BACKGROUND
the determination of complete genome sequences has allowed analysis by various statistical methods that have furthered understanding of the function of genomes.
analysis of promoter structure is one of the most important issues.
understanding of promoter structure allows predictions concerning promoter positions and expression profiles, and sheds light on hidden transcriptional networks.
several functional elements have been identified as promoter constituents for precise and regulated transcriptional initiation: tata box, initiator  motif, downstream promoter element , tfiib-recognition element , and so-called cis-regulatory elements  <cit> .
in addition, some mammalian promoters are associated with cpg islands  <cit> , which is related to the sp <dig> recognition site  <cit>  and have some relationship with gene regulation by dna-methylation  <cit> .
human transcriptional regulatory elements are reported to make clusters  at the promoter region as well as the 3' end of a gene  <cit> .
transcription start sites  in plant promoters have a cg-compositional strand bias, or gc-skew, where c is more frequently observed in the  strand than g  <cit> .
some of these features are well understood and some are not, but all these features are useful to understand individual promoters.
some of the above features have been utilized for promoter prediction  <cit> .
although these studies obtain certain success, our current knowledge of promoters is still insufficient  <cit> .
availability of microarray data on co-regulated gene expression on a genomic scale has enabled the prediction of novel cis-elements involved in gene regulation.
several approaches have been developed for this detection of consensus sequences in a co-regulated promoter set , and detection of over-represented sequence in co-regulated promoters with a set of reference sequences  <cit> .
these approaches are also applicable to chromatin immunoprecipitation  data  <cit> .
in addition, identification of conserved promoter sequences by comparative genomics supports the prediction of regulatory elements  <cit> .
studies on plant transcription factors and functional cis-regulatory elements have been summarized in several databases, and the collective information of cis-elements and/or transfactor-binding dna sequences are utilized for interpretation of plant promoters .
basis of these databases are published articles reporting analyses of individual promoters or transfactors, rather than large scale genomic analyses.
therefore, lack of large scale functional analyses of transcription factors in plant science is reflected in these databases as well.
in contrast to the above fact-based approaches, in silico prediction of plant promoter elements by survey of the arabidopsis genome is also reported.
molina and grotewold applied the meme and gibbs sampling methods to arabidopsis core promoter regions with genomic scale, and detected several motifs including a plant tata motif and microsatellites  <cit> .
recent studies on mammalian promoter elements have revealed that some of them have localized appearance along the promoter region, exemplified by the tata box  <cit> , and binding sites for nrf- <dig>  sp <dig>  creb, atf, and e2f  <cit> .
these studies evoke the idea that localized distribution is a signature of a functional element of the promoter.
recently, this feature was successfully utilized for extraction of functional sequences from human promoters  <cit> .
large-scale deletion analysis of human promoters suggested that there is some relationship between presence of functional elements and distance from tss  <cit> .
in this report, we have detected hundreds of short sequences showing localized distribution in plant promoters by comprehensive analyses of short sequences.
the extracted sequences are mentioned as "ldss -positive" in this work.
these sequences includes tata boxes, various regulatory sequences identified in previous studies, a novel sequence group that would be a general component of a core promoter, and also many novel sequences that share many characteristics with regulatory sequences.
our analyses have also revealed conservation of the promoter architecture between monocot and dicot plants.
RESULTS
patterns of distribution of peaks
typically, dna elements recognized by a protein  is within the range of  <dig> to  <dig> bp long  <cit> .
within this range, we decided to analyze localization patterns of hexamer and octamer sequences.
our results suggest that sequences longer than  <dig> bps would not provide enough number of appearance to survive statistical analysis.
for each hexamer sequence, a distribution profile in relation to distance from the tss was analyzed for arabidopsis thaliana.
looking through all the distribution profiles, we noticed that there are quite a few patterns.
most sequences have a flat distribution profile with no special tendency .
sometimes the base line has a slight slope with a higher frequency toward the tss.
there are also groups with peaks, and they can be classified according to the peak position.
we refer these sequences as ldss-positive.
one example of a ldss-positive sequence,  has a peak of appearance at the tss.
its complementary sequence  has a distinct distribution profile, showing that its appearance is sensitive to the direction of transcription.
although hexamers with this type of distribution profile tend to have only c and t in the sequence , there seems to be weak sequence preference, and not all the sequences filled with c and t show a peak-positive distribution .
a second example  is a tata box-related sequence.
this has a peak around - <dig> bp, and the peak is very sharp.
the complementary sequence showed a different pattern with no peak .
a third example  has a relatively wide and low peak.
complementary sequence of this sequence shows the same peak .
peak position and direction-insensitivity suggest that sequences with this type of distribution profile are so-called cis-regulatory sequences involved in transcriptional regulation  <cit> .
in fact, tgggcc in figure  <dig> is reported to be necessary for meristematic expression in arabidopsis, and mutation to tgaacc abolished the expression .
interestingly, distribution of the mutated sequence does not have any peaks , demonstrating a good correlation between functionality and peak distribution.
in addition, one base substitution, tgagcc, also caused the loss of the peak .
it is common that one base substitution drastically changes the distribution profile .
as controls, a set of random genomic sequences of  <dig> kb length was used for the distribution analysis instead of the promoter database.
when sequences with distribution patterns of peak-positive sequences were applied to this analysis, they were found to have no peaks in the random genome fragments .
beside ldss-positive elements, there are many ldss-negative sequences.
among them, frequently observed sequences beyond the theoretical occurrence rate  are rich in at and might promote promoter context, and rare sequences are rich in gc and they might disturb promoter function when located within the promoter region.
therefore, it might be possible to utilize these ldss-negative sequences as well for evaluation of promoter context.
parameters for peak evaluation
considering peak height, peak area, and fluctuation from the base line, we selected  <dig> sequences from all the hexamers as peak positive .
peak-positive hexamers can be classified according to their peak position
the ldss-positive hexamers identified were then classified into three major groups as mentioned above.
the first group, including ctcttc, in figure  <dig>  localize from - <dig> to - <dig> bp.
they typically have a peak at the most downstream region of the promoter , but peak positions distribute from - <dig> to - <dig>  most of their sequences are composed of only c and t, we refer to this group as y patch .
as shown in the table, y patch sequences are found in the majority of arabidopsis promoters.
