BACKGROUND
micrornas  are small rnas important to transcriptional and post-transcriptional regulation in animals, plants, and viruses.
mirnas bind complementarily to their target mrna sequences, leading to translational repression, rna degradation, or rna cleavage  <cit> .
most plant mirnas are encoded by gene families, and mature mirnas often have multiple target genes with similar complementary motifs in their mrnas.
the almost perfect complementarity between mirnas and targets facilitates computational prediction and could be due to their evolutionary origins.
one well-accepted model for mir gene evolution is the inverted duplication of target gene sequences in plant genomes.
these duplicated regions become mir genes over time through sequence variations, which permit the generation of hairpin-like transcripts to become substrates for dicer-like enzymes  <cit> .
another interesting model suggests that mir genes can randomly originate from various inverted repeats throughout the genome, independently of target gene duplications .
for instance, recent evidences indicate that mir genes may be generated as a result of transposon activity  <cit> .
it has been recently shown that evolutionary patterns of mirna genes  can potentially influence the evolution of their target genes  <cit> .
moreover, synonymous codons in target genes have been found near conserved mirna target sites in at least four plant genomes, indicating selection constraint on synonymous codons for efficient mirna binding and proper mirna function  <cit> .
several mirnas are conserved throughout large evolutionary distances from embryophyta to core rosids.
however, some mirnas appear to be species or lineage specific .
for instance, mir <dig> is conserved in all angiosperms studied thus far.
interestingly, mir <dig> is correlated at the nucleotide level with mir <dig>  sharing 14–16 nt  <cit> .
curiously, although both mirnas have a common ancestor in embryophytes, mir <dig> seems to have been lost in some taxonomic groups, including core eudicots such as arabidopsis thaliana .
both mirnas share target genes, which are members of the squamosa promoter-binding protein like  family  <cit> .
spl genes are plant-specific transcription factors defined by a highly conserved region of  <dig> amino acids called sbp domain  <cit> , and by their crucial and widespread functions in development .
spl genes can be roughly separated into two major groups–long and short–the latter containing responsive elements for mir <dig> and, in some species, for mir <dig> and mir <dig>  interestingly, sites for mir <dig> reside in coding as well as untranslated regions of target sequences, whereas mir <dig> binding sites are chiefly located in coding regions and overlap with mir <dig> sites  <cit> .
in plant lineages containing both mirna genes, differential expression of mir <dig> and mir <dig> in vegetative and reproductive organs/tissues might have favored lineage-specific retention of mir156/ <dig> sequence variants in their genomes  <cit> .
another possibility is that the combinatory action of mir <dig> and mir <dig> leads to the regulation of distinct targets in specific lineages, such as in physcomitrella patens  <cit> .
recently, the evolutionary differences between these two mirna families in monocot species have been investigated  <cit> , but the consequences of mir <dig> loss in core eudicots such as arabidopsis are not yet clear.
a broader evolutionary analysis of mir <dig> and mir <dig> genes and their targets including eudicot species should offer valuable insight into this issue.
in this study, we extended our knowledge regarding evolutionary and functional divergences between mir <dig> and mir <dig> regulation on their conserved targets.
through phylogenic reconstruction of target sequences, we confirmed that specific eudicot spl family members retained mir529-target sites, independently of the presence of mir <dig> genes in their genomes.
loss of mir <dig> function in eudicots is correlated with a more relaxed selection of the mirna529-specific target element, while mirna156-specific target sequence is under stronger selection.
additionally, we showed that a. thaliana plants overexpressing mir <dig> precursor from a monocot, but not from a basal eudicot, display similar phenotypes as the spl9;spl <dig> mutant due to the specific down-regulation of these mir156/529-targeted spls.
based upon functional and evolutionary analyses, we proposed that the loss of mir <dig> genes might have favored diversification of spls in eudicot species.
it is also possible that new mir <dig> family member have replaced mir <dig> functions in eudicots.
RESULTS
sequence and phylogenetic analyses reveal that a mir529-responsive element in spl genes is broadly conserved across land plants
mir <dig> genes are present in genomes of a number of species across land plants.
accordingly, in such species, transcripts of a subset of spl family have responsive elements for both mir <dig> and mir <dig>  <cit> .
to get a better view of the extent to which a mir529-responsive element is conserved in land plants, we searched for mir529-responsive elements in spl genes from species with publicly available genome sequences, including those species in which mirna <dig> is absent in their genomes or in transcribed sequences identified thus far.
high-confidence prediction of mirna targets was performed by psrnatarget based on sequence complementarity and evolutionary conservation  <cit> .
we collected conserved mature mir <dig> sequences from three species , which are available in mirbase v.  <dig>  and shown in fig.
<dig>  we then employed more stringent cut-off threshold for the maximum expectation  parameter  to minimize false positive target prediction .
surprisingly, we found spl genes from several species that retain a highly conserved and overlapping responsive element  for both mirnas , independently of the presence of mir <dig> genes in their genomes.
this suggests that, whereas mir <dig> and mir <dig> genes have undergone distinct evolutionary fates  <cit> , their mutual targets  have been more conservative even in species which apparently have lost mir <dig> genes.
for instance, the mir156/529-responsive element in eudicot spls resides only in coding regions, similarly to what is observed in monocots and bryophytes .fig.
<dig> alignment and phylogeny of spls containing the mir156/mir529-response element.
a alignments of the 25-nt mir156/529-response element were done using clustalw .
black color denotes conserved nucleotides.
b rooted ml phylogenetic tree depicts the relationship between spls containing the mir156/529-response element from representative species.
the numbers indicate branch support.
left numbers, posterior probability branch support.
right numbers, alrt branch support .
“-”, indicates that a particular branch is not observed in bayesian or maximum-likelihood trees.
blue line comprises moss spls and green line encompasses monocot and eudicot spls.
c target sites for mir <dig> and mir <dig> in a. thaliana spl <dig> and spl <dig> and a. lyrata spl transcripts.
at, arabidopsis thaliana; al, arabidopsis lyrata; th, thellungiella halophila; vv, vitis vinifera; sl, solanum lycopersium; md, malus domestica; fa, fragaria ananassa; pt, populus trichocarpa; gm, glycine max; mt, medicago truncatula; pont, poncirus trifoliata; am, antirrhinum majus; rc, ricinus communis; zm, zea mays; os, oryza sativa; bd, brachypodium distachyon; aqc, aquilegia coerulea; pp, physcomitrella patens.
accession numbers are given in additional file 1
to further explore the evolutionary relationship of mir156/ <dig> common targets, phylogenetic inference of sbp-box genes containing the mir156/529-responsive element was estimated using maximum-likelihood  and bayesian inference methods.
the percentage of pairwise identity of well-aligned sequence blocks  was  <dig>  %, and the substitution saturation test reported that the alignment was not saturated .
we observed two groups of spls in our consensus tree .
