BACKGROUND
the family asteraceae represents one of the largest evolutive radiations of flowering plants, including more than  <dig> · <dig> genera and  <dig> · <dig> species, comprising economically important as well as ornamental crops  <cit> .
among members of this dicotyledon family two crop species belonging to the cardueae tribe, carthamus tinctorius l. and cynara cardunculus var.
scolymus l., also hold a phyto-pharmaceutical interest.
the former, known as safflower, is the only member of this genus widely cultivated for industrial oil, as a livestock feed or for use in traditional medicine  <cit> .
the second species, the globe artichoke, apart from its importance as food, is popular for its dietary and therapeutic potentials, especially for hepato-biliary dysfunctions and digestive complaints  <cit> .
significant progress has been achieved in recent years on the mechanisms of gene regulation and expression in plant and animals  <cit>  and several studies already elucidated gene regulation in the model organisms caenorhabditis elegans and arabidopsis thaliana  <cit> .
few data are available thus far on gene expression  <cit>  or regulation based on small regulatory elements, on a wide scale, for c. cardunculus and c. tinctorius.
a class of tiny rna molecules, lin- <dig> and let- <dig>  both controlling the timing of juvenile development in c. elegans were the founding members of non-coding rnas called micrornas   <cit> .
plant mirnas came to light from a. thaliana studies  <cit> .
their discovery broadened the phylogenetic distribution of mirnas to plant genomes and highlighted their ancient origin and the important role played  <cit> .
recently, a significant advancement was achieved through the discovery that small rna molecules were not only active within the cell, but also as mediators at the cell-to-cell communication level  <cit> .
plant mirnas derive from long primary transcripts  giving rise to mature rnas products of 21- <dig> bp, fundamental in gene regulation  <cit> .
in plants, mirnas control the degradation of messengers or restrain translation, affecting development and response to biotic and abiotic stresses  <cit> .
the biogenesis of mirnas in plant is a multi step process: initially a mirna gene is transcribed in the nucleus  by means of polii enzyme  <cit> .
subsequently, the pri-mirna is cleaved in the mirna precursor or "pre-mirna".
in this step the precursor is organized in a typical stem loop structure.
a dicer-like enzyme is involved in the plant mirna maturation, functioning in concert with the dsrna binding protein   <cit> .
after this step, the processed mirna is transported to the cytoplasm by the hasty protein  <cit>  to build a complex, termed "rna-induced silencing complex ", with argonaute  proteins.
the risc complex guide has a functional activity of cleavage or translational repression of its cognate mrna target by base-paring.
in plants, mirnas have several roles involved in most biologic processes  <cit>  as well as in diverse signalling networks, like leaf  <cit>  and flower development  <cit> .
unlike animal mrna targets, plant targets show a single sequence motif displaying a near-perfect complementarity to their mirnas.
the imperfect but extensive correspondence of plant mirnas to their mrna targets provides a feature which makes a computational prediction possible  <cit> .
this approach is useful when data mining is performed on the basis of mirna:mrna targets conservation among different species, and was herein applied to the study of globe artichoke and safflower.
in this study, mirna identification and analysis were developed through a primary tool, rnahybrid  <cit> , in order to find all available targets.
in a subsequent step we looked for homologous expressed sequence tags , in both species.
finally, by means of a bioinformatics pipeline and a relational database, the presence of single targets in homologous ests regions was matched, in both species.
finally, some of the identified sequences were validated by means of experimental assays.
the overall procedure for mirna, target and homologous regions identification is explained in figure  <dig>
RESULTS
in silico mirnas identification
einverted program from emboss package  <cit> , was used to find stem loop regions on the complete est datasets of c. cardunculus and c. tinctorius, containing  <dig> and  <dig> entries respectively .
ests with inverted repeats were analysed to find the occurrence of putative mature mirnas, and their complementary units  sequences using  <dig> mature mirna related to  <dig> dicotyledon and  <dig> monocotyledon .
the ests with two match  were considered only if the sequence length between the matches is equivalent to the orthologous pre-mirnas, stored in mirbase  <cit> .
these ests were inferred as good candidates for mirna precursors.
the screening followed by analyses of the secondary structure  <cit>  of the putative pre-mirnas thus allowed identification of  <dig> mirna candidates within the two considered species.
in particular, using these criteria, three ests  coding for the homologous to mir <dig> were retrieved from the c. cardunculus dataset.
these entries showed a  <dig> bp homologous alignment region with identity > 95% and a less conserved, 3' region.
the mirna* strand and mature mirna of the ath-mir <dig> homolog were found in the conserved region, in reverse and complementary orientation .
when analysed by rnafold, these regions showed a secondary structure comparable to the arabidopsis mir398a/b/c  precursor .