1in this analysis, - <dig> is the position for an average from - <dig> to - <dig> that covers a region from - <dig> to - <dig>  so - <dig> is the most downstream position.
2peak width at the bottom of the peak.
3number of promoters containing the element out of  <dig>  arabidopsis promoters .
number of promoters containing an element within the peak area can be roughly estimated by #promoters × rpa.
for example, tataaa is found in approx.
<dig>  promoters within the peak area .
the second group contains tata box-related sequences.
an example is shown as ctataa in figure  <dig>  the characteristics of this group are high peak height, narrow peak width, and stringent peak position .
similar to y patch, the tata box group sequences are also found in the majority of arabidopsis promoters, although promoters with the tata box within the peak are is about  <dig>  or less for each sequence.
the third group, including tgggcc in figure  <dig>  is referred to as reg, for regulatory element group, in this study.
the peak positions of this group locate around - <dig> bp, and they have a wide peak width in comparison with that of the tata box group .
another feature of the group is high coverage of peak area against total area.
this means high specificity of localization within a promoter.
as shown in relative peak area  of the table, around 50% to 30% of a reg sequence is found in the peak area.
these ratios are much higher than those of the y patch  or tata box  groups.
compared to these, the number of promoters containing a reg sequence is smaller, consistent with the idea that each reg is not a component of the general core promoter but a specific regulator of gene expression.
in fact, table  <dig> contains several known cis-regulatory elements, including element ii of arabidopsis pcna- <dig>   <cit>  and g-box/abre   <cit> .
in addition to these three groups, there is also small number of exceptional hexamers with peak positions in the core promoter .
they might constitute a minor type within the core promoter .
the complete list of the extracted sequences is shown in table s <dig>  the table shows  <dig> y patch,  <dig> tata-related,  <dig> reg, and  <dig> unclassified hexamer sequences.
directional preference relative to transcription
subsequently, we examined if the orientation of the hexamers is critical.
the identified hexamers were tested to determine if their complementary sequences were also included or not.
if the complementary sequence was also found in this positive group, the original sequence is considered as direction-insensitive, and if not, direction-sensitive.
as shown in figure  <dig>  the downstream region from - <dig>  that is known to be the core promoter region  <cit>  and includes the y patch and tata box groups, is occupied with direction-sensitive sequences , while the upstream region, containing the reg group, is rich in direction-insensitive sequences .
these findings are consistent with the established idea that the core promoter determines position and direction of transcription, and cis-elements are direction insensitive.
these findings further support the idea that the y patch and tata box sequences are core promoter elements and reg sequences are the cis-elements  <cit> .
comparison of arabidopsis and rice promoters
subsequently, we analyzed the distribution of octamer sequences.
the average of octamer appearance rates is  <dig> -fold less than the one of hexamers, consistent with a mathematical expectation of 16-fold difference .
because rare sequences tend to show more fluctuations by chance, statistical evaluation was more critical for octamer analysis.
we prepared random distribution populations and used them for statistical evaluation of each octamer .
in this study, we have set a p value of  <dig> × 10- <dig> as a threshold.
in addition, data of the complementary sequences was merged only for reg detection to increase total count of an octamer in the database.
through the octamer analyses, we have identified  <dig> and  <dig> ldss-positive core elements , and  <dig> and  <dig> reg sequences from arabidopsis and rice, respectively .
sum of the p values for all the extracted octamers of individual species were around  <dig> × 10- <dig> each, so false-positive sequences by pure random distribution are not likely to be included in the lists.
for comparison of arabidopsis and rice elements, relative peak height  values of all the positive octamers in either of the two promoter databases were represented .
if a sequence has the same rph value in the arabidopsis and rice databases, a dot appears on the diagonal line.
as shown in the figure, we found that rph values are moderately conserved between arabidopsis and rice .
the figure also indicates that a considerable number of the sequences have a large difference in the parameter between arabidopsis and rice.
figure 4b shows venn diagram of the number of positive octamers in arabidopsis and rice.
as shown in the figure, approximately  <dig> to 50% of the octamers are conserved between arabidopsis and rice for both core groups of y patch and tata box, and the reg group.
presence of all the three categories in arabidopsis and rice, and sequential conservation as shown in the figure indicate that promoter architecture of these plant species is essentially conserved.
on the other hand, divergence of the positive sequences might reflect differentiation of the corresponding trans-factors between these species.
classification of arabidopsis ldss-positive octamers by distribution profiles
all the ldss-positive sequences from arabidopsis were subjected to clustering analysis according to their distribution profiles.
as expected from previous hexamer analyses, major clusters are regs, tata box, and y patch .
as shown in the figure, distribution profiles within each group  are quite similar, suggesting functional conservation within each group.
the observed clear classification of the ldss-positive sequences, represented in figure  <dig>  suggest that the local distribution is a quite useful feature in extraction of putative functional elements in the promoter.
clustering of arabidopsis regs based on presence and absence in promoters
subsequently, we did classification of  <dig> arabidopsis regs with the aid of the promoter database.