group i included known mir156/ <dig> spl targets in bryophyte, whereas group ii contained various monocot and core eudicot spls harboring conserved binding sites for mir156/ <dig>  this analysis indicated that spls containing mir156/ <dig> target sites have a common origin in land plants .
a. thaliana spl <dig> and spl <dig> are closely related and most likely form a pair of paralogous genes  <cit> .
accordingly, both spl <dig> and spl <dig> as well as their orthologs retained the mir156/529-responsive element .
it has been proposed for monocot species and p. patens that spls containing mir156/ <dig> sites evolved conservatively with a slow rate when compared with spls harboring only the mir156-responsive element  <cit> .
to further elucidate the evolutionary fates of eudicot spls containing the mir156/529-responsive element, we analyzed two blocks in spl sequences: “sbp domain” block, which contains nucleotides of the sbp domain  <cit>  plus few nucleotides upstream/downstream, and “target site” block, which contains nucleotides that comprise both mir156/529-responsive elements .
for the “sbp domain” block, we estimated nonsynonymous  and synonymous substitution  ratios  of representative spls.
we chose arabidopsis as a representative eudicot due to the fact that, even after extensive sequencing efforts, precursors or canonical mature sequences of mir <dig> have not been found in either a. thaliana or its closest relative a. lyrata  <cit> .
pairwise alignments of best-aligned blocks  among spl genes from a. thaliana, monocots, and p. patens indicated that the “sbp domain” block is under purifying selection for all comparisons, reflecting functional constraints, which is expected since sbp is likely a dna binding domain .
given that “target site” blocks are short  and could give rise to unrealistic ka/ks ratios, we decided to compare their alignment more directly .
considering the five first bases restricted to the mir <dig> target site, nonsynonymous substitutions including amino acids with different physicochemical features were found.
in contrast, such drastic changes did not occur for the nucleotides specifying mir <dig> target site .
these observations indicated that different regions of the “target site” block might be under distinct evolutionary constraints.
in fact, the bases that diverged more in the “target site” block specifically correspond to the mir <dig> target site .
therefore, it is possible that loss of mir <dig> regulation in eudicots allowed a more relaxed or low purifying selection of the mirna-responsive element region, which reflects its loss of functionality as a mir <dig> target.
in line with this observation, the percentage of nucleotide identity within the “target site” block  is lower among eudicot spl genes  than among monocot and bryophyte ones  .
in monocots and p. patens, the “target site” block is more conserved, likely because it is functionally relevant as mir <dig> is still present in these species  <cit> .fig.
<dig> dna alignments of mir <dig> and mirna156/ <dig> target sites.
amino acids coded by mir156/ <dig> target site are shown.
amino acids are colored according to their hydrophobicity; red box, eudicot sequences; green box, monocot sequences; blue box, bryophyte sequences.
percentage of nucleotide identity for the 5′ four nucleotides is shown at the side of each colored box.
black lines underneath the topmost logo indicate mirna binding sites.
box at the bottom shows logos constructed based on alignments of each plant group.
accession numbers are given in additional file 1
overexpression of a basal eudicot microrna <dig> precursor in a. thaliana phenocopies mir <dig> overexpressor
although mir <dig> is highly conserved, being present in all plant species assessed thus far, mir <dig> seems to be restricted to particular taxonomic groups  <cit> .
to get a better view of the mir156/ <dig> gene evolution, the phylogenetic relationship of these mirnas was accessed using the maximum-likelihood  approach.
for phylogenetic analyses, we included mir <dig> and mir <dig> precursors from physcomitrella patens, monocot species , a basal eudicot , and precursors of mir <dig> genes from arabidopsis thaliana.
a consensus ml tree was generated in which two general, broad groups were readily observed .
group i comprised mir <dig> precursors from different species, while group ii contained mir <dig> precursors from monocots and moss.
monocot mir <dig> precursors were grouped into a distinct subset of group ii , suggesting that evolutionary divergence occurred in a common ancestor of land plants, which led to the split between mir <dig> genes of moss and flowering plants.
it had been proposed earlier that a key feature of mirna evolution is that, once evolved, families and family members are rarely lost  <cit> .
however, not all mirnas are equally conserved and it has been recently shown that several mirna losses occurred in families that evolved prior to the origin of spermatophytes  <cit> .
our data suggest that mir <dig> and mir <dig> families experienced dynamic duplications and losses across embryophytes, through which clade- or species-specific mirna gene subgroups have arisen or were eliminated.
for instance, a. thaliana has at least  <dig> mir <dig> loci and  <dig> mir156-targeted spls, whereas rice has at least  <dig> mir <dig> loci, two mir <dig> loci, and eight mir156-targeted and four mir156/mir529-targeted spls  <cit> .
interestingly, the predicted mir <dig> precursor from the basal eudicot a. coerulea  <cit>  was grouped into group i, with a. thaliana mir156h and aquilegia mir156a and b precursors, indicating a common origin of these mirnas .
moreover, aquilegia mir <dig> seems to be highly conserved with arabidopsis mir156h at both nucleotide and secondary structure levels .
these observations raised the question of whether this mir precursor of aquilegia defined as mir <dig> is indeed a mir <dig> homolog.
to test this hypothesis, we investigated whether loci flanking aquilegia pre-mir <dig> are localized into syntenic blocks when comparing with monocot species.
we firstly searched for such syntenic groups among distinct monocot species, including z. mays, o. sativa, b. distachyon, and s. bicolor .
the colinearity of genes around pre-mir <dig> locus is relatively conserved, therefore defining conserved syntenic block in monocots.
conversely, we did not detect any conservation of syntenic block including pre-mir <dig> from aquilegia, although some orthologous genes were found to be present in this species .
moreover, there is no detectable synteny between mir156h-associated regions from eudicots  and the mir529-associated region from basal eudicot a. coerulea .
this complete loss of synteny, along with our phylogenetic and sequence analyses, suggests that the basal eudicot aquilegia lost a bona-fide mir <dig> gene, perhaps after the regulatory role of mir <dig> was perturbed due to mutations in the mir <dig> sequence as recently proposed  <cit> .
to determine whether aqcmir <dig> could be properly processed and could give rise to functional mirnas, we constitutively expressed its foldback in a. thaliana plants under control of the viral 35s promoter, which confers strong and near-ubiquitous expression .
we compared vegetative phenotypes among col- <dig> or wild type, 35s:: aqcmir <dig> lines, transgenic plants overexpressing the atmir156a precursor , and the double mutant spl9;spl <dig> .
this mutant contains t-dna insertions in both atspl <dig> and atspl <dig> loci, which resulted in an accentuated reduction in their expression and function, although other atspls are still functional.
transgenic a. thaliana plants transformed with the aquilegia-overexpressing construct had phenotypes similar to 35s::mir156a plants, such as a high number of smaller, more rounded rosette leaves .
moreover, overexpression of the mir <dig> precursor from aguilegia in a. thaliana led to the down-regulation not only of atspl <dig> and atspl <dig> genes , but also other spl genes .
thus, 35s::aqcmir <dig> lines showed a stronger tendency toward the phenotype of 35s::mir156a plants .fig.
