BACKGROUND
micrornas  are a large class of recently discovered short non-coding rnas  involved in post-transcriptional regulation of protein-coding genes.
in plants they repress gene expression by catalytic mrna degradation on the basis of sequence homology between the microrna itself and a target sequence.
through this function they act as major players in the regulation of a series of fundamental processes in plant growth and development, in response to biotic and abiotic stress and in the regulation of components of the plant silencing machinery itself  <cit> .
in plants, rna polymerase ii produces a long primary transcript  folded in a typical stem-loop structure  <cit>  that is processed by a dicer-like rnase iii ribonuclease , first in a shorter mirna precursor  and then in the mirna:mirna* duplex  <cit> .
the mirna:mirna* duplex is transported to the cytoplasm and the mature mirna is incorporated in the rna-induced silencing complex  where it drives the slicer argonaute <dig>  to silence the target mrna  <cit> .
plant mirnas have been found in a wide variety of species and several mirna families are evolutionarily highly conserved, ranging from mosses and ferns to dicots  <cit> .
the members of each mirna family normally retain a complete or almost complete conservation of mirna and mirna* sequences and of the structure formed by their pairing.
generally strong conservation constraints characterize the sequences and structure of the pre-mirna hairpin structure, whereas the conservation constraints on loop and flanking sequences are less tight  <cit> .
this is due to the fact that in plants mirna processing depends on pre-mirna structure rather than on sequence and in particular on the structure of the flanking sequences  rather than on the mature mirna itself  <cit> .
a detailed analysis of mir <dig> biogenesis has revealed that the release of the mature microrna requires at least three dcl <dig> cleavage steps spaced by  <dig> nucleotide intervals each, starting from the base of its unusually long lower stem  <cit> .
similar studies in animals have shown that structural features of the lower stem are essential for cleavage of pri-mirna by drosha .
in contrast to the complexity that regulatory cascades of transcription factors can reach  <cit> , plant micrornas are organized according to a simple, two-level hierarchy: only three of them, mir <dig>  mir <dig> and mir <dig>  <cit> , control their own expression and that of the other mirnas by targeting specific proteins involved in the post-transcriptional gene silencing pathway.
in particular, mir <dig> regulates the function of all mirnas by targeting ago <dig> expression, therefore modulating its actual levels and consequently risc activity  <cit> .
mir <dig> is present in a low copy number in different plant species  <cit>  and in the arabidopsis genome two mir <dig> paralogs  are present.
only mir168a, for which the primary transcript has been isolated  <cit> , was shown to be involved in ago <dig> post-transcriptional gene silencing in arabidopsis.
a mir168a-resistant version of ago <dig> showed increased levels of ago <dig> mrna, the over-accumulation of mir <dig> and developmental defects partially overlapping with those observed in dcl <dig>  hen <dig> and hyl <dig> mutants  <cit> .
a complex feedback loop, involving on the one hand cleavage of ago <dig> transcripts directed by mir <dig> and on the other hand stabilization of mir <dig> through ago <dig> association, was shown to maintain ago <dig> homeostasis which is pivotal for mirna-mediated post-transcriptional gene silencing  <cit> .
the overlapping expression patterns of mir168a and ago <dig> and the restored development and fertility in ago <dig> mutants expressing mir168a-promoter:ago <dig> fusion support this model  <cit> .
despite the relevance of mir168a in plant development, up to now no detailed comparative study has been carried out to characterize its evolution, nor has the function of its paralog mir168b been determined.
in this study we applied phylogenetic footprinting to the characterization of the genomic organization, and structural and functional evolution of mir <dig> sets of orthologs in brassicaceae.
we found that, despite having originated before brassicaceae radiation, mir168a and mir168b paralogs have been maintained, most likely as functional, throughout brassicacea evolution, with mir168b having undergone a partial sub-functionalization.
we also provide evolutionary evidence that the lower stem in the pre-mirna structure  is significantly longer than lower stems in animals and propose the hypothesis that, similarly to mir <dig>  it may play a relevant role in multi-step mir <dig> biogenesis.
RESULTS
synteny of mir168a and mir168b loci in a. thaliana and p. trichocarpa
in the genomes of both a. thaliana  and p. trichocarpa  two mir <dig> loci have been identified, called mir168a and mir168b, located respectively on chromosome  <dig> and  <dig> in arabidopsis and on linkage_group_iii and scaffold_ <dig> in poplar  <cit> .
analyses of synteny conservation were carried out by searching in poplar for the putative orthologs of the  <dig> arabidopsis genes flanking mir168a and mir168b by screening for reciprocal best matches  in blastp searches  <cit>  .
the queries from the former analyses were then used to identify recent segmental duplications .
assuming orthology among the arabidopsis and poplar genomic regions encompassing the mir <dig> loci, the surrounding rbm pairs should be found mainly among the same pair of chromosomes.
the uneven distribution of loci forming rbm pairs, however, indicated that the mir <dig> loci may have been the result of independent duplication events.
dating of duplication events
only two arabidopsis paralogs formed rbm pairs in poplar .
to determine the chronological order of these duplications, we carried out a phylogenetic reconstruction of all the genes that are homologous to the rbm pairs in the two genomes.
the results show that the splitting of the two species predated two duplication events that took place independently in the arabidopsis and poplar lineages.
the two arabidopsis paralogs, at4g <dig> and at5g <dig>  displayed a rate of synonymous substitution  of  <dig> , a higher value than that observed for paralogs resulting from the arabidopsis alpha whole genome duplication  <cit> .
the two poplar paralogs, eugene <dig>  and estext_fgenesh4_pg.c_ <dig>  were confirmed to have diverged more recently .
the divergence between poplar and arabidopsis homologs ranged between ks =  <dig>  and ks =  <dig> .