for each promoter, number of appearance for each reg was scored, and two-dimensional reg-promoter clustering was performed.
this reg-promoter association has revealed that  <dig>  out of  <dig>  arabidopsis promoters have at least one reg at the region of - <dig> to - <dig> bp.
this high coverage  is due to the long list of reg sequences.
this 2d clustering puts co-localized regs proximal, and promoters with similar reg compositions also come close.
two promoter clusters are shown in figure 6a and 6b.
one cluster of promoters  are rich in gccca-containing regs, and another cluster  have acgt-containing regs.
gccca-containing regs is the same kind as tgggcc  and known to show cell cycle-dependent expression and meristematic expression .
interestingly, this promoter group is rich in ribosomal proteins.
as shown in figure 6a, as high as 38%  of the annotated promoters are for ribosomal proteins .
in contrast, ribosomal promoters are not included in the acgt-containing promoter clusters .
instead, the latter cluster is rich in photosynthesis-related genes and stress-responsive genes, both of which would show environmental responses.
in fact, as many as  <dig> out of  <dig> genes in this cluster with expression data are responsive to light  or abiotic stress including salt, drought, and cold , according to public microarray data  <cit> .
although this clustering is not so accurate as to distinguish between light and stress responses, it has been proved to classify genes with respect to gene expression with a certain range of accuracy.
the results are reasonable because cis-elements for light response  and stress response  are related sequences both of which belong to the acgt motif for environmental responses .
therefore, clustering of promoters appears reasonable, although the accuracy may not be enough for pinpoint speculation of gene function.
1number of octamer sequences.
this classification is not completely mutually exclusive.
clustering of regs turned out to be reliable as well, and thus useful for reg classification.
according to this 2d method, overlapping regs  have a bias toward coexistence by chance.
however, similar but mutually exclusive sequences  are also clustered into the same group, suggesting that regs with the same role are clustered together.
this is explained by existence of multiple copies of the same kind of a cis-element in a promoter as different octamer expression.
figure  <dig> shows the whole tree of arabidopsis regs.
this figure demonstrates that regs with related sequences are clustered together with high reliability.
according to these results,  <dig> motifs have been extracted from arabidopsis regs , and are summarized in table  <dig>
one group has a ggccca core sequence that is known as site iia or element ii .
element ii is necessary for cell cycle-related expression and for meristematic expression  <cit> .
many sequences containing ggccca in the center of an octamer were found in reg group of both arabidopsis and rice .
as seen in the table, this group is a good indicator of conservation.
regs found in both arabidopsis and rice are indicated with a sharp  symbol.
an asterisk indicates any base and is used to restrict the position of the motif in the octamer sequence.
another group shown in the table has the bzip protein-binding motif containing acgt core sequence.
this group mediates various environmental signals  <cit> .
both species have this group in common, but arabidopsis has wider variations than rice .
classification of arabidopsis and rice regs are shown in table  <dig>  the largest group is the group  <dig>  which includes element ii of the arabidopsis pcna- <dig> involved in cell cycle-related expression, as mentioned above.
as shown in the table, this group is well conserved between arabidopsis and rice and has many members for both species.
there are several other reg groups, some of which are rich in only arabidopsis and some are found from both .
comparison between arabidopsis and rice suggests both conserved and differentiated types of regs.
the identified arabidopsis reg sequences were referred to the place database that is a collection of reported plant cis-regulatory elements  <cit> .
the comparison revealed that  <dig> out of  <dig> arabidopsis regs show  <dig> % match with at least one of the arabidopsis place entries, giving an estimation that  <dig> % of the regs are of established cis-regulatory elements .
these results again provide strong evidence for biologically meaningful extraction of sequences by the ldss method.
from another point of view,  <dig> out of  <dig> arabidopsis place entries have been found in the reg list .
comparison with another cis-element database, agris  <cit> , resulted in lower match than place  among arabidopsis motif entries shorter than  <dig> bps .
these results suggest that not all of the cis-regulatory elements are detected by the ldss strategy.
one of the valuable finding of this analysis is the identification of a large number of novel regs.
characterization of transcription start site
we then analyzed sequence characteristics around the tss.
in this region, the initiator motif yy, tss is underlined) is known in some mammalian promoters  <cit> , and it is also functional in plants  <cit> .
a survey of arabidopsis tss revealed that a limited number of promoters  have the inr motif around the tss.
thus, we looked for a more general rule.
we surveyed which base is preferred at the -1/+ <dig> position among arabidopsis tss.
the most frequently observed sequence was ca , and ta was the second.
as summarized in figure 8a, there is a strong preference of a dimer sequence at the -1/+ <dig> position.
the graph clearly shows most of the tss is a or g, and the - <dig> position is likely to be c or t. this "yr rule"  applies to as many as 77% of the arabidopsis promoters that is a much higher frequency than expected random appearance .
similar analysis for the -2/- <dig> and +1/+ <dig> positions did not reveal clear extension of the rule.
when the yr rule was applied to the -6/- <dig> to +4/+ <dig> positions, we found that the ratio of yr rule-positive is highest at the -1/+ <dig> position in the local region examined .
the figure shows that this rule is also applicable to rice tss .
these analyses have revealed that sequence preference at tss is well conserved between arabidopsis and rice.
an example of arabidopsis promoter
our simple ldss analysis has successfully revealed three distinct groups consisting of hundreds of short sequences.
figure 9a illustrates the architecture of plant promoters based on these findings.
tight positioning of the tata boxes relative to the tss fits with the general idea that the tata boxes determine the position of the tss.
in addition, the yr rule of arabidopsis would be another important determinant as well.
the y patches locate between the tata boxes and the tss, but they can be upstream of the tata boxes, considering the wide distribution profiles .