after a blast  <cit>  search these sequences did not exhibit any significant similarity with known proteins from the uniprot database  pointing out that they might not code for proteins, and suggesting that they could code for a putative mir <dig> like or homologue in c. cardunculus.
in the following est embl:ge <dig>  embl:el <dig>  embl:el <dig>  embl:el <dig> using the same procedure we identified orthologous for mir <dig>  .
furthermore, were identified mir167d in embl:ge <dig> , mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir4387c, mir <dig> families corresponding to embl:ge <dig>  embl:ge <dig>  embl:ge <dig>  embl:el <dig>  embl:el <dig>  embl:ge <dig> and embl:ge <dig>  embl:el <dig>  in addition we used sequences from ngs study  <cit>  find out that sequence, embl:el <dig> represents the precursor of cat-m <dig>
blastx analysis showed at least three mirnas within coding regions, , corresponding to mir168a and mir <dig> in c. tinctorius, and to mir <dig> and mir <dig> in cynara.
the latter was detected within embl:ge <dig>  that is homologous to the burp domain-containing proteins , identified in many plants, putatively involved in a variety of vegetative and reproductive developmental functions, as well as in responses to environmental stresses  <cit> .
mir <dig>  identified in embl:ge <dig>  showed a high similarity level with chaperonin family tcp-1/cpn <dig>  <cit> , .
all sequences of precursors of the above mentioned pre-mirnas, fold into a perfect hairpin structure with low free energy levels .
experimental identification of mature mirnas
an experimental assay was developed for in-vitro isolation of mature mirnas from c. cardunculus.
primers were designed for  <dig> mirnas, based on the alignment of available a. thaliana mature mirnas and est sequences of c. cardunculus and c. tinctorius, considering variable nucleotides at divergent sites .
assays were performed on total rna from artichoke leaves.
the first strand  was synthesized using an oligo-dt primer carrying an adaptor.
a 3' primer complementary to the adaptor and a 5' primer corresponding to the mirnas, annealing to their reverse transcribed target, were used for polymerase chain reaction .
in six reactions out of ten, mirna-rt-pcr produced the predicted  <dig> base pair fragments, including the adaptor sequences and the stabilisation tails.
cloning and sequencing the pcr products confirmed the identity of four amplified artichoke mirnas, corresponding to mir171b.c , mir833-5p , mir <dig> , and mir390b .
the latter sequence matched the reported mirna of glycine max that differs from that of a. thaliana in the 19th nucleotide, in which there is an a instead of g .
the two mirnas not represented in the sequenced clones, together with the not amplified mirnas, suggested either a lack of expression in the artichoke leaves tested, or a very low abundance preventing isolation or misidentification.
three sequenced mirnas  appeared highly conserved in plants, due to their occurrence in several species.
however, this is the first retrieval within the asteraceae family of mir833-5p, thus far considered specific for arabidopsis only.
in spite of the pcr reaction sensitivity, this method allowed the validation of a first set of mirnas from artichoke leaves.
to increase their number, a further step is needed to enrich the fractions of small rnas.
a wider range of plant developmental stages is also required to allow the identification of further mirnas, involved in other differential regulatory pathways.
§ degenerated bases are indicated by the following one-letter code: c+t = y; a+c+g = v; a+c+g+t = n.
motif cgccgc corresponds to stabilisation tail.
* common reverse primers for all mirnas
identification of homologous regions between c. cardunculus and c. tinctorius, and the conservation of identical putative targets in both analysed sequences, provided a guide for specific primer design to further assess c. cardunculus mirnas in vivo.
this approach allowed the experimental validation of  <dig> out of  <dig> selected mirnas, using as template rnas from young c. cardunculus leaves.
however, to be able to capture all expressed mirnas and considering that only young leaves were assayed, it appeared necessary to test various phenological stages or plant developmental phases, in order to get a more complete scenario of the mirna expression patterns.
however, this study revealed that some sequences considered specific only for arabidopsis are also present in carduoideae .
all analyses carried out in this study highlighted and confirmed the broad conservation and homology of plant mature mirnas.
results herein shown strengthen the hypothesis that mirnas play a fundamental role at the cellular level, and that they are nearly always conserved in related organisms  <cit> .
in particular, their conservation suggests a feedback mechanism of genome stabilization and protection, through selective pressures active at the gene expression level.
mutations in mirna sequences have in fact a very low probability of being mirrored by complementary changes occurring at their target loci or vice versa, thus setting in place a simple but efficient mechanism of control  <cit> .
however, as for orphan genes, during evolution some structures persist even if their function was lost.
since a. thaliana and carduoideae do not share the same evolutionary line, caution is needed when inferring the functionality of the mirna-target links observed.
target prediction
rnahybrid results showed that  <dig> and  <dig> ests in c. tinctorius and c. cardunculus, respectively, have at least one predicted mirna target .