based on the estimated divergence time between cleomaceae and brassicaceae , this should correspond to a poplar-arabidopsis divergence time of about  <dig> million years, in full agreement with the 100– <dig> million year range provided by previous reports  <cit> .
this dating agrees with the observation that synteny conservation between arabidopsis and poplar is higher than between arabidopsis chromosomes.
taken together, these results indicate that no orthologous relationship can be inferred between arabidopsis and poplar mir <dig> homologs.
genomic characterization of mir <dig> loci in brassicaceae species
on the basis of these results we focused on analysis of the evolution and conservation between species of the two mir <dig> homologs in a group of  <dig> brassicaceae species .
amplification of the intergenic regions upstream and downstream of mir168a and mir168b in brassicaceae species.
ir: intergenic region; kbp: kilo-basepair.
n.d.: not determined.
a, b: whole mir168a or mir168b loci obtained from the upstream to the downstream gene.
mir168a and mir168b homologs were amplified through a gene-to-gene amplification based on their up- and downstream genes in arabidopsis.
the intergenic region downstream of mir168a was amplified from all the species with an amplification rate double than that of the upstream intergenic region .
in the case of mir168b the intergenic regions were fully isolated  in most of the species.
the taxonomic distance of the single species from arabidopsis did not significantly affect the isolation of intergenic regions.
the isolation of intergenic regions and the level of sequence conservation between species highlighted by their multiple alignments indicate:  general micro-synteny conservation in the regions surrounding mir168a and mir168b and  conservation of the orthologous relationship of all isolated mir168a and mir168b genes at the family level .
mir168a and mir168b phylogenetic footprinting
a clear phylogenetic footprint was identified in all species ~100– <dig> bp upstream of the mature mir168a  in correspondence with arabidopsis mir168a transcription start site .
on the contrary, the use of different alignment programs failed to identify a highly conserved footprint corresponding to mir168b tss.
the location of mir168b tss in arabidopsis was therefore determined by sequencing  <dig> race products obtained from pmir168b1::gfp-gus transgenic lines.
the 5' end of all clones mapped in three points of a region ~60– <dig> bp upstream of the mature mir168b proximal to a tata-like motif  conserved in both paralogs .
the three tss conformed in all cases to the ta class of dinucleotides identified by the yr rule  <cit> .
this poorly conserved footprint could be identified by manual editing of a multiple sequence alignment performed with clustalw, thus indicating a lower functional constraint on mir168b as compared to mir168a transcription.
detailed analysis of pre-mir168a and pre-mir168b and flanking sequences revealed a considerable conservation of the pre-mirna sequences at both loci .
both mir <dig> and mir168* were completely or almost completely conserved between orthologs and paralogs in all species .
the ~ <dig> bp flanking regions preceding the mature mir <dig> and following the mir168* showed a significant level of sequence conservation between orthologs and also, although to a lower extent, between paralogs .
a completely conserved  <dig> bp long motif  was isolated in both mir168a and mir168b just downstream of the pre-microrna .
despite being a palindromic structure, it was not involved in any predicted secondary structure.
searches for this motif in the athamap database  <cit>  showed a high quality match with the binding site of the tobacco agp <dig> transcription factor  <cit> .
no significant over-representation of the  <dig> bp motif downstream of microrna loci was detected as compared with coding genes .
an identical pattern was also detected in mir396a downstream of, but at a higher distance as compared with mir <dig>  to check for over-representation of this motif in specific groups of micrornas,  <dig> microrna superfamilies were defined based on the classification of their targets.
the application of random permutation resampling approach led to the identification of only one superfamily which showed an enrichment in this motif .
this superfamily encompasses both mir <dig> paralogs and mir <dig>  a microrna targeting argonaute <dig>  that is a member of the argonaute family of slicers responsible for mrna cleavage in ptgs.
a footprint specific to mir168b was located about  <dig> bp downstream of the tss .
the footprint matched the binding sites of agamous like  <dig>  and agamous like  <dig> , two mads-box domain transcription factors involved in floral organ identity and meristem determinacy  <cit> .
the presence of a  <dig> bp insertion in the basal species aethionema grandiflora prompted us to separately consider two sub-motifs .
their occurrence upstream of arabidopsis micrornas was not significantly over-represented compared to 5'utrs of all arabidopsis coding genes .
no statistical support for their preferential occurrence in the 5' region of specific microrna superfamilies was found at the  <dig>  level .
phylogenetic reconstruction of mir168a and mir168b
phylogenetic reconstruction with all brassicaceae mir <dig> homologs confirmed the successful isolation of orthologs of arabidopsis mir168a and mir168b.
the limited amount of parsimony-informative sites, however, could not provide a phylogenetic reconstruction resolved enough to compare the evolutionary rates of the single mir <dig> loci .
two data partitions were created by concatenating mir168a with mir168b and its with eif3e  <cit> .
the resulting phylogenetic reconstructions of mir <dig> as compared with the its-eif3e neutral markers showed slightly incongruent topologies that are the consequence of the overall lower resolution provided by the mir <dig> partition .
comparative analysis of predicted pre-mir168a and pre-mir168b structures
secondary structures for pre-mir168a and pre-mir168b plus  <dig> bp of flanking sequences on each side were predicted based on free energy minimization  <cit> .
the consensus of the most conserved portion of these regions, including about  <dig> bp upstream of mature mir <dig> and downstream of mir168*, is shown in figure  <dig>  the mature microrna-microrna* secondary structure  was completely conserved in the case of mir168a and almost completely conserved in the case of mir168b .
the structure adjacent to the upper stem  was also highly conserved in mir168a and mir168b.
in mir168a it ranged from  <dig> to  <dig> bp, with two mismatches and one bulge loop .
the lower stem of the predicted mir168b structure was  <dig> to  <dig> bp long and presented three mismatches usually at positions - <dig>  - <dig> and - <dig> .
the lower stem flanking sequences distal to the upper stem were single stranded.