the role of the y patch is not known.
the above three elements are orientation-sensitive, and constituents of a core promoter.
regs appear upstream of the tata box, and they exist in an orientation-insensitive manner.
rice promoters share the above characteristics, showing architectural conservation between dicots and monocots.
an example of an arabidopsis promoter that has the y patch and tata box is shown in figure 9b.
octamer analysis of the promoter revealed one cluster of group  <dig> regs , one cluster of y patches, one cluster of tata box, and yr rule.
an interesting feature of the figure is the multiple hits of a locus, detecting a longer element.
this demonstrates that octamer analysis can detect long functional units as clusters of octamers.
discussion
characteristics of ldss analysis
in this study, we have identified hundreds of novel sequences solely based on local distribution in the promoter region of arabidopsis and rice.
biological information, such as microarray data, was not used at all for sequence extraction, and it becomes useful only during interpretation of the extracted sequence.
this method is equally sensitive in detection of major and minor motifs in a promoter population as demonstrated by simultaneous detection of major tata elements and minor reg elements.
this feature is an advantage of the ldss method over other methods of detection of consensus sequences among promoter populations, such as gibbs sampling method.
we successfully applied the ldss method to arabidopsis and rice promoters, and of course, it is applicable to bacterial and mammalian research as well.
the observed localized distribution is a direct result of the selection pressure.
while the localization is an indication of a beneficial role for the organism, the relationship between local distribution of a sequence and its functionality is indirect.
therefore, the question arises if all regulatory elements can be picked up by the ldss strategy.
when we compared reg sequences with established cis-elements in the place database, it was found that  <dig> out of  <dig> arabidopsis place entries are absent in the extracted regs .
these results indicate that not all of the functional elements are ldss-positive, and thus some would not be detected by this method.
there are two possibilities for the presence of cis-elements that do not show local distribution.
one possibility is that these elements are relatively "new" so there has not been selection pressure for a long enough period.
another possibility is that there has not been any selection pressure because of functional differences from the ldss-positive elements.
the latter idea suggests localization-insensitive classes of regulatory elements that are distinct from regs.
so called long range-regulators  <cit>  might be one of the classes.
generally, any functional sequences in the genome are recognized by trans-acting factors that are dna-binding proteins.
promoter elements and their trans-factors have a relationship of co-evolution.
therefore, differentiation of regs in the two species would reflect a different status of the corresponding trans-factors.
functional comparison of dna-binding proteins of arabidopsis and rice is expected to give some answers as to why these two species have differentiated reg sequences.
as for the conserved regs, it is reasonable that cell cycle-related elements  comprise the most conserved group, because the cell cycle is one of the most conserved activities in organisms.
reg sequences can be extracted form mammalian promoters as well.
however, our preliminary analyses suggest that the ldss method can detect much less regs than of plants .
this may be reflected by different promoter architecture between plants and animals.
y patch
the discovery that the y patch is conserved in monocots and dicots is one of the major achievements of this study.
a related motif is reported by molina and grotewold from arabidopsis core promoter analysis using the gibbs-sampling method .
the biochemical role of y patch is not known, but its position, direction sensitivity, and its abundant nature strongly suggest that it is a general component of the core promoter.
our ldss analyses suggest that human and mouse do not share this element with plants and thus this is a plant-specific core element .
yr rule
at the tss, the initiator  motif  is known as a recognition site by tfiid  <cit> .
following their rules, the yr rule can be considered as a less stringent form of inr.
according to this point of view, the yr rule might be recognized by tfiid.
the high coverage of the yr rule is a useful feature for prediction of tss.
recently, carninci et al., have reported the same rule is applicable to mouse and human promoters as well  <cit> , revealing conservation of yr rule between plants and mammals.
this rule is not an artifact by the cap-trapper method that is the basis of tss mapping of this study and mammalian studies mentioned above  <cit> , because it is applicable to human tss determined by another method  as well .
a plant consensus around tss  is reported by shahmuradov et al based on  <dig> dicot promoters .
this consensus also largely overlaps with yr rule.
the tfiib-recognition element  is another core promoter element of animal genes.
it is located just upstream of the tata box and has a gc-rich sequence, cgcc  <cit> .
our analysis did not detect the bre as a ldss-positive element, although cc is preferred at the neighboring sequence of the tata box at the upstream side in both arabidopsis and rice promoters .
ldss analysis provides useful information toward precise promoter prediction
the hundreds of octamer sequences identified by the ldss analysis can be used for promoter prediction.
the presence of the tata box is an important feature of a promoter, but there are many false-positives in the genome.
for example, a tata octamer sequence with the highest specific localization is found within the peak area 30% of times in the promoter region, meaning that 70% are found outside of the peak area.
this is essentially consistent with a previous study, where more than  <dig>  putative tbp-binding sites were detected from the arabidopsis genome  <cit> .
utilization of preferential sequence around the tata box, and coexistence with the y patch and reg are expected to elevate accuracy of prediction.
although such a combinational approach is incorporated into several promoter prediction programs  <cit> , motifs to be detected have been limited so far.
our long list of the ldss-positive octamers is expected to serve as a thick dictionary for precise interpretation of plant genomes.
CONCLUSIONS
in this report, we showed that ldss can be applied to plant genomes.
we have successfully extracted hundreds of promoter elements as ldss-positive octamers.
all the observed behaviors of the isolated elements suggest functionality of these elements.
promoter architectures of monocot and dicot revealed in this study are well conserved, but there are moderate variations in the utilized sequences.