the selection parameters used in the analysis  were: maximum one loop, one mismatch, and overhangs not longer than  <dig> nucleotides and mfe cut off value > 70% of the perfect match obtained by rnahybrid, and calculated for each mirna mature sequence  <cit>  .
data also showed a pool of  <dig> ests,  <dig> for c. scolymus and  <dig> for c. tinctorius, in which repeated  targets occur in the same est.
the prediction of multiple targets within the same est in several positions, suggests the possibility of evolutive mechanisms related to co-regulation or amplification.
data indicate that, in asteraceae, several types of mirnas could be involved in this mechanism, in a way similar to the multiple and different targets on the same mrnas proposed in arabidopsis, i.e.
mir <dig> and mir <dig> that target tair:at1g <dig>  tair:at1g <dig> and tair:at1g <dig> mrnas  <cit> .
blast results and cos identification
the comparative approach herein followed allowed the identification of conserved homologous/orthologous sequences  in est datasets of c. tinctorius and c. cardunculus, pointing out the unique features , present in this phylogenetic group.
a systematic search for aligned regions between c. tinctorius and c. cardunculus ests, through blast analyses, resulted in the identification of  <dig> homologous sequences.
we inferred that about 75% of c. tinctorius and c. cardunculus ests shared at least one homologous region .
the aligned regions longer than  <dig> bp , were considered as significant .
they covered about 50% of the complete est dataset  and were considered to represent sequences that code for proteins with similar functions in these two species.
about  <dig> c. cardunculus and  <dig> c. tinctorius est sequences did not show a common region, probably due to incomplete transcriptome sequencing data.
conserved target in cos region
analyses carried out in cos regions resulted in the identification of mirna targets, conserved in c. cardunculus and c. tinctorius, obtained after aligning of  <dig> and  <dig> ests of cynara and carthamus respectively.
following a statistical evaluation of the predicted targets, a pool of  <dig>  different mature mirna targets with a signal to noise ratio >  <dig> and specificity >  <dig>  was identified.
the complete est sequences analyses, including target and relative functions, are reported in additional file  <dig>  ests embl:ge <dig> and embl:el <dig>  showed a  <dig> bp homologous region in which a mir157a/b/c target was present.
members of this family are ubiquitous within plants, as they were also found in fabaceae, vitaceae, solanaceae and now in cardueae .
blastx analysis showed a significant similarity of these accessions to arabidopsis cyclophillin , an important protein involved in variety of cellular processes in plants, including protein trafficking and maturation, receptor signalling and complex stabilization  <cit> .
a target for mir397a was predicted on a cos region , similar to laccase , a multi-copper containing glycoprotein, member of a multigene family present in plants as well as other organisms  <cit> .
furthermore, mir <dig> was predicted in cos region with specificity >  <dig> ; comparison analysis of these ests  against the arabidopsis proteome showed significant similarity to copper/zinc superoxide dismutase  .
in arabidopsis, down-regulation of cu,zn-sod expression involves mir <dig>  that directs the degradation of its mrna at cu++ low concentrations  <cit> .
three additional mirna families, mir <dig> , mir <dig> and mir <dig> were predicted in arabidopsis and other plants to target transcripts for the cu++ protein plastocyanin and members of the laccase family, as shown by their accumulation in response to low cu++ concentrations, demonstrating a microrna-mediated down regulation  <cit> .
furthermore, the four mirnas above mentioned are known to perform important functions in cu++ homeostasis within the plant cells  <cit> .
moreover, other blastx analyses against the arabidopsis proteome showed similarity of c. cardunculus with a nucleotide binding protein and to another protein with unknown function .
in this study, potential targets for mir <dig> were found within  <dig> ests of c. cardunculus, whereas only  <dig> were conserved in c. tinctorius cos regions.
in arabidopsis, mature ath-mir390a is obtained by two precursors .
the mature sequence of ath-mir390a is ubiquitous in plants, since it is shared with other  <dig> species of eudicotyledons, monocotyledons as well as physcomitrella patens .
a homologous sequence is also known in coniferophyta  with a g→u substitution in position  <dig>  <cit> .
ath-mir <dig> drives the cleavage of the non-protein-coding primary transcripts of trans-acting  genes directing the formation of small-interfering ta-sirnas.
these mirnas are involved in the cleavage of ago proteins, with rnaseh-like activity cleaving ta-sirnas single-stranded rna transcripts in the region complementary to small rnas  <cit> .
two mir <dig> target sites  were shown to be necessary in arabidopsis for ta-sirnas  <dig> precursor rna cleavage, dependent on a specific interaction between ago <dig> and mir <dig>  suggesting a similar putative role of mir <dig> in the cardueae .
within the c. cardunculus transcriptome, this pool of mirnas conserved in cos regions was considered trustable for subsequent in vivo validation, since their identification was experimentally more probable than the vertical prevision carried out by rnahybrid in each of the two datasets, separately.
eight c. cardunculus est sequences  were found as possible targets of ath-mir <dig> .
so far, ath-mir <dig> was considered specific in arabidopsis .
blastx analysis of these ests showed a significant similarity with an arabidopsis  <dig> proteasome subunit-like protein .
in arabidopsis, mir <dig> is predicted to target more than  <dig> genes of the pentatricopeptide repeat  protein family, characterized by tandem repeats of a degenerate  <dig> amino acid motif.
some proteins play a role in post-transcriptional processes within organelles, current evidence suggesting that ppr proteins bind rna as well as other proteins  <cit> .