thermodynamic profiles and patterns of nucleotide substitutions
the average thermodynamic profile calculated from the predicted minimum free energy  structure of each species was nearly identical at the level of the upper stem and more variable for the lower stem of both micrornas .
a common feature of both the upper and lower stem was that the secondary structure was less stable  at the 5' side with an increase in stability in the central part and at the 3' side.
the level of nucleotidic conservation across species, however, did not correlate with the dg values, indicating that the observed footprints could not be explained by a simple increase in the stability of the corresponding secondary structure .
on the contrary, the comparison of mir168a and mir168b thermodynamic profiles and the classification of their nucleotide substitutions with respect to base pairing indicated a clear positional effect concerning the lower stem: the central region was more variable than the 3– <dig> bp close to each end of both stems.
in particular the nucleotidic stretch of 5– <dig> bp connecting upper and lower stems of both micrornas  were extremely conserved despite having an average free energy of - <dig>  kcal/mole, which is the average free energy of both stems.
the highest number of both structurally conservative  and non-conservative nucleotide substitutions  was found in the central portion of mir168a lower stem.
this was in stark contrast with the whole upper stem and the neighboring  <dig> bp of the lower stem in mir168a, where no nucleotide substitutions were observed, indicating the effect of a strong purifying selection.
on the contrary, an overall lower number of substitutions  were spread all along the stem of mir168b, with a clear depletion towards the ends of both upper and lower stems.
expression pattern of mir168a and mir168b
the high conservation of mir168b suggests that it could be expressed and functional, even though, up to now, no experimental evidence has been reported.
the arabidopsis intergenic region upstream of the mature mir168b is only approximately  <dig> bp long.
therefore, we used two genomic regions including the whole intergenic region plus  <dig> or  <dig> bp upstream to functionally characterize the mir168b promoter and ascertain if some regulatory elements may be present in the upstream gene.
these two regions were used to drive the expression of a reporter egfp-uida fusion gene  in stably transformed arabidopsis transgenic lines.
a construct encompassing the mir168a promoter was used as a control .
both pmir168b1::gfp-gus and pmir168b2::gfp-gus constructs produced the same expression pattern .
this result indicates that the intergenic region used in the shortest construct contains all the regulatory information to drive mir168b expression.
similarly to what was observed for mir168a, the expression of mir168b was localized in emerging leaves and in a region underneath the shoot apical meristem corresponding to leaf primordia .
none of the mir168b transgenic lines, in contrast to mir168a, displayed expression in correspondence with vascular tissues.
discussion
since the first reports about the presence of micrornas in plants  <cit>  a number of mirna families have been identified.
while attention has been devoted mostly to their discovery, both in silico and experimentally, relatively little is as yet known about plant microrna evolution and biogenesis.
in this study we applied a phylogenetic footprinting approach to the comparative study of the evolutionary patterns of two paralogous microrna loci, mir168a and mir168b, in the brassicaceae family.
the presence of highly conserved phylogenetic footprints, in fact, is an indication of selective constraints acting on specific sequences  <cit> .
if, as in the case of mir <dig>  the divergence time among genes can be demonstrated to be sufficiently high, parallel phylogenetic footprinting of paralogs provides a powerful tool to yield evolutionary evidence for the functionality of a locus as a whole or of its parts.
evolution of mir <dig> in brassicaceae
based on the analysis of synteny conservation and on the phylogenetic reconstruction of a set of closely linked homologs, we dated the origin of arabidopsis mir <dig> paralogs to shortly before the divergence between the sister families brassicaceae and cleomaceae, about  <dig> million years ago  <cit> .
applying a genome walking method based on microsyntenic conservation, we were able to ascertain reliably the presence of and isolate both mir168a and mir168b paralogs in all analyzed species.
the successful isolation of both mir168a and mir168b from the most basal crucifer, aethionema grandiflora, provides demonstration that the origin of mir <dig> paralogs predates both arabidopsis alpha polyploidization, which took place approximately  <dig> million years ago   <cit> , and brassicaceae radiation which took place between  <dig> and  <dig> mya  <cit> .
the limited synteny conservation observed in arabidopsis further suggests that the mir168a and mir168b paralogs escaped the extensive diploidization resulting in the maintenance of only one homeolog per locus in the surrounding regions.
similarly to mir319a  <cit> , we identified phylogenetic footprints that corresponded to functionally relevant regions, such as the tss and the mature mir and mir* sequences, that indicate a functional conservation of both mir168a and mir168b throughout the brassicacea family.
additionally, in the present study a novel  <dig> nt highly conserved region has been identified immediately downstream of the lower stem.
the palindromic structure of this phylogenetic footprint and its pattern nearly perfectly matched the consensus-binding site of apg <dig>  the tobacco putative ortholog of a. thaliana bme <dig>  this would suggest its function as a homodimeric transcription factor binding site  <cit> .
the functional complementation with mir168a promoter, however, indicates that this motif is not necessary for normal mir <dig> expression  <cit> .
it may, instead, have a functional relevance for rna processing or stability even while not being involved in any of the predicted pre-mir <dig> secondary structures.
the lack of a significant over-representation downstream of other microrna gene families in arabidopsis indicates that this motif is not involved in a general mechanism of microrna biogenesis or regulation.
however, the occurrence of the same motif dowstream of mir <dig>  a microrna predicted to target ago <dig>  raises the interesting possibility that it may be specifically involved in the regulation of ago genes by micrornas.
further studies are, therefore, required to clarify the functional relevance of this phylogenetic footprint.
role of the lower stem in mir <dig> biogenesis
based on the combination of phylogenetic footprinting and secondary structure predictions, the only secondary structures conserved in mir <dig> during the approximately  <dig> million years of brassicaceae evolution were the stem containing the mir-mir* pairing  and its distal extension .