 the determination of complete genome sequences has allowed analysis by various statistical methods that have furthered understanding of the function of genomes. analysis of promoter structure is one of the most important issues. understanding of promoter structure allows predictions concerning promoter positions and expression profiles, and sheds light on hidden transcriptional networks. several functional elements have been identified as promoter constituents for precise and regulated transcriptional initiation: tata box, initiator motif, downstream promoter element, tfiib-recognition element, and so-called cis-regulatory elements. in addition, some mammalian promoters are associated with cpg islands, which is related to the sp recognition site and have some relationship with gene regulation by dna-methylation. human transcriptional regulatory elements are reported to make clusters at the promoter region as well as the 3' end of a gene. transcription start sites in plant promoters have a cg-compositional strand bias, or gc-skew, where c is more frequently observed in the strand than g. some of these features are well understood and some are not, but all these features are useful to understand individual promoters. some of the above features have been utilized for promoter prediction. although these studies obtain certain success, our current knowledge of promoters is still insufficient. availability of microarray data on co-regulated gene expression on a genomic scale has enabled the prediction of novel cis-elements involved in gene regulation. several approaches have been developed for this detection of consensus sequences in a co-regulated promoter set, and detection of over-represented sequence in co-regulated promoters with a set of reference sequences. these approaches are also applicable to chromatin immunoprecipitation data. in addition, identification of conserved promoter sequences by comparative genomics supports the prediction of regulatory elements. studies on plant transcription factors and functional cis-regulatory elements have been summarized in several databases, and the collective information of cis-elements and/or transfactor-binding dna sequences are utilized for interpretation of plant promoters. basis of these databases are published articles reporting analyses of individual promoters or transfactors, rather than large scale genomic analyses. therefore, lack of large scale functional analyses of transcription factors in plant science is reflected in these databases as well. in contrast to the above fact-based approaches, in silico prediction of plant promoter elements by survey of the arabidopsis genome is also reported. molina and grotewold applied the meme and gibbs sampling methods to arabidopsis core promoter regions with genomic scale, and detected several motifs including a plant tata motif and microsatellites. recent studies on mammalian promoter elements have revealed that some of them have localized appearance along the promoter region, exemplified by the tata box, and binding sites for nrf- sp creb, atf, and e2f. these studies evoke the idea that localized distribution is a signature of a functional element of the promoter. recently, this feature was successfully utilized for extraction of functional sequences from human promoters. large-scale deletion analysis of human promoters suggested that there is some relationship between presence of functional elements and distance from tss. in this report, we have detected hundreds of short sequences showing localized distribution in plant promoters by comprehensive analyses of short sequences. the extracted sequences are mentioned as "ldss -positive" in this work. these sequences includes tata boxes, various regulatory sequences identified in previous studies, a novel sequence group that would be a general component of a core promoter, and also many novel sequences that share many characteristics with regulatory sequences. our analyses have also revealed conservation of the promoter architecture between monocot and dicot plants.  patterns of distribution of peaks typically, dna elements recognized by a protein is within the range of to bp long. within this range, we decided to analyze localization patterns of hexamer and octamer sequences. our results suggest that sequences longer than bps would not provide enough number of appearance to survive statistical analysis. for each hexamer sequence, a distribution profile in relation to distance from the tss was analyzed for arabidopsis thaliana. looking through all the distribution profiles, we noticed that there are quite a few patterns. most sequences have a flat distribution profile with no special tendency. sometimes the base line has a slight slope with a higher frequency toward the tss. there are also groups with peaks, and they can be classified according to the peak position. we refer these sequences as ldss-positive. one example of a ldss-positive sequence, has a peak of appearance at the tss. its complementary sequence has a distinct distribution profile, showing that its appearance is sensitive to the direction of transcription. although hexamers with this type of distribution profile tend to have only c and t in the sequence, there seems to be weak sequence preference, and not all the sequences filled with c and t show a peak-positive distribution. a second example is a tata box-related sequence. this has a peak around - bp, and the peak is very sharp. the complementary sequence showed a different pattern with no peak. a third example has a relatively wide and low peak. complementary sequence of this sequence shows the same peak. peak position and direction-insensitivity suggest that sequences with this type of distribution profile are so-called cis-regulatory sequences involved in transcriptional regulation. in fact, tgggcc in figure is reported to be necessary for meristematic expression in arabidopsis, and mutation to tgaacc abolished the expression. interestingly, distribution of the mutated sequence does not have any peaks, demonstrating a good correlation between functionality and peak distribution. in addition, one base substitution, tgagcc, also caused the loss of the peak. it is common that one base substitution drastically changes the distribution profile. as controls, a set of random genomic sequences of kb length was used for the distribution analysis instead of the promoter database. when sequences with distribution patterns of peak-positive sequences were applied to this analysis, they were found to have no peaks in the random genome fragments. beside ldss-positive elements, there are many ldss-negative sequences. among them, frequently observed sequences beyond the theoretical occurrence rate are rich in at and might promote promoter context, and rare sequences are rich in gc and they might disturb promoter function when located within the promoter region. therefore, it might be possible to utilize these ldss-negative sequences as well for evaluation of promoter context. parameters for peak evaluation considering peak height, peak area, and fluctuation from the base line, we selected sequences from all the hexamers as peak positive. peak-positive hexamers can be classified according to their peak position the ldss-positive hexamers identified were then classified into three major groups as mentioned above. the first group, including ctcttc, in figure localize from - to - bp. they typically have a peak at the most downstream region of the promoter, but peak positions distribute from - to - most of their sequences are composed of only c and t, we refer to this group as y patch. as shown in the table, y patch sequences are found in the majority of arabidopsis promoters. 