<dig> phenotypic, expression, and race analyses of arabidopsis 35s::aqcmir <dig> plants.
a morphology of 25-day-old plants .
scale bar represents 1 cm.
b stem–loop pulsed rt-pcr to detect aqcmir <dig> precursor and some atspl transcripts in arabidopsis leaf tissues.
reactions without rt  and without cdna  were used as negative controls.
numbers between brackets indicate pcr cycles.
actin- <dig> was used as an internal control.
c modified 5′-race analyzes of cleaved spl <dig> transcripts in 35s::aqcmir <dig> leaf tissues.
the 5′-ends of the spl <dig> cleavage products preferentially map to mir156
in line with the observed phenotypes and expression analyses, race analysis of spl <dig> cleavage sites demonstrated that transcripts are chiefly targeted by mir <dig> in 35s::aqcmir <dig> plants .
based on these observations and given that aqcmir <dig> precursor is more conserved with mir156-like precursors , we propose at least two non-exclusive possibilities:  the predicted aqcmir <dig> precursor is more likely paralogous to aqcmir156a and b genes and would produce a mirna156-like small rna;  aqcmir <dig> precursor might have accumulated mutations in the mir <dig> sequence as recently proposed  <cit> , leading to a loss of mir <dig> biogenesis and/or function.
as other basal eudicots seem to accumulate mir529-like small rnas  <cit> , we cannot rule out the possibility that mir529-like small rnas still accumulate in specific a. coerulea tissues, since there is no available information regarding large-scale identification of small rna populations in this basal eudicot.
mir <dig> and mir <dig> show overlapping expression patterns during rice vegetative development
distinct from the basal eudicot aquilegia, it is well documented that monocot species retained both mir <dig> and mir <dig> precursors in their genomes  <cit> , raising the question of whether mir <dig> regulation has been retained in monocot species because it is essential or it is just a classical case of redundancy reflecting subfunctionalization in which mir <dig> has a limited effect as compared with mir <dig>  rice has two copies of mir <dig> precursors  in its genome.
it has been shown that osmir529a*  is preferentially expressed in panicle, whereas osmir529b is ubiquitously expressed in roots, shoots, and panicle  <cit> .
the authors, however, did not evaluate the expression of osmir <dig> or osmir <dig> in vegetative apices and young leaves, organs in which spls define an endogenous flowering pathway and control leaf maturation and initiation, respectively  <cit> .
to get more insights into the possible roles of osmir <dig>  <cit> , we analyzed transcript accumulation patterns of osmir529b, osmir156a-j, and one of their common targets  in vegetative apices, juvenile leaves , and young panicles.
osmir529b and osmir156a-j were expressed in all tissues/organs evaluated, though at variable levels .
osmir156a-j transcripts accumulated at high levels in juvenile leaf tissues, whereas osspl <dig> was down-regulated in leaf and young panicle tissues .
osspl <dig> is targeted by both mirnas in seedlings, whereas it seems to be predominantly targeted by mir529a-5p in panicle  <cit> .
likewise, osmir529b might also have a more prominent effect on the post-transcriptional regulation of osspl <dig> expression in young panicle.fig.
<dig> expression patterns of mir <dig> and mir <dig> in oryza sativa.
a stem–loop pulsed rt-pcr to detect osmir529b, osmir156a-j, and osspl <dig> transcripts in vegetative apices , leaf , and young panicle tissues.
reactions without rt  and without cdna  were used as negative controls.
numbers between brackets indicate pcr cycles.
dag, days after germination.
rice ubiquitin  was used as an internal control.
b stem–loop pulsed rt-pcr to detect osmir529b transcripts in leaf  tissues  from rice seedlings and tillering developmental stages.
reactions without rt  and without cdna  were used as negative controls.
numbers between brackets indicate pcr cycles.
rice ubiquitin  was used as an internal control.
c spatiotemporal expression patterns of mir <dig> and mir <dig> in rice shoot apical meristem .
probes of 3′-labelled lna-modified oligonucleotides detecting mir <dig> and mir <dig> as described  <cit>  were hybridized with longitudinal sections of the sam from 25-dag rice seedlings.
a scramble-mirna 3′-labelled lna probe was used as a negative control.
purple staining shows probe localization.
m, meristem; p, leaf primordia.
bars: 10 μm
osmir <dig> is dynamically expressed during rice leaf development, and a gradual increase of osmir <dig> expression might be essential for regulating the temporal expression of target genes, including osspl <dig>  <cit> .
we evaluated the expression pattern of osmir529b in similar developed leaves in seedling  and tillering stages.
in contrast with the observed osmir <dig> expression patterns  <cit> , osmir529b was ubiquitously expressed in all leaf developmental stages , suggesting that this mirna has a minor or negligible contribution for temporal control of the expression of spl genes during rice leaf maturation.
nevertheless, it is also possible that mir <dig> function as a dampening mirna to establish the correct balance of spl targets during temporal leaf development in monocots.
given that osmir529b and osmir156a-j transcripts accumulated in the vegetative apex , we decided to evaluate their spatial expression patterns in the shoot apical meristem  via in situ hybridization using specific probes as described .
both mirnas are expressed in incipient  and developing leaf primordia, but not in the meristem proper  .
such expression pattern strengthened the data from arabidopsis, which showed that mirna regulation of spls occurs mainly in leaf primordia and that spl activity may nonautonomously inhibit initiation of new leaves at the sam, perhaps via auxin signaling pathways  <cit> .
our in situ data showed that osmir529a-b and osmir156a-j have overlapping spatial expression patterns in leaf primordia, which suggest that these mirnas can redundantly regulate or collaborate to fine-tune regulation of target expression in these organs.
however, based on reported higher levels of osmir <dig> expression compared with osmir <dig> expression  <cit> , the contribution for spl expression modulation is unlikely to be comparable for both mirnas, mainly during early stages of rice vegetative development.
modified 5′-race procedure can be used to access cleavage products of mirna targets as well as the processing of mirna precursors  <cit> .
parallel analysis of rna end  signatures that are derived from rice degradome and that only mapped to the pre-mirnas can give additional evidences of roles of mir <dig> and mir <dig> in rice development.
we therefore collected pare signatures of osmir156a-l and osmir529a-b precursors from publicly available resources .
based on available data, it seems that rice mir <dig> and mir <dig> precursors are differentially processed, which may lead to differential mirna accumulation across rice tissues/organs .
even within the osmir <dig> family, specific members are differentially processed.
for example, osmir156k and –l showed fewer pare signatures when compared with the remaining mir <dig> precursors.
likewise, osmir529a and b precursors have a smaller amount of signatures when compared with most osmir <dig> precursors .
it is possible that differential precursor processing also account for the distinct functions of osmir <dig> and osmir <dig> in development.