recently it has been demonstrated that correct animal pri-mirna processing depends on the length of the lower stem  <cit> .
in agreement with this, our results indicate that the lower stem is particularly conserved in mir <dig>  with the difference that the phylogenetic footprint identified in plants  is significantly longer than the  <dig> base pair lower stem reported for animals  <cit> .
in animals, the drosha-pasha  complex required for pre-mir processing is responsible for conversion of pri-mirna to pre-mirna  <cit> .
in plants, this function is carried out by a functionally analogous complex involving dcl <dig>  hyl <dig> and se  <cit> .
the observed difference in length of the lower stems may, therefore, indicate a general difference in the mechanisms of mirna biogenesis in plants and animals.
the phylogenetic footprints identified in this study are consistent with two step pri-mirna processing analogous to that described for mir <dig> in arabidopsis  <cit> .
the recent origin of mir <dig> and the extensive base complementarity of its inverted repeats  <cit>  may indicate that the multi-step processing of this microrna could be more an exception than the rule.
our finding that a clearly detectable selective pressure has been acting on mir <dig> lower stem throughout brassicaceae radiation indicates that multi-step pri-mirna processing is not peculiar to mir <dig> or to newly formed micrornas.
hyl <dig> has been recently shown to interact with dcl <dig> for the correct processing of mir <dig>  assuming a common processing mechanism, it is possible that the highly conserved regions we identified in mir <dig> at the ends of both lower and upper stems may be the footprints of the dcl1/hyl <dig> complex  <cit> .
the phylogenetic reconstruction carried out on concatenated mir168a and mir168b sequences indicates that mir <dig> evolution did not depart from that of the analyzed species.
interestingly, however, while a large difference in purifying selection is evident in mir168a, the distribution of nucleotide substitutions turns out to be much more uniform in the case of mir168b, as also reflected by their thermodynamic profiles.
this may indicate that the lower stem has a function in fine-tuning the pri-mir <dig> precursor processing efficiency.
function of mir <dig> paralogs in arabidopsis
the high conservation of mir168a and mir168b sequences, rna predicted secondary structures and thermodynamic profiles observed in all the species we analyzed indicates that constant selective pressure has been acting on both loci throughout the brassicaceae evolution.
interestingly, these results point to the fact that mir168b has most likely been functionally conserved in all of the tested species.
former attempts to confirm mir168b expression by race were not successful, possibly due to tissue specific expression  <cit> .
in contrast to the extreme conservation observed in both mir168a and mir319a  <cit> , mir168b tss identified in a. thaliana by race mapped to a phylogenetic footprint only partly conserved in the examined species, thus leaving open the possibility that the second footprint identified may function as a primary or alternative tss in other species.
this lower conservation indicates a lower selective pressure acting on the expression of mir168b as compared with mir168a, consistent with an accessory function of this locus  <cit> .
however, the clear staining we observed in a. thaliana transformed with a uida reporter gene driven by the whole intergenic regions of mir168b and part of its upstream gene confirms mir168b expression.
taken together, these results and the presence in the mir168b stem-loop structure of the sequence information necessary for processing the mature microrna  <cit> , provide evidence for the functionality of this locus.
the similar but more circumscribed expression pattern of mir168b as compared with mir168a is consistent with either neo- or sub-functionalization of duplicated genes previously reported for other microrna loci  <cit> - <cit> .
in light of the nearly overlapping expression patterns of mir168a and ago <dig>  <cit> , the difference in expression in the leaf vasculature observed between mir <dig> paralogs is most likely due to sub-functionalization of mir168b than to neo-functionalization of mir168a.
CONCLUSIONS
phylogenetic footprinting is a powerful technique for the identification of regions that, being functionally relevant, have been maintained under selective constraint during evolution  <cit> .
we used a comparative phylogenetic footprinting approach to identify the structural and functional constraints that shaped the evolution of mir <dig> paralogs in brassicaceae.
previous studies in arabidopsis demonstrated the functionality of mir168a  <cit> , but left open the possibility that mir168b may be either non-functional or functionally redundant with respect to its paralog.
although their duplication happened at least  <dig> million years ago, we found evidence that both mir <dig> paralogs have been maintained throughout brassicaceae evolution.
the extremely high conservation of regions functionally relevant for microrna expression and biogenesis, predicted secondary structure and thermodynamic profile also provide evolutionary evidence of functionality of both loci, as further supported by the expression of mir168b in arabidopsis.
interestingly, the expression pattern of mir168b indicates partial sub-functionalization based on the expression patterns of both mir168a and ago <dig>  the identification of a highly conserved mir168b-specific footprint downstream of the tss matching the binding sites for the agl <dig> and agl <dig> transcription factors  <cit> - <cit> , provides the indication for a first candidate motif possibly involved in the regulation of mir168b at specific developmental stages.
the phylogenetic footprinting carried out on the mir <dig> paralogs finally points to the fact that the mir <dig> lower stem  is significantly longer than animal lower stems and possibly indicates a multistep mir <dig> biogenesis process analogous to the one for mir <dig> maturation.
the application of phylogenetic footprinting to more microrna and plant families holds the promise of furthering our understanding of the regulation of biogenesis, the function and evolution of these intriguing regulators of both animal and plant gene expression.
the design of artificial micrornas  <cit>  and its application to both basic and applied research may also greatly benefit from a more detailed identification of the determinants for efficient mirna biogenesis.