1in this analysis, - is the position for an average from - to - that covers a region from - to - so - is the most downstream position. 2peak width at the bottom of the peak. 3number of promoters containing the element out of arabidopsis promoters. number of promoters containing an element within the peak area can be roughly estimated by #promoters × rpa. for example, tataaa is found in approx. promoters within the peak area. the second group contains tata box-related sequences. an example is shown as ctataa in figure the characteristics of this group are high peak height, narrow peak width, and stringent peak position. similar to y patch, the tata box group sequences are also found in the majority of arabidopsis promoters, although promoters with the tata box within the peak are is about or less for each sequence. the third group, including tgggcc in figure is referred to as reg, for regulatory element group, in this study. the peak positions of this group locate around - bp, and they have a wide peak width in comparison with that of the tata box group. another feature of the group is high coverage of peak area against total area. this means high specificity of localization within a promoter. as shown in relative peak area of the table, around 50% to 30% of a reg sequence is found in the peak area. these ratios are much higher than those of the y patch or tata box groups. compared to these, the number of promoters containing a reg sequence is smaller, consistent with the idea that each reg is not a component of the general core promoter but a specific regulator of gene expression. in fact, table contains several known cis-regulatory elements, including element ii of arabidopsis pcna- and g-box/abre. in addition to these three groups, there is also small number of exceptional hexamers with peak positions in the core promoter. they might constitute a minor type within the core promoter. the complete list of the extracted sequences is shown in table s the table shows y patch, tata-related, reg, and unclassified hexamer sequences. directional preference relative to transcription subsequently, we examined if the orientation of the hexamers is critical. the identified hexamers were tested to determine if their complementary sequences were also included or not. if the complementary sequence was also found in this positive group, the original sequence is considered as direction-insensitive, and if not, direction-sensitive. as shown in figure the downstream region from - that is known to be the core promoter region and includes the y patch and tata box groups, is occupied with direction-sensitive sequences, while the upstream region, containing the reg group, is rich in direction-insensitive sequences. these findings are consistent with the established idea that the core promoter determines position and direction of transcription, and cis-elements are direction insensitive. these findings further support the idea that the y patch and tata box sequences are core promoter elements and reg sequences are the cis-elements. comparison of arabidopsis and rice promoters subsequently, we analyzed the distribution of octamer sequences. the average of octamer appearance rates is -fold less than the one of hexamers, consistent with a mathematical expectation of 16-fold difference. because rare sequences tend to show more fluctuations by chance, statistical evaluation was more critical for octamer analysis. we prepared random distribution populations and used them for statistical evaluation of each octamer. in this study, we have set a p value of × 10- as a threshold. in addition, data of the complementary sequences was merged only for reg detection to increase total count of an octamer in the database. through the octamer analyses, we have identified and ldss-positive core elements, and and reg sequences from arabidopsis and rice, respectively. sum of the p values for all the extracted octamers of individual species were around × 10- each, so false-positive sequences by pure random distribution are not likely to be included in the lists. for comparison of arabidopsis and rice elements, relative peak height values of all the positive octamers in either of the two promoter databases were represented. if a sequence has the same rph value in the arabidopsis and rice databases, a dot appears on the diagonal line. as shown in the figure, we found that rph values are moderately conserved between arabidopsis and rice. the figure also indicates that a considerable number of the sequences have a large difference in the parameter between arabidopsis and rice. figure 4b shows venn diagram of the number of positive octamers in arabidopsis and rice. as shown in the figure, approximately to 50% of the octamers are conserved between arabidopsis and rice for both core groups of y patch and tata box, and the reg group. presence of all the three categories in arabidopsis and rice, and sequential conservation as shown in the figure indicate that promoter architecture of these plant species is essentially conserved. on the other hand, divergence of the positive sequences might reflect differentiation of the corresponding trans-factors between these species. classification of arabidopsis ldss-positive octamers by distribution profiles all the ldss-positive sequences from arabidopsis were subjected to clustering analysis according to their distribution profiles. as expected from previous hexamer analyses, major clusters are regs, tata box, and y patch. as shown in the figure, distribution profiles within each group are quite similar, suggesting functional conservation within each group. the observed clear classification of the ldss-positive sequences, represented in figure suggest that the local distribution is a quite useful feature in extraction of putative functional elements in the promoter. clustering of arabidopsis regs based on presence and absence in promoters subsequently, we did classification of arabidopsis regs with the aid of the promoter database. for each promoter, number of appearance for each reg was scored, and two-dimensional reg-promoter clustering was performed. this reg-promoter association has revealed that out of arabidopsis promoters have at least one reg at the region of - to - bp. this high coverage is due to the long list of reg sequences. this 2d clustering puts co-localized regs proximal, and promoters with similar reg compositions also come close. two promoter clusters are shown in figure 6a and 6b. one cluster of promoters are rich in gccca-containing regs, and another cluster have acgt-containing regs. gccca-containing regs is the same kind as tgggcc and known to show cell cycle-dependent expression and meristematic expression. interestingly, this promoter group is rich in ribosomal proteins. as shown in figure 6a, as high as 38% of the annotated promoters are for ribosomal proteins. in contrast, ribosomal promoters are not included in the acgt-containing promoter clusters. instead, the latter cluster is rich in photosynthesis-related genes and stress-responsive genes, both of which would show environmental responses. in fact, as many as out of genes in this cluster with expression data are responsive to light or abiotic stress including salt, drought, and cold, according to public microarray data. although this clustering is not so accurate as to distinguish between light and stress responses, it has been proved to classify genes with respect to gene expression with a certain range of accuracy. the results are reasonable because cis-elements for light response and stress response are related sequences both of which belong to the acgt motif for environmental responses. therefore, clustering of promoters appears reasonable, although the accuracy may not be enough for pinpoint speculation of gene function. 