it would be interesting in the future to address the question of whether mir <dig> regulation of spls is crucial for rice vegetative development.
in contrast to mir <dig>  the effects of over-expressing or down-regulating mir <dig> have yet to be examined in transgenic/mutant monocot plants.
conserved mir529-responsive element is functional in atspl <dig> and atspl <dig> genes
as discussed above, mir <dig> has been lost in eudicots like a. thaliana, yet the closely related atspl <dig> and atspl <dig> genes  <cit>  harbor a conserved mir529-responsive element, similarly to their homolog in a. lyrata .
given that mirna::target gene interactions have been comprehensively identified in a. thaliana  <cit> , we asked whether mir <dig> binding site is functional in this core eudicot.
to test this hypothesis, we generated transgenic arabidopsis plants harboring the well-annotated rice mir529b precursor under the control of viral 35s promoter .
as atspl <dig> and atspl <dig> are involved in regulating leaf initiation and flowering time, we compared vegetative and flowering phenotypes among col- <dig>  35s::osmir529b lines, transgenic plants overexpressing the atmir156a precursor, and the double mutant spl9;spl <dig> .
at least three independent 35s::osmir529b lines displayed similar phenotypes as the double mutant spl9;spl <dig>  such as an increased number of rosette leaves in combination with a slight delay in flowering, albeit these phenotypes are less pronounced than those from 35s::atmir156a plants .fig.
<dig> phenotypic and expression analyses of 35s::osmir529b arabidopsis plants.
a morphology of six-week-old  and 25-day-old  plants .
scale bar represents 1 cm.
b stem–loop pulsed rt-pcr to detect osmir529b precursor, osmir529b, mir <dig>  and atspl transcripts in arabidopsis leaf tissues.
reactions without rt  and without cdna  were used as negative controls.
numbers between brackets indicate pcr cycles.
actin- <dig> was used as an internal control.
c modified 5′-race analyses of cleaved spl <dig> transcripts in col- <dig> and 35s::osmir529b leaf tissues.
the 5′-ends of the spl <dig> cleavage products preferentially map to mir <dig>  and mir529b binding sites
to further confirm the phenotypic similarities between 35s::osmir529b and spl9;spl <dig> plants, we evaluated the average number of juvenile and rosette leaves .
under our long-day  growing conditions the 35s::mir156a line produced  <dig>  times more juvenile leaves than col- <dig> , similarly to data previously reported  <cit> , whereas 35s::osmir529b and spl9;spl <dig> plants produced, on average, only  <dig>  times more.
likewise, the production of rosette leaves of 35s::osmir529b lines showed a stronger tendency toward the phenotype of the spl9;spl <dig> mutant .table  <dig> phenotypic evaluation of 35s::osmir529b lines in comparison with col- <dig>  spl9;spl <dig>  and 35s::mir156a plants under ld growing conditions
fifteen plants per genotype were used for determination.
dag, days after germination.
average values followed by the same letter do not differ statistically
1number of rosette leaves formed before the appearance of the first leaf with abaxial trichomes
2opening of the first flower
in addition to estimating the number of leaves formed before the appearance of the first flowers, we also determined the time that 35s::osmir529b lines needed to bolt as well as to anthesis .
on average, transgenic plants overexpressing osmir529b showed a slight delay in the transition to flowering  when compared with col- <dig> .
in line with the observations of schwarz and co-workers  <cit> , we also observed that the double mutant spl9;spl <dig> showed an intermediate behavior between col- <dig> and mir <dig> overexpressor .
importantly, spl9;spl <dig> plants did not differ statistically from 35s::osmir529b lines in terms of transition to flowering and leaf development .
these data reinforced the observation that osmir529b overexpressors display similar vegetative and reproductive phenotypes as spl9;spl <dig> mutant, likely due to the low levels of spl <dig> and spl <dig> transcripts in both genotypes.
mir <dig> targets, besides spl <dig> and spl <dig>  exclusively other eight spl family members  <cit>  and these were shown to be down-regulated in atmir156b-overexpressing plants  <cit> .
in comparison with spl9;spl <dig> double mutant and 35s::osmir529b lines, 35s::mir156a line displays more severe, aberrant vegetative and reproductive phenotypes , which is likely due to the fact that additional mir156-targeted spl genes act redundantly to regulate leaf initiation and phase change  <cit> .
conversely, as in the spl9;spl <dig> double mutant, only atspl <dig> and atspl <dig> genes may be repressed in 35s::osmir529b lines, rendering them a less aberrant phenotype .
to confirm this hypothesis, we evaluated the expression patterns of several spl family members in leaf tissues of 35s::osmir529b lines, spl9;spl <dig> mutant, 35s::mir156a line, and col- <dig>  we also evaluated the presence of transcripts from osmir529b precursor and the accumulation of its respective mature mirna .
as expected, several spl genes were down-regulated in the 35s::mir156a plants .
however, in line with our phylogenetic  and phenotypic data , only transcript levels of atspl <dig> and atspl <dig> showed a severe reduction in 35s::osmir529b plants.
moreover, levels of mir <dig> transcripts were similar in col- <dig> and 35s::osmir529b plants , suggesting that the accentuated reduction in spl <dig> and spl <dig> expression in osmir529b overexpressors is most likely due to the accumulation of rice mir529b transcripts.
together, these results substantiated the fact that monocot mir <dig> precursor is correctly processed in a core eudicot to generate mature mir <dig> transcripts and down-regulate specific spl genes.
to confirm the post-transcriptional regulation of atspl genes by mir529b, we mapped cleavage sites in spl <dig> transcripts in 35s::osmir529b plants employing the modified 5′-race approach  <cit> .
race analyses showed that cleavage sites occurred between the base  <dig> and  <dig> of the mir529b , similarly to what has been described for monocots  <cit> .
these results robustly showed that spl <dig> is mainly regulated by osmir529b in arabidopsis 35s::osmir529b plants, demonstrating a conserved function of mir <dig> in post-transcriptionally regulating specific spl family members.
importantly, our data imply that the mir529-responsive element is conserved and functional in arabidopsis spl <dig> and spl <dig> genes, likely due to the selective constraint on the amino acid or rna secondary structure of the region surrounding mir156/529-responsive element.
CONCLUSIONS
we have shown that, although mir <dig> genes have been lost in arabidopsis and perhaps in all eudicot species, particular spl genes in these species retained the mir529-responsive element, possibly due to the maintenance of synonymous codons for efficient mir <dig> binding and proper function  <cit> .
more specifically, a. thaliana spl <dig> and spl <dig> genes retained a functional mir529-responsive element, even in the absence of a mir529-generated locus.
similarly to monocot spls, eudicot spl genes containing the mir156/529-responsive element appear to be under evolutionary constraints distinct from those containing only the mir156-responsive element.
such tendency would be indicative of target evolution constrained by mirna-mediated regulation.
it is possible that the interplay between mir <dig> and mir <dig> regulation of specific spls be important to fine-tune flower architecture development in monocots, particularly in grasses  <cit> .
as arabidopsis does not have mir <dig>  perhaps particular mir <dig> family members  functionally replace mir <dig>  it is conceivable that other core rosids and/or closely related species of a. thaliana share similar mir156/mir529/spl evolutionary history, though such confirmation requires future studies.