 micrornas are a large class of recently discovered short non-coding rnas involved in post-transcriptional regulation of protein-coding genes. in plants they repress gene expression by catalytic mrna degradation on the basis of sequence homology between the microrna itself and a target sequence. through this function they act as major players in the regulation of a series of fundamental processes in plant growth and development, in response to biotic and abiotic stress and in the regulation of components of the plant silencing machinery itself. in plants, rna polymerase ii produces a long primary transcript folded in a typical stem-loop structure that is processed by a dicer-like rnase iii ribonuclease, first in a shorter mirna precursor and then in the mirna:mirna* duplex. the mirna:mirna* duplex is transported to the cytoplasm and the mature mirna is incorporated in the rna-induced silencing complex where it drives the slicer argonaute to silence the target mrna. plant mirnas have been found in a wide variety of species and several mirna families are evolutionarily highly conserved, ranging from mosses and ferns to dicots. the members of each mirna family normally retain a complete or almost complete conservation of mirna and mirna* sequences and of the structure formed by their pairing. generally strong conservation constraints characterize the sequences and structure of the pre-mirna hairpin structure, whereas the conservation constraints on loop and flanking sequences are less tight. this is due to the fact that in plants mirna processing depends on pre-mirna structure rather than on sequence and in particular on the structure of the flanking sequences rather than on the mature mirna itself. a detailed analysis of mir biogenesis has revealed that the release of the mature microrna requires at least three dcl cleavage steps spaced by nucleotide intervals each, starting from the base of its unusually long lower stem. similar studies in animals have shown that structural features of the lower stem are essential for cleavage of pri-mirna by drosha. in contrast to the complexity that regulatory cascades of transcription factors can reach, plant micrornas are organized according to a simple, two-level hierarchy: only three of them, mir mir and mir, control their own expression and that of the other mirnas by targeting specific proteins involved in the post-transcriptional gene silencing pathway. in particular, mir regulates the function of all mirnas by targeting ago expression, therefore modulating its actual levels and consequently risc activity. mir is present in a low copy number in different plant species and in the arabidopsis genome two mir paralogs are present. only mir168a, for which the primary transcript has been isolated, was shown to be involved in ago post-transcriptional gene silencing in arabidopsis. a mir168a-resistant version of ago showed increased levels of ago mrna, the over-accumulation of mir and developmental defects partially overlapping with those observed in dcl hen and hyl mutants. a complex feedback loop, involving on the one hand cleavage of ago transcripts directed by mir and on the other hand stabilization of mir through ago association, was shown to maintain ago homeostasis which is pivotal for mirna-mediated post-transcriptional gene silencing. the overlapping expression patterns of mir168a and ago and the restored development and fertility in ago mutants expressing mir168a-promoter:ago fusion support this model. despite the relevance of mir168a in plant development, up to now no detailed comparative study has been carried out to characterize its evolution, nor has the function of its paralog mir168b been determined. in this study we applied phylogenetic footprinting to the characterization of the genomic organization, and structural and functional evolution of mir sets of orthologs in brassicaceae. we found that, despite having originated before brassicaceae radiation, mir168a and mir168b paralogs have been maintained, most likely as functional, throughout brassicacea evolution, with mir168b having undergone a partial sub-functionalization. we also provide evolutionary evidence that the lower stem in the pre-mirna structure is significantly longer than lower stems in animals and propose the hypothesis that, similarly to mir it may play a relevant role in multi-step mir biogenesis.  synteny of mir168a and mir168b loci in a. thaliana and p. trichocarpa in the genomes of both a. thaliana and p. trichocarpa two mir loci have been identified, called mir168a and mir168b, located respectively on chromosome and in arabidopsis and on linkage_group_iii and scaffold_ in poplar. analyses of synteny conservation were carried out by searching in poplar for the putative orthologs of the arabidopsis genes flanking mir168a and mir168b by screening for reciprocal best matches in blastp searches. the queries from the former analyses were then used to identify recent segmental duplications. assuming orthology among the arabidopsis and poplar genomic regions encompassing the mir loci, the surrounding rbm pairs should be found mainly among the same pair of chromosomes. the uneven distribution of loci forming rbm pairs, however, indicated that the mir loci may have been the result of independent duplication events. dating of duplication events only two arabidopsis paralogs formed rbm pairs in poplar. to determine the chronological order of these duplications, we carried out a phylogenetic reconstruction of all the genes that are homologous to the rbm pairs in the two genomes. the results show that the splitting of the two species predated two duplication events that took place independently in the arabidopsis and poplar lineages. the two arabidopsis paralogs, at4g and at5g displayed a rate of synonymous substitution of, a higher value than that observed for paralogs resulting from the arabidopsis alpha whole genome duplication. the two poplar paralogs, eugene and estext_fgenesh4_pg.c_ were confirmed to have diverged more recently. the divergence between poplar and arabidopsis homologs ranged between ks = and ks =. based on the estimated divergence time between cleomaceae and brassicaceae, this should correspond to a poplar-arabidopsis divergence time of about million years, in full agreement with the 100– million year range provided by previous reports. this dating agrees with the observation that synteny conservation between arabidopsis and poplar is higher than between arabidopsis chromosomes. taken together, these results indicate that no orthologous relationship can be inferred between arabidopsis and poplar mir homologs. genomic characterization of mir loci in brassicaceae species on the basis of these results we focused on analysis of the evolution and conservation between species of the two mir homologs in a group of brassicaceae species. amplification of the intergenic regions upstream and downstream of mir168a and mir168b in brassicaceae species. ir: intergenic region; kbp: kilo-basepair. n.d.: not determined. a, b: whole mir168a or mir168b loci obtained from the upstream to the downstream gene. mir168a and mir168b homologs were amplified through a gene-to-gene amplification based on their up- and downstream genes in arabidopsis. the intergenic region downstream of mir168a was amplified from all the species with an amplification rate double than that of the upstream intergenic region. in the case of mir168b the intergenic regions were fully isolated in most of the species. the taxonomic distance of the single species from arabidopsis did not significantly affect the isolation