1number of octamer sequences. this classification is not completely mutually exclusive. clustering of regs turned out to be reliable as well, and thus useful for reg classification. according to this 2d method, overlapping regs have a bias toward coexistence by chance. however, similar but mutually exclusive sequences are also clustered into the same group, suggesting that regs with the same role are clustered together. this is explained by existence of multiple copies of the same kind of a cis-element in a promoter as different octamer expression. figure shows the whole tree of arabidopsis regs. this figure demonstrates that regs with related sequences are clustered together with high reliability. according to these results, motifs have been extracted from arabidopsis regs, and are summarized in table one group has a ggccca core sequence that is known as site iia or element ii. element ii is necessary for cell cycle-related expression and for meristematic expression. many sequences containing ggccca in the center of an octamer were found in reg group of both arabidopsis and rice. as seen in the table, this group is a good indicator of conservation. regs found in both arabidopsis and rice are indicated with a sharp symbol. an asterisk indicates any base and is used to restrict the position of the motif in the octamer sequence. another group shown in the table has the bzip protein-binding motif containing acgt core sequence. this group mediates various environmental signals. both species have this group in common, but arabidopsis has wider variations than rice. classification of arabidopsis and rice regs are shown in table the largest group is the group which includes element ii of the arabidopsis pcna- involved in cell cycle-related expression, as mentioned above. as shown in the table, this group is well conserved between arabidopsis and rice and has many members for both species. there are several other reg groups, some of which are rich in only arabidopsis and some are found from both. comparison between arabidopsis and rice suggests both conserved and differentiated types of regs. the identified arabidopsis reg sequences were referred to the place database that is a collection of reported plant cis-regulatory elements. the comparison revealed that out of arabidopsis regs show % match with at least one of the arabidopsis place entries, giving an estimation that % of the regs are of established cis-regulatory elements. these results again provide strong evidence for biologically meaningful extraction of sequences by the ldss method. from another point of view, out of arabidopsis place entries have been found in the reg list. comparison with another cis-element database, agris, resulted in lower match than place among arabidopsis motif entries shorter than bps. these results suggest that not all of the cis-regulatory elements are detected by the ldss strategy. one of the valuable finding of this analysis is the identification of a large number of novel regs. characterization of transcription start site we then analyzed sequence characteristics around the tss. in this region, the initiator motif yy, tss is underlined) is known in some mammalian promoters, and it is also functional in plants. a survey of arabidopsis tss revealed that a limited number of promoters have the inr motif around the tss. thus, we looked for a more general rule. we surveyed which base is preferred at the -1/+ position among arabidopsis tss. the most frequently observed sequence was ca, and ta was the second. as summarized in figure 8a, there is a strong preference of a dimer sequence at the -1/+ position. the graph clearly shows most of the tss is a or g, and the - position is likely to be c or t. this "yr rule" applies to as many as 77% of the arabidopsis promoters that is a much higher frequency than expected random appearance. similar analysis for the -2/- and +1/+ positions did not reveal clear extension of the rule. when the yr rule was applied to the -6/- to +4/+ positions, we found that the ratio of yr rule-positive is highest at the -1/+ position in the local region examined. the figure shows that this rule is also applicable to rice tss. these analyses have revealed that sequence preference at tss is well conserved between arabidopsis and rice. an example of arabidopsis promoter our simple ldss analysis has successfully revealed three distinct groups consisting of hundreds of short sequences. figure 9a illustrates the architecture of plant promoters based on these findings. tight positioning of the tata boxes relative to the tss fits with the general idea that the tata boxes determine the position of the tss. in addition, the yr rule of arabidopsis would be another important determinant as well. the y patches locate between the tata boxes and the tss, but they can be upstream of the tata boxes, considering the wide distribution profiles. the role of the y patch is not known. the above three elements are orientation-sensitive, and constituents of a core promoter. regs appear upstream of the tata box, and they exist in an orientation-insensitive manner. rice promoters share the above characteristics, showing architectural conservation between dicots and monocots. an example of an arabidopsis promoter that has the y patch and tata box is shown in figure 9b. octamer analysis of the promoter revealed one cluster of group regs, one cluster of y patches, one cluster of tata box, and yr rule. an interesting feature of the figure is the multiple hits of a locus, detecting a longer element. this demonstrates that octamer analysis can detect long functional units as clusters of octamers. discussion characteristics of ldss analysis in this study, we have identified hundreds of novel sequences solely based on local distribution in the promoter region of arabidopsis and rice. biological information, such as microarray data, was not used at all for sequence extraction, and it becomes useful only during interpretation of the extracted sequence. this method is equally sensitive in detection of major and minor motifs in a promoter population as demonstrated by simultaneous detection of major tata elements and minor reg elements. this feature is an advantage of the ldss method over other methods of detection of consensus sequences among promoter populations, such as gibbs sampling method. we successfully applied the ldss method to arabidopsis and rice promoters, and of course, it is applicable to bacterial and mammalian research as well. the observed localized distribution is a direct result of the selection pressure. while the localization is an indication of a beneficial role for the organism, the relationship between local distribution of a sequence and its functionality is indirect. therefore, the question arises if all regulatory elements can be picked up by the ldss strategy. when we compared reg sequences with established cis-elements in the place database, it was found that out of arabidopsis place entries are absent in the extracted regs. these results indicate that not all of the functional elements are ldss-positive, and thus some would not be detected by this method. there are two possibilities for the presence of cis-elements that do not show local distribution. one possibility is that these elements are relatively "new" so there has not been selection pressure for a long enough period. another possibility is that there has not been any selection pressure because of functional differences from the ldss-positive elements. the