 micrornas are small rnas important to transcriptional and post-transcriptional regulation in animals, plants, and viruses. mirnas bind complementarily to their target mrna sequences, leading to translational repression, rna degradation, or rna cleavage. most plant mirnas are encoded by gene families, and mature mirnas often have multiple target genes with similar complementary motifs in their mrnas. the almost perfect complementarity between mirnas and targets facilitates computational prediction and could be due to their evolutionary origins. one well-accepted model for mir gene evolution is the inverted duplication of target gene sequences in plant genomes. these duplicated regions become mir genes over time through sequence variations, which permit the generation of hairpin-like transcripts to become substrates for dicer-like enzymes. another interesting model suggests that mir genes can randomly originate from various inverted repeats throughout the genome, independently of target gene duplications. for instance, recent evidences indicate that mir genes may be generated as a result of transposon activity. it has been recently shown that evolutionary patterns of mirna genes can potentially influence the evolution of their target genes. moreover, synonymous codons in target genes have been found near conserved mirna target sites in at least four plant genomes, indicating selection constraint on synonymous codons for efficient mirna binding and proper mirna function. several mirnas are conserved throughout large evolutionary distances from embryophyta to core rosids. however, some mirnas appear to be species or lineage specific. for instance, mir is conserved in all angiosperms studied thus far. interestingly, mir is correlated at the nucleotide level with mir sharing 14–16 nt. curiously, although both mirnas have a common ancestor in embryophytes, mir seems to have been lost in some taxonomic groups, including core eudicots such as arabidopsis thaliana. both mirnas share target genes, which are members of the squamosa promoter-binding protein like family. spl genes are plant-specific transcription factors defined by a highly conserved region of amino acids called sbp domain, and by their crucial and widespread functions in development. spl genes can be roughly separated into two major groups–long and short–the latter containing responsive elements for mir and, in some species, for mir and mir interestingly, sites for mir reside in coding as well as untranslated regions of target sequences, whereas mir binding sites are chiefly located in coding regions and overlap with mir sites. in plant lineages containing both mirna genes, differential expression of mir and mir in vegetative and reproductive organs/tissues might have favored lineage-specific retention of mir156/ sequence variants in their genomes. another possibility is that the combinatory action of mir and mir leads to the regulation of distinct targets in specific lineages, such as in physcomitrella patens. recently, the evolutionary differences between these two mirna families in monocot species have been investigated, but the consequences of mir loss in core eudicots such as arabidopsis are not yet clear. a broader evolutionary analysis of mir and mir genes and their targets including eudicot species should offer valuable insight into this issue. in this study, we extended our knowledge regarding evolutionary and functional divergences between mir and mir regulation on their conserved targets. through phylogenic reconstruction of target sequences, we confirmed that specific eudicot spl family members retained mir529-target sites, independently of the presence of mir genes in their genomes. loss of mir function in eudicots is correlated with a more relaxed selection of the mirna529-specific target element, while mirna156-specific target sequence is under stronger selection. additionally, we showed that a. thaliana plants overexpressing mir precursor from a monocot, but not from a basal eudicot, display similar phenotypes as the spl9;spl mutant due to the specific down-regulation of these mir156/529-targeted spls. based upon functional and evolutionary analyses, we proposed that the loss of mir genes might have favored diversification of spls in eudicot species. it is also possible that new mir family member have replaced mir functions in eudicots.  sequence and phylogenetic analyses reveal that a mir529-responsive element in spl genes is broadly conserved across land plants mir genes are present in genomes of a number of species across land plants. accordingly, in such species, transcripts of a subset of spl family have responsive elements for both mir and mir. to get a better view of the extent to which a mir529-responsive element is conserved in land plants, we searched for mir529-responsive elements in spl genes from species with publicly available genome sequences, including those species in which mirna is absent in their genomes or in transcribed sequences identified thus far. high-confidence prediction of mirna targets was performed by psrnatarget based on sequence complementarity and evolutionary conservation. we collected conserved mature mir sequences from three species, which are available in mirbase v. and shown in fig.  we then employed more stringent cut-off threshold for the maximum expectation parameter to minimize false positive target prediction. surprisingly, we found spl genes from several species that retain a highly conserved and overlapping responsive element for both mirnas, independently of the presence of mir genes in their genomes. this suggests that, whereas mir and mir genes have undergone distinct evolutionary fates, their mutual targets have been more conservative even in species which apparently have lost mir genes. for instance, the mir156/529-responsive element in eudicot spls resides only in coding regions, similarly to what is observed in monocots and bryophytes.fig. alignment and phylogeny of spls containing the mir156/mir529-response element. a alignments of the 25-nt mir156/529-response element were done using clustalw. black color denotes conserved nucleotides. b rooted ml phylogenetic tree depicts the relationship between spls containing the mir156/529-response element from representative species. the numbers indicate branch support. left numbers, posterior probability branch support. right numbers, alrt branch support. “-”, indicates that a particular branch is not observed in bayesian or maximum-likelihood trees. blue line comprises moss spls and green line encompasses monocot and eudicot spls. c target sites for mir and mir in a. thaliana spl and spl and a. lyrata spl transcripts. at, arabidopsis thaliana; al, arabidopsis lyrata; th, thellungiella halophila; vv, vitis vinifera; sl, solanum lycopersium; md, malus domestica; fa, fragaria ananassa; pt, populus trichocarpa; gm, glycine max; mt, medicago truncatula; pont, poncirus trifoliata; am, antirrhinum majus; rc, ricinus communis; zm, zea mays; os, oryza sativa; bd, brachypodium distachyon; aqc, aquilegia coerulea; pp, physcomitrella patens. accession numbers are given in additional file 1 to further explore the evolutionary relationship of mir156/ common targets, phylogenetic inference of sbp-box genes containing the mir156/529-responsive element was estimated using maximum-likelihood and bayesian inference methods. the percentage of pairwise identity of well-aligned sequence blocks was  %, and the substitution saturation test reported that the alignment was not saturated. we observed two groups of spls in our consensus tree. group i included known mir156/ spl targets in bryophyte, whereas group ii contained various monocot and core eudicot spls harboring conserved binding sites for mir156/ this analysis indicated that spls containing mir156/ target sites have a common origin in land plants. a. thaliana spl and spl are closely related and most likely form a pair of paralogous genes. accordingly, both spl and spl as well as their orthologs retained the mir156/529-responsive element. it has been proposed for monocot species and p. patens that spls containing mir156/ sites evolved conservatively with a slow rate when compared with spls harboring only the mir156-responsive element. to further elucidate the evolutionary fates of eudicot spls containing the mir156/529-responsive