of intergenic regions. the isolation of intergenic regions and the level of sequence conservation between species highlighted by their multiple alignments indicate: general micro-synteny conservation in the regions surrounding mir168a and mir168b and conservation of the orthologous relationship of all isolated mir168a and mir168b genes at the family level. mir168a and mir168b phylogenetic footprinting a clear phylogenetic footprint was identified in all species ~100– bp upstream of the mature mir168a in correspondence with arabidopsis mir168a transcription start site. on the contrary, the use of different alignment programs failed to identify a highly conserved footprint corresponding to mir168b tss. the location of mir168b tss in arabidopsis was therefore determined by sequencing race products obtained from pmir168b1::gfp-gus transgenic lines. the 5' end of all clones mapped in three points of a region ~60– bp upstream of the mature mir168b proximal to a tata-like motif conserved in both paralogs. the three tss conformed in all cases to the ta class of dinucleotides identified by the yr rule. this poorly conserved footprint could be identified by manual editing of a multiple sequence alignment performed with clustalw, thus indicating a lower functional constraint on mir168b as compared to mir168a transcription. detailed analysis of pre-mir168a and pre-mir168b and flanking sequences revealed a considerable conservation of the pre-mirna sequences at both loci. both mir and mir168* were completely or almost completely conserved between orthologs and paralogs in all species. the ~ bp flanking regions preceding the mature mir and following the mir168* showed a significant level of sequence conservation between orthologs and also, although to a lower extent, between paralogs. a completely conserved bp long motif was isolated in both mir168a and mir168b just downstream of the pre-microrna. despite being a palindromic structure, it was not involved in any predicted secondary structure. searches for this motif in the athamap database showed a high quality match with the binding site of the tobacco agp transcription factor. no significant over-representation of the bp motif downstream of microrna loci was detected as compared with coding genes. an identical pattern was also detected in mir396a downstream of, but at a higher distance as compared with mir to check for over-representation of this motif in specific groups of micrornas, microrna superfamilies were defined based on the classification of their targets. the application of random permutation resampling approach led to the identification of only one superfamily which showed an enrichment in this motif. this superfamily encompasses both mir paralogs and mir a microrna targeting argonaute that is a member of the argonaute family of slicers responsible for mrna cleavage in ptgs. a footprint specific to mir168b was located about bp downstream of the tss. the footprint matched the binding sites of agamous like and agamous like, two mads-box domain transcription factors involved in floral organ identity and meristem determinacy. the presence of a bp insertion in the basal species aethionema grandiflora prompted us to separately consider two sub-motifs. their occurrence upstream of arabidopsis micrornas was not significantly over-represented compared to 5'utrs of all arabidopsis coding genes. no statistical support for their preferential occurrence in the 5' region of specific microrna superfamilies was found at the level. phylogenetic reconstruction of mir168a and mir168b phylogenetic reconstruction with all brassicaceae mir homologs confirmed the successful isolation of orthologs of arabidopsis mir168a and mir168b. the limited amount of parsimony-informative sites, however, could not provide a phylogenetic reconstruction resolved enough to compare the evolutionary rates of the single mir loci. two data partitions were created by concatenating mir168a with mir168b and its with eif3e. the resulting phylogenetic reconstructions of mir as compared with the its-eif3e neutral markers showed slightly incongruent topologies that are the consequence of the overall lower resolution provided by the mir partition. comparative analysis of predicted pre-mir168a and pre-mir168b structures secondary structures for pre-mir168a and pre-mir168b plus bp of flanking sequences on each side were predicted based on free energy minimization. the consensus of the most conserved portion of these regions, including about bp upstream of mature mir and downstream of mir168*, is shown in figure the mature microrna-microrna* secondary structure was completely conserved in the case of mir168a and almost completely conserved in the case of mir168b. the structure adjacent to the upper stem was also highly conserved in mir168a and mir168b. in mir168a it ranged from to bp, with two mismatches and one bulge loop. the lower stem of the predicted mir168b structure was to bp long and presented three mismatches usually at positions - - and -. the lower stem flanking sequences distal to the upper stem were single stranded. thermodynamic profiles and patterns of nucleotide substitutions the average thermodynamic profile calculated from the predicted minimum free energy structure of each species was nearly identical at the level of the upper stem and more variable for the lower stem of both micrornas. a common feature of both the upper and lower stem was that the secondary structure was less stable at the 5' side with an increase in stability in the central part and at the 3' side. the level of nucleotidic conservation across species, however, did not correlate with the dg values, indicating that the observed footprints could not be explained by a simple increase in the stability of the corresponding secondary structure. on the contrary, the comparison of mir168a and mir168b thermodynamic profiles and the classification of their nucleotide substitutions with respect to base pairing indicated a clear positional effect concerning the lower stem: the central region was more variable than the 3– bp close to each end of both stems. in particular the nucleotidic stretch of 5– bp connecting upper and lower stems of both micrornas were extremely conserved despite having an average free energy of - kcal/mole, which is the average free energy of both stems. the highest number of both structurally conservative and non-conservative nucleotide substitutions was found in the central portion of mir168a lower stem. this was in stark contrast with the whole upper stem and the neighboring bp of the lower stem in mir168a, where no nucleotide substitutions were observed, indicating the effect of a strong purifying selection. on the contrary, an overall lower number of substitutions were spread all along the stem of mir168b, with a clear depletion towards the ends of both upper and lower stems. expression pattern of mir168a and mir168b the high conservation of mir168b suggests that it could be expressed and functional, even though, up to now, no experimental evidence has been reported. the arabidopsis intergenic region upstream of the mature mir168b is only approximately bp long. therefore, we used two genomic regions including the whole intergenic region plus or bp upstream to functionally characterize the mir168b promoter and ascertain if some regulatory elements may be present in the upstream gene. these two regions were used to drive the expression of a reporter egfp-uida fusion gene in stably transformed arabidopsis transgenic lines. a construct encompassing the mir168a promoter was used as a control. both pmir168b1::gfp-gus