latter idea suggests localization-insensitive classes of regulatory elements that are distinct from regs. so called long range-regulators might be one of the classes. generally, any functional sequences in the genome are recognized by trans-acting factors that are dna-binding proteins. promoter elements and their trans-factors have a relationship of co-evolution. therefore, differentiation of regs in the two species would reflect a different status of the corresponding trans-factors. functional comparison of dna-binding proteins of arabidopsis and rice is expected to give some answers as to why these two species have differentiated reg sequences. as for the conserved regs, it is reasonable that cell cycle-related elements comprise the most conserved group, because the cell cycle is one of the most conserved activities in organisms. reg sequences can be extracted form mammalian promoters as well. however, our preliminary analyses suggest that the ldss method can detect much less regs than of plants. this may be reflected by different promoter architecture between plants and animals. y patch the discovery that the y patch is conserved in monocots and dicots is one of the major achievements of this study. a related motif is reported by molina and grotewold from arabidopsis core promoter analysis using the gibbs-sampling method. the biochemical role of y patch is not known, but its position, direction sensitivity, and its abundant nature strongly suggest that it is a general component of the core promoter. our ldss analyses suggest that human and mouse do not share this element with plants and thus this is a plant-specific core element. yr rule at the tss, the initiator motif is known as a recognition site by tfiid. following their rules, the yr rule can be considered as a less stringent form of inr. according to this point of view, the yr rule might be recognized by tfiid. the high coverage of the yr rule is a useful feature for prediction of tss. recently, carninci et al., have reported the same rule is applicable to mouse and human promoters as well, revealing conservation of yr rule between plants and mammals. this rule is not an artifact by the cap-trapper method that is the basis of tss mapping of this study and mammalian studies mentioned above, because it is applicable to human tss determined by another method as well. a plant consensus around tss is reported by shahmuradov et al based on dicot promoters. this consensus also largely overlaps with yr rule. the tfiib-recognition element is another core promoter element of animal genes. it is located just upstream of the tata box and has a gc-rich sequence, cgcc. our analysis did not detect the bre as a ldss-positive element, although cc is preferred at the neighboring sequence of the tata box at the upstream side in both arabidopsis and rice promoters. ldss analysis provides useful information toward precise promoter prediction the hundreds of octamer sequences identified by the ldss analysis can be used for promoter prediction. the presence of the tata box is an important feature of a promoter, but there are many false-positives in the genome. for example, a tata octamer sequence with the highest specific localization is found within the peak area 30% of times in the promoter region, meaning that 70% are found outside of the peak area. this is essentially consistent with a previous study, where more than putative tbp-binding sites were detected from the arabidopsis genome. utilization of preferential sequence around the tata box, and coexistence with the y patch and reg are expected to elevate accuracy of prediction. although such a combinational approach is incorporated into several promoter prediction programs, motifs to be detected have been limited so far. our long list of the ldss-positive octamers is expected to serve as a thick dictionary for precise interpretation of plant genomes.  in this report, we showed that ldss can be applied to plant genomes. we have successfully extracted hundreds of promoter elements as ldss-positive octamers. all the observed behaviors of the isolated elements suggest functionality of these elements. promoter architectures of monocot and dicot revealed in this study are well conserved, but there are moderate variations in the utilized sequences. 
sequences of the tata box group are consistent with the ones reported in previous studies. ldss profiles of short sequences along the plant promoter have been analyzed in silico, and hundreds of hexamer and octamer sequences have been identified as having localized distributions within promoters of arabidopsis thaliana and rice. in addition, a dimer motif named the yr rule has been identified at the transcription start site. several promoter elements including tata box, and several types of transcriptional regulatory elements have been found to show local distribution within promoters, and this feature has been successfully utilized for extraction of promoter constituents from human genome. ldss was successfully applied to plant genomes and hundreds of putative promoter elements have been extracted as ldss-positive octamers. identified promoter architecture of monocot and dicot are well conserved, but there are moderate variations in the utilized sequences. this rule also fits both arabidopsis and rice promoters. the reg group includes more than sequences, and half of them correspond to known cis-elements. based on their localization patterns, the identified sequences could be classified into three groups, pyrimidine patch, tata box, and reg. plant promoter architecture is important for understanding regulation and evolution of the promoters, but our current knowledge about plant promoter structure, especially with respect to the core promoter, is insufficient. comparison of ldss-positive sequences between arabidopsis and rice has revealed moderate conservation of elements and common promoter architecture.
 plant promoter architecture is important for understanding regulation and evolution of the promoters, but our current knowledge about plant promoter structure, especially with respect to the core promoter, is insufficient. several promoter elements including tata box, and several types of transcriptional regulatory elements have been found to show local distribution within promoters, and this feature has been successfully utilized for extraction of promoter constituents from human genome.  ldss profiles of short sequences along the plant promoter have been analyzed in silico, and hundreds of hexamer and octamer sequences have been identified as having localized distributions within promoters of arabidopsis thaliana and rice. based on their localization patterns, the identified sequences could be classified into three groups, pyrimidine patch, tata box, and reg. sequences of the tata box group are consistent with the ones reported in previous studies. the reg group includes more than sequences, and half of them correspond to known cis-elements. the other reg subgroups, together with about a hundred uncategorized sequences, are suggested to be novel cis-regulatory elements. comparison of ldss-positive sequences between arabidopsis and rice has revealed moderate conservation of elements and common promoter architecture. in addition, a dimer motif named the yr rule has been identified at the transcription start site. this rule also fits both arabidopsis and rice promoters.  ldss was successfully applied to plant genomes and hundreds of putative promoter elements have been extracted as ldss-positive octamers. identified promoter architecture of monocot and dicot are well conserved, but there are moderate variations in the utilized sequences. 