element, we analyzed two blocks in spl sequences: “sbp domain” block, which contains nucleotides of the sbp domain plus few nucleotides upstream/downstream, and “target site” block, which contains nucleotides that comprise both mir156/529-responsive elements. for the “sbp domain” block, we estimated nonsynonymous and synonymous substitution ratios of representative spls. we chose arabidopsis as a representative eudicot due to the fact that, even after extensive sequencing efforts, precursors or canonical mature sequences of mir have not been found in either a. thaliana or its closest relative a. lyrata. pairwise alignments of best-aligned blocks among spl genes from a. thaliana, monocots, and p. patens indicated that the “sbp domain” block is under purifying selection for all comparisons, reflecting functional constraints, which is expected since sbp is likely a dna binding domain. given that “target site” blocks are short and could give rise to unrealistic ka/ks ratios, we decided to compare their alignment more directly. considering the five first bases restricted to the mir target site, nonsynonymous substitutions including amino acids with different physicochemical features were found. in contrast, such drastic changes did not occur for the nucleotides specifying mir target site. these observations indicated that different regions of the “target site” block might be under distinct evolutionary constraints. in fact, the bases that diverged more in the “target site” block specifically correspond to the mir target site. therefore, it is possible that loss of mir regulation in eudicots allowed a more relaxed or low purifying selection of the mirna-responsive element region, which reflects its loss of functionality as a mir target. in line with this observation, the percentage of nucleotide identity within the “target site” block is lower among eudicot spl genes than among monocot and bryophyte ones. in monocots and p. patens, the “target site” block is more conserved, likely because it is functionally relevant as mir is still present in these species.fig. dna alignments of mir and mirna156/ target sites. amino acids coded by mir156/ target site are shown. amino acids are colored according to their hydrophobicity; red box, eudicot sequences; green box, monocot sequences; blue box, bryophyte sequences. percentage of nucleotide identity for the 5′ four nucleotides is shown at the side of each colored box. black lines underneath the topmost logo indicate mirna binding sites. box at the bottom shows logos constructed based on alignments of each plant group. accession numbers are given in additional file 1 overexpression of a basal eudicot microrna precursor in a. thaliana phenocopies mir overexpressor although mir is highly conserved, being present in all plant species assessed thus far, mir seems to be restricted to particular taxonomic groups. to get a better view of the mir156/ gene evolution, the phylogenetic relationship of these mirnas was accessed using the maximum-likelihood approach. for phylogenetic analyses, we included mir and mir precursors from physcomitrella patens, monocot species, a basal eudicot, and precursors of mir genes from arabidopsis thaliana. a consensus ml tree was generated in which two general, broad groups were readily observed. group i comprised mir precursors from different species, while group ii contained mir precursors from monocots and moss. monocot mir precursors were grouped into a distinct subset of group ii, suggesting that evolutionary divergence occurred in a common ancestor of land plants, which led to the split between mir genes of moss and flowering plants. it had been proposed earlier that a key feature of mirna evolution is that, once evolved, families and family members are rarely lost. however, not all mirnas are equally conserved and it has been recently shown that several mirna losses occurred in families that evolved prior to the origin of spermatophytes. our data suggest that mir and mir families experienced dynamic duplications and losses across embryophytes, through which clade- or species-specific mirna gene subgroups have arisen or were eliminated. for instance, a. thaliana has at least mir loci and mir156-targeted spls, whereas rice has at least mir loci, two mir loci, and eight mir156-targeted and four mir156/mir529-targeted spls. interestingly, the predicted mir precursor from the basal eudicot a. coerulea was grouped into group i, with a. thaliana mir156h and aquilegia mir156a and b precursors, indicating a common origin of these mirnas. moreover, aquilegia mir seems to be highly conserved with arabidopsis mir156h at both nucleotide and secondary structure levels. these observations raised the question of whether this mir precursor of aquilegia defined as mir is indeed a mir homolog. to test this hypothesis, we investigated whether loci flanking aquilegia pre-mir are localized into syntenic blocks when comparing with monocot species. we firstly searched for such syntenic groups among distinct monocot species, including z. mays, o. sativa, b. distachyon, and s. bicolor. the colinearity of genes around pre-mir locus is relatively conserved, therefore defining conserved syntenic block in monocots. conversely, we did not detect any conservation of syntenic block including pre-mir from aquilegia, although some orthologous genes were found to be present in this species. moreover, there is no detectable synteny between mir156h-associated regions from eudicots and the mir529-associated region from basal eudicot a. coerulea. this complete loss of synteny, along with our phylogenetic and sequence analyses, suggests that the basal eudicot aquilegia lost a bona-fide mir gene, perhaps after the regulatory role of mir was perturbed due to mutations in the mir sequence as recently proposed. to determine whether aqcmir could be properly processed and could give rise to functional mirnas, we constitutively expressed its foldback in a. thaliana plants under control of the viral 35s promoter, which confers strong and near-ubiquitous expression. we compared vegetative phenotypes among col- or wild type, 35s:: aqcmir lines, transgenic plants overexpressing the atmir156a precursor, and the double mutant spl9;spl. this mutant contains t-dna insertions in both atspl and atspl loci, which resulted in an accentuated reduction in their expression and function, although other atspls are still functional. transgenic a. thaliana plants transformed with the aquilegia-overexpressing construct had phenotypes similar to 35s::mir156a plants, such as a high number of smaller, more rounded rosette leaves. moreover, overexpression of the mir precursor from aguilegia in a. thaliana led to the down-regulation not only of atspl and atspl genes, but also other spl genes. thus, 35s::aqcmir lines showed a stronger tendency toward the phenotype of 35s::mir156a plants.fig. phenotypic, expression, and race analyses of arabidopsis 35s::aqcmir plants. a morphology of 25-day-old plants. scale bar represents 1 cm. b stem–loop pulsed rt-pcr to detect aqcmir precursor and some atspl transcripts in arabidopsis leaf tissues. reactions without rt and without cdna were used as negative controls. numbers between brackets indicate pcr cycles. actin- was used as an internal control. c modified 5′-race analyzes of cleaved spl transcripts in 35s::aqcmir leaf tissues. the 5′-ends of the spl cleavage products preferentially map to mir156 in line with the observed phenotypes and expression analyses, race analysis of spl cleavage sites demonstrated that transcripts are chiefly targeted by mir in 35s::aqcmir plants. based on these observations and given that aqcmir precursor is more conserved with mir156-like precursors, we propose at least two non-exclusive possibilities: the predicted aqcmir precursor is more likely paralogous to aqcmir156a and b genes and would produce a mirna156-like small rna; aqcmir precursor might have accumulated mutations in the mir sequence as recently proposed, leading to a loss of mir biogenesis and/or function. as other basal eudicots seem to accumulate mir529-like small rnas, we cannot rule out the possibility that mir529-like small rnas still accumulate in specific a. coerulea tissues, since there is no available information regarding large-scale identification of small rna populations in this basal