and pmir168b2::gfp-gus constructs produced the same expression pattern. this result indicates that the intergenic region used in the shortest construct contains all the regulatory information to drive mir168b expression. similarly to what was observed for mir168a, the expression of mir168b was localized in emerging leaves and in a region underneath the shoot apical meristem corresponding to leaf primordia. none of the mir168b transgenic lines, in contrast to mir168a, displayed expression in correspondence with vascular tissues. discussion since the first reports about the presence of micrornas in plants a number of mirna families have been identified. while attention has been devoted mostly to their discovery, both in silico and experimentally, relatively little is as yet known about plant microrna evolution and biogenesis. in this study we applied a phylogenetic footprinting approach to the comparative study of the evolutionary patterns of two paralogous microrna loci, mir168a and mir168b, in the brassicaceae family. the presence of highly conserved phylogenetic footprints, in fact, is an indication of selective constraints acting on specific sequences. if, as in the case of mir the divergence time among genes can be demonstrated to be sufficiently high, parallel phylogenetic footprinting of paralogs provides a powerful tool to yield evolutionary evidence for the functionality of a locus as a whole or of its parts. evolution of mir in brassicaceae based on the analysis of synteny conservation and on the phylogenetic reconstruction of a set of closely linked homologs, we dated the origin of arabidopsis mir paralogs to shortly before the divergence between the sister families brassicaceae and cleomaceae, about million years ago. applying a genome walking method based on microsyntenic conservation, we were able to ascertain reliably the presence of and isolate both mir168a and mir168b paralogs in all analyzed species. the successful isolation of both mir168a and mir168b from the most basal crucifer, aethionema grandiflora, provides demonstration that the origin of mir paralogs predates both arabidopsis alpha polyploidization, which took place approximately million years ago, and brassicaceae radiation which took place between and mya. the limited synteny conservation observed in arabidopsis further suggests that the mir168a and mir168b paralogs escaped the extensive diploidization resulting in the maintenance of only one homeolog per locus in the surrounding regions. similarly to mir319a, we identified phylogenetic footprints that corresponded to functionally relevant regions, such as the tss and the mature mir and mir* sequences, that indicate a functional conservation of both mir168a and mir168b throughout the brassicacea family. additionally, in the present study a novel nt highly conserved region has been identified immediately downstream of the lower stem. the palindromic structure of this phylogenetic footprint and its pattern nearly perfectly matched the consensus-binding site of apg the tobacco putative ortholog of a. thaliana bme this would suggest its function as a homodimeric transcription factor binding site. the functional complementation with mir168a promoter, however, indicates that this motif is not necessary for normal mir expression. it may, instead, have a functional relevance for rna processing or stability even while not being involved in any of the predicted pre-mir secondary structures. the lack of a significant over-representation downstream of other microrna gene families in arabidopsis indicates that this motif is not involved in a general mechanism of microrna biogenesis or regulation. however, the occurrence of the same motif dowstream of mir a microrna predicted to target ago raises the interesting possibility that it may be specifically involved in the regulation of ago genes by micrornas. further studies are, therefore, required to clarify the functional relevance of this phylogenetic footprint. role of the lower stem in mir biogenesis based on the combination of phylogenetic footprinting and secondary structure predictions, the only secondary structures conserved in mir during the approximately million years of brassicaceae evolution were the stem containing the mir-mir* pairing and its distal extension. recently it has been demonstrated that correct animal pri-mirna processing depends on the length of the lower stem. in agreement with this, our results indicate that the lower stem is particularly conserved in mir with the difference that the phylogenetic footprint identified in plants is significantly longer than the base pair lower stem reported for animals. in animals, the drosha-pasha complex required for pre-mir processing is responsible for conversion of pri-mirna to pre-mirna. in plants, this function is carried out by a functionally analogous complex involving dcl hyl and se. the observed difference in length of the lower stems may, therefore, indicate a general difference in the mechanisms of mirna biogenesis in plants and animals. the phylogenetic footprints identified in this study are consistent with two step pri-mirna processing analogous to that described for mir in arabidopsis. the recent origin of mir and the extensive base complementarity of its inverted repeats may indicate that the multi-step processing of this microrna could be more an exception than the rule. our finding that a clearly detectable selective pressure has been acting on mir lower stem throughout brassicaceae radiation indicates that multi-step pri-mirna processing is not peculiar to mir or to newly formed micrornas. hyl has been recently shown to interact with dcl for the correct processing of mir assuming a common processing mechanism, it is possible that the highly conserved regions we identified in mir at the ends of both lower and upper stems may be the footprints of the dcl1/hyl complex. the phylogenetic reconstruction carried out on concatenated mir168a and mir168b sequences indicates that mir evolution did not depart from that of the analyzed species. interestingly, however, while a large difference in purifying selection is evident in mir168a, the distribution of nucleotide substitutions turns out to be much more uniform in the case of mir168b, as also reflected by their thermodynamic profiles. this may indicate that the lower stem has a function in fine-tuning the pri-mir precursor processing efficiency. function of mir paralogs in arabidopsis the high conservation of mir168a and mir168b sequences, rna predicted secondary structures and thermodynamic profiles observed in all the species we analyzed indicates that constant selective pressure has been acting on both loci throughout the brassicaceae evolution. interestingly, these results point to the fact that mir168b has most likely been functionally conserved in all of the tested species. former attempts to confirm mir168b expression by race were not successful, possibly due to tissue specific expression. in contrast to the extreme conservation observed in both mir168a and mir319a, mir168b tss identified in a. thaliana by race mapped to a phylogenetic footprint only partly conserved in the examined species, thus leaving open the possibility that the second footprint identified may function as a primary or alternative tss in other species. this lower conservation indicates a lower selective pressure acting on the expression of mir168b as compared with mir168a, consistent with an accessory function of this locus. however, the clear staining we observed in a. thaliana transformed with a uida reporter gene driven by the