similar results were obtained in arabidopsis and rice, where eight out of the top-ten scoring arabidopsis promoters are associated with ssp genes and the top-ten scoring rice promoters are all associated with ssp genes. gene id, gene identifier pwm rank, total pwm matching score rank c-b rank, total cluster-buster score rank hit id, hit identifier description, hit description e value, hit alignment expectation value. the promoters of soybean genes coding for different seed storage protein subunits vary in motif composition although genes coding for different soybean ssp subunits have been shown to be expressed specifically in seeds during maturation, some subunits are differentially expressed. we investigated whether there were also differences in promoter motif composition. soybean major ssp sequences from the swiss-prot database, namely glycinin subunits, β-conglycinin subunits and basic 7s globulins, were aligned against all soybean predicted peptides. we identified soybean peptide sequences with high similarity, and an additional two sequences with moderate similarity. figure shows the pwm scores for each fabaceae ssp promoter motif in soybean ssp gene promoters compared with a baseline. the promoters of genes coding respectively for glycinins gy, gy, gy, gy and gy scored relatively high for the presence of fabaceae ssp gene promoter motifs. the promoters of all genes coding for the β-conglycinin subunits, namely α', α and β were among the top- scoring promoters out of the soybean promoters. the promoters of the gene coding for the basic 7s globulin was also among the top-ten promoters, while that of the gene coding for the basic 7s globulin scored lower. the products of two genes flanking gene glyma g on chromosome are equivalently good matches to the three β-conglycinin subunits, making a precise annotation difficult for those two genes. interestingly, the promoter of glyma g scored very low while that of glyma g was among the top- scoring promoters. discussion we have applied the seeder discriminative dna motif discovery algorithm to an in-depth analysis of ssp gene promoters from brassicaceae, fabaceae and poaceae. most discovered motifs match experimentally characterized cis-regulatory element consensus sequences, which strongly supports the validity of the discovered motifs. the analysis of brassicaceae ssp gene promoters highlighted the presence of three significant motifs corresponding to two ry motifs and one acgt motif. it is interesting to contrast this result with that obtained from the analysis of promoters of arabidopsis seed-specific marker genes where one ry motif and one acgt motif were significantly enriched. the three motifs match components of the ry/g complex experimentally characterized in the rapeseed napa promoter. the analysis of brassicaceae ssp gene promoter sequences using the seeder algorithm did not initially reveal enrichment in a tata-box motif. this could be explained by the proportion of promoters containing a tata-box in the background set of sequences, or by the relatively low complexity of tata-box motifs which makes them hard to discriminate from background, particularly if we take into account the fact that promoter sequences are generally a/t rich. we used a pwm corresponding to a putative fabaceae tata-box motif to retrieve, in brassicaceae ssp gene promoter sequences, a motif highly localized around position - to - relative to the transcriptional start site. the localization, the information content of the motif and the fact that it is very similar to tata-box motifs found in fabaceae and poaceae ssp gene promoters suggest that this motif indeed corresponds to a brassicaceae ssp gene promoter tata-box motif, in accordance with reported occurrences of tata-box motifs in the promoters of e.g. napa and napb. fabaceae ssp gene promoters have also revealed enrichment in two ry motifs. the ry motif has long been known to be conserved in legume seed-protein gene promoters and ry cres have been proven to be functional e.g. in soybean and broad bean. a novel, e2fb-like motif was discovered in fabaceae ssp gene promoters. e2f transcription factors are involved in the control of cell cycle. the role of this e2fb-like motif in seed-specific gene expression will require further experimental verification. position weight matrices corresponding to motifs discovered in fabaceae, arabidopsis and rice ssp gene promoters were used to score the respective whole genome sets of promoter sequences. the top-ten scoring promoters are associated with ssp-coding genes in soybean and rice, as are eight out of the top-ten scoring promoters in arabidopsis. this combination of a few motifs is thus sufficient to constitute a signature of ssp gene promoters. the fact that the promoter of some soybean genes coding for ssp protein subunits did score relatively low to the combination of fabaceae ssp gene promoter motifs may indicate alternative regulatory mechanisms for those genes. furthermore, the promoters of other soybean ssp protein genes such as those coding for albumin- and 2s albumin did also score relatively low and could be regulated by a different set of tfs. in soybean, experimental ssp gene promoter analyses have focused on the α' and β subunits of β-conglycinin. experimental analyses have revealed the importance of the proximal region and the presence of several factors binding the promoters and the presence of a ry cis-regulatory element. the study by fujiwara and beachy disproved a cis-regulatory role for the binding sites of sef and sef located within the proximal promoter and confirmed the role of the ry element in seed-specific gene regulation. the work by yoshino et al. on the promoter of the α subunit of β-conglycinin also suggests a role for ry elements in seed-specific gene regulation. the promoters of genes coding for glycinin subunits gy and gy have also been analyzed experimentally yet although an a/t-rich sef-binding sequence has been identified, the only clearly confirmed cis-regulatory element therein is a ry element. our results suggest that soybean ssp promoters may be characterized