eudicot. mir and mir show overlapping expression patterns during rice vegetative development distinct from the basal eudicot aquilegia, it is well documented that monocot species retained both mir and mir precursors in their genomes, raising the question of whether mir regulation has been retained in monocot species because it is essential or it is just a classical case of redundancy reflecting subfunctionalization in which mir has a limited effect as compared with mir rice has two copies of mir precursors in its genome. it has been shown that osmir529a* is preferentially expressed in panicle, whereas osmir529b is ubiquitously expressed in roots, shoots, and panicle. the authors, however, did not evaluate the expression of osmir or osmir in vegetative apices and young leaves, organs in which spls define an endogenous flowering pathway and control leaf maturation and initiation, respectively. to get more insights into the possible roles of osmir, we analyzed transcript accumulation patterns of osmir529b, osmir156a-j, and one of their common targets in vegetative apices, juvenile leaves, and young panicles. osmir529b and osmir156a-j were expressed in all tissues/organs evaluated, though at variable levels. osmir156a-j transcripts accumulated at high levels in juvenile leaf tissues, whereas osspl was down-regulated in leaf and young panicle tissues. osspl is targeted by both mirnas in seedlings, whereas it seems to be predominantly targeted by mir529a-5p in panicle. likewise, osmir529b might also have a more prominent effect on the post-transcriptional regulation of osspl expression in young panicle.fig. expression patterns of mir and mir in oryza sativa. a stem–loop pulsed rt-pcr to detect osmir529b, osmir156a-j, and osspl transcripts in vegetative apices, leaf, and young panicle tissues. reactions without rt and without cdna were used as negative controls. numbers between brackets indicate pcr cycles. dag, days after germination. rice ubiquitin was used as an internal control. b stem–loop pulsed rt-pcr to detect osmir529b transcripts in leaf tissues from rice seedlings and tillering developmental stages. reactions without rt and without cdna were used as negative controls. numbers between brackets indicate pcr cycles. rice ubiquitin was used as an internal control. c spatiotemporal expression patterns of mir and mir in rice shoot apical meristem. probes of 3′-labelled lna-modified oligonucleotides detecting mir and mir as described were hybridized with longitudinal sections of the sam from 25-dag rice seedlings. a scramble-mirna 3′-labelled lna probe was used as a negative control. purple staining shows probe localization. m, meristem; p, leaf primordia. bars: 10 μm osmir is dynamically expressed during rice leaf development, and a gradual increase of osmir expression might be essential for regulating the temporal expression of target genes, including osspl. we evaluated the expression pattern of osmir529b in similar developed leaves in seedling and tillering stages. in contrast with the observed osmir expression patterns, osmir529b was ubiquitously expressed in all leaf developmental stages, suggesting that this mirna has a minor or negligible contribution for temporal control of the expression of spl genes during rice leaf maturation. nevertheless, it is also possible that mir function as a dampening mirna to establish the correct balance of spl targets during temporal leaf development in monocots. given that osmir529b and osmir156a-j transcripts accumulated in the vegetative apex, we decided to evaluate their spatial expression patterns in the shoot apical meristem via in situ hybridization using specific probes as described. both mirnas are expressed in incipient and developing leaf primordia, but not in the meristem proper. such expression pattern strengthened the data from arabidopsis, which showed that mirna regulation of spls occurs mainly in leaf primordia and that spl activity may nonautonomously inhibit initiation of new leaves at the sam, perhaps via auxin signaling pathways. our in situ data showed that osmir529a-b and osmir156a-j have overlapping spatial expression patterns in leaf primordia, which suggest that these mirnas can redundantly regulate or collaborate to fine-tune regulation of target expression in these organs. however, based on reported higher levels of osmir expression compared with osmir expression, the contribution for spl expression modulation is unlikely to be comparable for both mirnas, mainly during early stages of rice vegetative development. modified 5′-race procedure can be used to access cleavage products of mirna targets as well as the processing of mirna precursors. parallel analysis of rna end signatures that are derived from rice degradome and that only mapped to the pre-mirnas can give additional evidences of roles of mir and mir in rice development. we therefore collected pare signatures of osmir156a-l and osmir529a-b precursors from publicly available resources. based on available data, it seems that rice mir and mir precursors are differentially processed, which may lead to differential mirna accumulation across rice tissues/organs. even within the osmir family, specific members are differentially processed. for example, osmir156k and –l showed fewer pare signatures when compared with the remaining mir precursors. likewise, osmir529a and b precursors have a smaller amount of signatures when compared with most osmir precursors. it is possible that differential precursor processing also account for the distinct functions of osmir and osmir in development. it would be interesting in the future to address the question of whether mir regulation of spls is crucial for rice vegetative development. in contrast to mir the effects of over-expressing or down-regulating mir have yet to be examined in transgenic/mutant monocot plants. conserved mir529-responsive element is functional in atspl and atspl genes as discussed above, mir has been lost in eudicots like a. thaliana, yet the closely related atspl and atspl genes harbor a conserved mir529-responsive element, similarly to their homolog in a. lyrata. given that mirna::target gene interactions have been comprehensively identified in a. thaliana, we asked whether mir binding site is functional in this core eudicot. to test this hypothesis, we generated transgenic arabidopsis plants harboring the well-annotated rice mir529b precursor under the control of viral 35s promoter. as atspl and atspl are involved in regulating leaf initiation and flowering time, we compared vegetative and flowering phenotypes among col- 35s::osmir529b lines, transgenic plants overexpressing the atmir156a precursor, and the double mutant spl9;spl. at least three independent 35s::osmir529b lines displayed similar phenotypes as the double mutant spl9;spl such as an increased number of rosette leaves in combination with a slight delay in flowering, albeit these phenotypes are less pronounced than those from 35s::atmir156a plants.fig. phenotypic and expression analyses of 35s::osmir529b arabidopsis plants. a morphology of six-week-old and 25-day-old plants. scale bar represents 1 cm. b stem–loop pulsed rt-pcr to detect osmir529b precursor, osmir529b, mir and atspl transcripts in arabidopsis leaf tissues. reactions without rt and without cdna were used as negative controls. numbers between brackets indicate pcr cycles. actin- was used as an internal control. c modified 5′-race analyses of cleaved spl transcripts in col- and 35s::osmir529b leaf tissues. the 5′-ends of the spl cleavage products preferentially map to mir and mir529b binding sites to further confirm the phenotypic similarities between 35s::osmir529b and spl9;spl plants, we evaluated the average number of juvenile and rosette leaves. under our long-day growing conditions the 35s::mir156a line produced times more juvenile leaves than col-, similarly to data previously reported, whereas 35s::osmir529b and spl9;spl plants produced, on average, only times more. likewise, the production of rosette leaves of 35s::osmir529b lines showed a stronger tendency toward the phenotype of the spl9;spl mutant.table phenotypic evaluation of 35s::osmir529b lines in comparison with col- spl9;spl and 35s::mir156a plants under ld growing conditions fifteen plants per genotype were used for determination. dag, days after