whole intergenic regions of mir168b and part of its upstream gene confirms mir168b expression. taken together, these results and the presence in the mir168b stem-loop structure of the sequence information necessary for processing the mature microrna, provide evidence for the functionality of this locus. the similar but more circumscribed expression pattern of mir168b as compared with mir168a is consistent with either neo- or sub-functionalization of duplicated genes previously reported for other microrna loci -. in light of the nearly overlapping expression patterns of mir168a and ago, the difference in expression in the leaf vasculature observed between mir paralogs is most likely due to sub-functionalization of mir168b than to neo-functionalization of mir168a.  phylogenetic footprinting is a powerful technique for the identification of regions that, being functionally relevant, have been maintained under selective constraint during evolution. we used a comparative phylogenetic footprinting approach to identify the structural and functional constraints that shaped the evolution of mir paralogs in brassicaceae. previous studies in arabidopsis demonstrated the functionality of mir168a, but left open the possibility that mir168b may be either non-functional or functionally redundant with respect to its paralog. although their duplication happened at least million years ago, we found evidence that both mir paralogs have been maintained throughout brassicaceae evolution. the extremely high conservation of regions functionally relevant for microrna expression and biogenesis, predicted secondary structure and thermodynamic profile also provide evolutionary evidence of functionality of both loci, as further supported by the expression of mir168b in arabidopsis. interestingly, the expression pattern of mir168b indicates partial sub-functionalization based on the expression patterns of both mir168a and ago the identification of a highly conserved mir168b-specific footprint downstream of the tss matching the binding sites for the agl and agl transcription factors -, provides the indication for a first candidate motif possibly involved in the regulation of mir168b at specific developmental stages. the phylogenetic footprinting carried out on the mir paralogs finally points to the fact that the mir lower stem is significantly longer than animal lower stems and possibly indicates a multistep mir biogenesis process analogous to the one for mir maturation. the application of phylogenetic footprinting to more microrna and plant families holds the promise of furthering our understanding of the regulation of biogenesis, the function and evolution of these intriguing regulators of both animal and plant gene expression. the design of artificial micrornas and its application to both basic and applied research may also greatly benefit from a more detailed identification of the determinants for efficient mirna biogenesis. 
interestingly, the expression patterns observed in arabidopsis indicate that mir168b underwent partial subfunctionalization as determined by the experimental characterization of its expression pattern provided in this study. we applied different approaches to studying the genomic organization and the structural and functional evolution of mir homologs in brassicaeae. while orthology was lacking between arabidopsis and poplar mir genes, we successfully isolated orthologs of both loci present in arabidopsis from all the brassicaceae species analyzed, including the basal species aethionema grandiflora, thus indicating that independent duplication events took place in arabidopsis and poplar lineages and the origin of mir paralogs predates both the brassicaceae radiation and the arabidopsis alpha polyploidization. a whole genome comparison of arabidopsis and poplar, phylogenetic footprinting and phylogenetic reconstruction were used to date the duplication events originating mir homologs in these genomes. we found further evolutionary evidence that pre-mir lower stem is significantly longer than animal lower stems and probably plays a relevant role in multi-step mir biogenesis. different phylogenetic footprints, corresponding to known functionally relevant regions or for which functions could be proposed, were found to be highly conserved among mir homologs. although their duplication happened at least million years ago, we found evidence that both mir paralogs have been maintained throughout the evolution of brassicaceae, most likely functionally as indicated by the extremely high conservation of functionally relevant regions, predicted secondary structure and thermodynamic profile. we used a comparative phylogenetic footprinting approach to identify the structural and functional constraints that shaped mir evolution in brassicaceae. comparative predictions of the identified micrornas also indicate extreme conservation of secondary structure and thermodynamic stability. in plants, expression of argonaute, the catalytic subunit of the rna-induced silencing complex responsible for post-transcriptional gene silencing, is controlled through a feedback loop involving the mir microrna.
 in plants, expression of argonaute, the catalytic subunit of the rna-induced silencing complex responsible for post-transcriptional gene silencing, is controlled through a feedback loop involving the mir microrna. this complex auto-regulatory loop, composed of mir168-guided ago1-catalyzed cleavage of ago mrna and ago1-mediated stabilization of mir was shown to ensure the maintenance of ago homeostasis that is pivotal for the correct functioning of the mirna pathway.  we applied different approaches to studying the genomic organization and the structural and functional evolution of mir homologs in brassicaeae. a whole genome comparison of arabidopsis and poplar, phylogenetic footprinting and phylogenetic reconstruction were used to date the duplication events originating mir homologs in these genomes. while orthology was lacking between arabidopsis and poplar mir genes, we successfully isolated orthologs of both loci present in arabidopsis from all the brassicaceae species analyzed, including the basal species aethionema grandiflora, thus indicating that independent duplication events took place in arabidopsis and poplar lineages and the origin of mir paralogs predates both the brassicaceae radiation and the arabidopsis alpha polyploidization. different phylogenetic footprints, corresponding to known functionally relevant regions or for which functions could be proposed, were found to be highly conserved among mir homologs. comparative predictions of the identified micrornas also indicate extreme conservation of secondary structure and thermodynamic stability.  we used a comparative phylogenetic footprinting approach to identify the structural and functional constraints that shaped mir evolution in brassicaceae. although their duplication happened at least million years ago, we found evidence that both mir paralogs have been maintained throughout the evolution of brassicaceae, most likely functionally as indicated by the extremely high conservation of functionally relevant regions, predicted secondary structure and thermodynamic profile. interestingly, the expression patterns observed in arabidopsis indicate that mir168b underwent partial subfunctionalization as determined by the experimental characterization of its expression pattern provided in this study. we found further evolutionary evidence that pre-mir lower stem is significantly longer than animal lower stems and probably plays a relevant role in multi-step mir biogenesis. 