by four cis-regulatory motifs, in addition to a tata-box motif. motifs enriched in the promoters of poaceae ssp genes were all good matches to experimentally characterized plant seed-specific cis-regulatory elements including a glm motif, two prolamin-box-like motifs, a skn-1-like motif and a tata-box motif. a recent study has identified a barley protein homologous to the arabidopsis fusca that regulates ssp genes and binds ry boxes; this was the first report of a possible implication of the ry motif in seed-specific gene regulation in a monocotyledonous plant species. our computational analysis did not reveal significant enrichment in ry motifs among poaceae ssp gene promoters. this however does not necessarily refute a possible role for b3-type transcription factors and ry-like elements in the transcriptional regulation of some poaceae ssp genes, which could be an attribute of a limited number of genes only, and not a general feature of poaceae ssp gene promoters. on the other hand, motifs containing the aaag core of dof transcription factor binding sites were found only in poaceae ssp gene promoters. soybean dof-type transcription factor have been reported to be involved in the regulation of the lipid content in soybean seeds, and a prolamin-box motif has been reported in pea. however, prolamin-box motifs have been reported mostly in poaceae promoters. indeed, our results suggest that prolamin-box-like motifs are conserved in poaceae ssp gene promoters, but are not featured in brassicaceae or fabaceae ssp gene promoters.  presented results highlight motifs that are conserved in ssp gene promoters within three plant families. promoter/motif combinations generated in this analysis can be further validated experimentally, e.g. in a framework such as that used by. most motifs conserved in ssp gene promoters have a high degree of similarity with experimentally characterized cis-regulatory elements; this is an indicator that they are indeed functional in seed-specific gene regulation. the same methodology can be applied to analyze various data sets and decipher transcriptional regulation mechanisms in plants and other eukaryotes. 
conserved motifs identified in poaceae ssp gene promoters include a gcn4-like motif, two prolamin-box-like motifs and an skn-1-like motif. we have used a discriminative seeding dna motif discovery algorithm for an in-depth analysis of seed storage protein gene promoters from three plant families, namely brassicaceae, fabaceae and poaceae using backgrounds based on complete sets of promoters from a representative species in each family, namely arabidopsis heynh. evidence of the presence of a variant of the tata-box is found in the ssp gene promoters from the three plant families. seed storage protein gene promoter motifs are conserved in diverse species, and different plant families are characterized by a distinct combination of conserved motifs. we have identified three conserved motifs in brassicaceae and fabaceae ssp gene promoters that are similar to experimentally characterized seed-specific cis-regulatory elements. the majority of discovered motifs match experimentally characterized cis-regulatory elements. these results provide a good starting point for further experimental analysis of plant seed-specific promoters and our methodology can be used to unravel more transcriptional regulatory mechanisms in plants and other eukaryotes. the highest-scoring promoters are associated with genes coding for different subunits or precursors of seed storage proteins. accurate computational identification of cis-regulatory motifs is difficult, particularly in eukaryotic promoters, which typically contain multiple short and degenerate dna sequences bound by several interacting factors. this provides a basis for the computational identification of cis-regulatory motifs. motifs discovered in ssp gene promoters were used to score whole-genome sets of promoters from arabidopsis, soybean and rice.
 accurate computational identification of cis-regulatory motifs is difficult, particularly in eukaryotic promoters, which typically contain multiple short and degenerate dna sequences bound by several interacting factors. enrichment in combinations of rare motifs in the promoter sequence of functionally or evolutionarily related genes among several species is an indicator of conserved transcriptional regulatory mechanisms. this provides a basis for the computational identification of cis-regulatory motifs.  we have used a discriminative seeding dna motif discovery algorithm for an in-depth analysis of seed storage protein gene promoters from three plant families, namely brassicaceae, fabaceae and poaceae using backgrounds based on complete sets of promoters from a representative species in each family, namely arabidopsis heynh.), soybean merr.) and rice respectively. we have identified three conserved motifs in brassicaceae and fabaceae ssp gene promoters that are similar to experimentally characterized seed-specific cis-regulatory elements. fabaceae ssp gene promoter sequences are also enriched in a novel, seed-specific e2fb-like motif. conserved motifs identified in poaceae ssp gene promoters include a gcn4-like motif, two prolamin-box-like motifs and an skn-1-like motif. evidence of the presence of a variant of the tata-box is found in the ssp gene promoters from the three plant families. motifs discovered in ssp gene promoters were used to score whole-genome sets of promoters from arabidopsis, soybean and rice. the highest-scoring promoters are associated with genes coding for different subunits or precursors of seed storage proteins.  seed storage protein gene promoter motifs are conserved in diverse species, and different plant families are characterized by a distinct combination of conserved motifs. the majority of discovered motifs match experimentally characterized cis-regulatory elements. these results provide a good starting point for further experimental analysis of plant seed-specific promoters and our methodology can be used to unravel more transcriptional regulatory mechanisms in plants and other eukaryotes. 