germination. average values followed by the same letter do not differ statistically 1number of rosette leaves formed before the appearance of the first leaf with abaxial trichomes 2opening of the first flower in addition to estimating the number of leaves formed before the appearance of the first flowers, we also determined the time that 35s::osmir529b lines needed to bolt as well as to anthesis. on average, transgenic plants overexpressing osmir529b showed a slight delay in the transition to flowering when compared with col-. in line with the observations of schwarz and co-workers, we also observed that the double mutant spl9;spl showed an intermediate behavior between col- and mir overexpressor. importantly, spl9;spl plants did not differ statistically from 35s::osmir529b lines in terms of transition to flowering and leaf development. these data reinforced the observation that osmir529b overexpressors display similar vegetative and reproductive phenotypes as spl9;spl mutant, likely due to the low levels of spl and spl transcripts in both genotypes. mir targets, besides spl and spl exclusively other eight spl family members and these were shown to be down-regulated in atmir156b-overexpressing plants. in comparison with spl9;spl double mutant and 35s::osmir529b lines, 35s::mir156a line displays more severe, aberrant vegetative and reproductive phenotypes, which is likely due to the fact that additional mir156-targeted spl genes act redundantly to regulate leaf initiation and phase change. conversely, as in the spl9;spl double mutant, only atspl and atspl genes may be repressed in 35s::osmir529b lines, rendering them a less aberrant phenotype. to confirm this hypothesis, we evaluated the expression patterns of several spl family members in leaf tissues of 35s::osmir529b lines, spl9;spl mutant, 35s::mir156a line, and col- we also evaluated the presence of transcripts from osmir529b precursor and the accumulation of its respective mature mirna. as expected, several spl genes were down-regulated in the 35s::mir156a plants. however, in line with our phylogenetic and phenotypic data, only transcript levels of atspl and atspl showed a severe reduction in 35s::osmir529b plants. moreover, levels of mir transcripts were similar in col- and 35s::osmir529b plants, suggesting that the accentuated reduction in spl and spl expression in osmir529b overexpressors is most likely due to the accumulation of rice mir529b transcripts. together, these results substantiated the fact that monocot mir precursor is correctly processed in a core eudicot to generate mature mir transcripts and down-regulate specific spl genes. to confirm the post-transcriptional regulation of atspl genes by mir529b, we mapped cleavage sites in spl transcripts in 35s::osmir529b plants employing the modified 5′-race approach. race analyses showed that cleavage sites occurred between the base and of the mir529b, similarly to what has been described for monocots. these results robustly showed that spl is mainly regulated by osmir529b in arabidopsis 35s::osmir529b plants, demonstrating a conserved function of mir in post-transcriptionally regulating specific spl family members. importantly, our data imply that the mir529-responsive element is conserved and functional in arabidopsis spl and spl genes, likely due to the selective constraint on the amino acid or rna secondary structure of the region surrounding mir156/529-responsive element.  we have shown that, although mir genes have been lost in arabidopsis and perhaps in all eudicot species, particular spl genes in these species retained the mir529-responsive element, possibly due to the maintenance of synonymous codons for efficient mir binding and proper function. more specifically, a. thaliana spl and spl genes retained a functional mir529-responsive element, even in the absence of a mir529-generated locus. similarly to monocot spls, eudicot spl genes containing the mir156/529-responsive element appear to be under evolutionary constraints distinct from those containing only the mir156-responsive element. such tendency would be indicative of target evolution constrained by mirna-mediated regulation. it is possible that the interplay between mir and mir regulation of specific spls be important to fine-tune flower architecture development in monocots, particularly in grasses. as arabidopsis does not have mir perhaps particular mir family members functionally replace mir it is conceivable that other core rosids and/or closely related species of a. thaliana share similar mir156/mir529/spl evolutionary history, though such confirmation requires future studies. 
in this study, through phylogenic reconstruction of mir156/ target sequences from several taxonomic groups, we have found that specific eudicot spls, despite mirna loss, retained the corresponding target site. our results suggest loss of functionality of mir genes in the evolutionary history of eudicots and show that the mir529-responsive element present in some eudicot spls is still functional. for instance, mirna is evolutionarily related to mirna, but it is lost in arabidopsis thaliana. similarly to coding genes, mirna genes follow a birth and death pattern of evolution likely reflecting functional relevance and divergence. our data support the notion that particular mirna family members might have compensated for the loss of mir regulation in eudicot species, which concomitantly may have favored diversification of eudicot spls. detailed molecular evolutionary analyses of mir156/mir529-target sequence showed that loss of mir in core eudicots, such as arabidopsis, is correlated with a more relaxed selection of the mirna specific target element, while mirna156-specific target sequence is under stronger selection, indicating that these two target sites might be under distinct evolutionary constraints. importantly, over-expression in arabidopsis of mir precursor from a monocot, but not from a basal eudicot, demonstrates specific mir regulation of atspl and atspl genes, which contain conserved responsive elements for both mir and mir interestingly, both mirnas target sequences overlap in some members of the squamosa promoter-binding protein like family, raising important questions regarding the diversification of the mir156/mir529-associated regulatory network in land plants.
 micrornas are important regulatory elements of gene expression. similarly to coding genes, mirna genes follow a birth and death pattern of evolution likely reflecting functional relevance and divergence. for instance, mirna is evolutionarily related to mirna, but it is lost in arabidopsis thaliana. interestingly, both mirnas target sequences overlap in some members of the squamosa promoter-binding protein like family, raising important questions regarding the diversification of the mir156/mir529-associated regulatory network in land plants.  in this study, through phylogenic reconstruction of mir156/ target sequences from several taxonomic groups, we have found that specific eudicot spls, despite mirna loss, retained the corresponding target site. detailed molecular evolutionary analyses of mir156/mir529-target sequence showed that loss of mir in core eudicots, such as arabidopsis, is correlated with a more relaxed selection of the mirna specific target element, while mirna156-specific target sequence is under stronger selection, indicating that these two target sites might be under distinct evolutionary constraints. importantly, over-expression in arabidopsis of mir precursor from a monocot, but not from a basal eudicot, demonstrates specific mir regulation of atspl and atspl genes, which contain conserved responsive elements for both mir and mir  our results suggest loss of functionality of mir genes in the evolutionary history of eudicots and show that the mir529-responsive element present in some eudicot spls is still functional. our data support the notion that particular mirna family members might have compensated for the loss of mir regulation in eudicot species, which concomitantly may have favored diversification of eudicot spls. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords mir156mir529evolutionarabidopsiseudicotscnpqhttp://dx.doi.org/ /501100001807fundação de amparo à pesquisa do estado de são paulo2012/51146-2nogueira fabio tebaldi siveira issue-copyright-statement© the author 2016 