CONCLUSIONS
the confirmed role played by mirnas in both physiological and pathological processes make them an interesting object of study  <cit> .
herein we followed a comparative transcriptome in silico approach that let us mining for putative mirnas and their possible targets in globe artichoke and safflower.
using this procedure we identified  <dig> mirnas in both species and after statistical analysis  <dig> conserved targets, found on the cos regions and belonging to  <dig> mirna families, with a signal to noise ratio >  <dig>  and ≥  <dig>  specificity, allowing for their in vivo functional analyses.
the identification of  <dig> ests codifying for mirna precursors in asteraceae family, showed that the rate of precursors retrieve is estimated at  <dig> %, alike to the real rate found in arabidopsis , highlighting the validity of the developed method; moreover catm <dig>  a novel mirna predicted from sufflower  <cit> , let us to retrieve a est that codified the entire precursor .
the complementarity between mirnas, targets and sequence conservation in cynara and carthamus helped us predict regulated genes, as well.
the bioinformatic approach identified ests whose sequences were maintained in both species as most probable targets.
the occurrence and mining of cos regions from both species validated the analysed targets, suggesting that many true positives were also possibly missed.
in fact, any real target identified in one species was ignored if the counterpart sequence was not found in the other species, a problem occurring when the ests in public databases do not represent the whole transcriptome.
the presence of multiple targets within a gene agrees with the concept of cooperatively acting mirnas.
finally, a possible outcome of our approach would be the development of bioinformatic tools detecting multi-species cos regions using publicly available ests, altogether with an evaluation of their level of conservation.
in fact, recent data concerning mirnas identified in safflower  <cit>  confirmed the validity of the in-silico approach herein applied, since 52% of the mirna groups identified in this study were also present in the deep sequencing data produced from different organs of this plant.

 the family asteraceae represents one of the largest evolutive radiations of flowering plants, including more than · genera and · species, comprising economically important as well as ornamental crops. among members of this dicotyledon family two crop species belonging to the cardueae tribe, carthamus tinctorius l. and cynara cardunculus var. scolymus l., also hold a phyto-pharmaceutical interest. the former, known as safflower, is the only member of this genus widely cultivated for industrial oil, as a livestock feed or for use in traditional medicine. the second species, the globe artichoke, apart from its importance as food, is popular for its dietary and therapeutic potentials, especially for hepato-biliary dysfunctions and digestive complaints. significant progress has been achieved in recent years on the mechanisms of gene regulation and expression in plant and animals and several studies already elucidated gene regulation in the model organisms caenorhabditis elegans and arabidopsis thaliana. few data are available thus far on gene expression or regulation based on small regulatory elements, on a wide scale, for c. cardunculus and c. tinctorius. a class of tiny rna molecules, lin- and let- both controlling the timing of juvenile development in c. elegans were the founding members of non-coding rnas called micrornas. plant mirnas came to light from a. thaliana studies. their discovery broadened the phylogenetic distribution of mirnas to plant genomes and highlighted their ancient origin and the important role played. recently, a significant advancement was achieved through the discovery that small rna molecules were not only active within the cell, but also as mediators at the cell-to-cell communication level. plant mirnas derive from long primary transcripts giving rise to mature rnas products of 21- bp, fundamental in gene regulation. in plants, mirnas control the degradation of messengers or restrain translation, affecting development and response to biotic and abiotic stresses. the biogenesis of mirnas in plant is a multi step process: initially a mirna gene is transcribed in the nucleus by means of polii enzyme. subsequently, the pri-mirna is cleaved in the mirna precursor or "pre-mirna". in this step the precursor is organized in a typical stem loop structure. a dicer-like enzyme is involved in the plant mirna maturation, functioning in concert with the dsrna binding protein. after this step, the processed mirna is transported to the cytoplasm by the hasty protein to build a complex, termed "rna-induced silencing complex ", with argonaute proteins. the risc complex guide has a functional activity of cleavage or translational repression of its cognate mrna target by base-paring. in plants, mirnas have several roles involved in most biologic processes as well as in diverse signalling networks, like leaf and flower development. unlike animal mrna targets, plant targets show a single sequence motif displaying a near-perfect complementarity to their mirnas. the imperfect but extensive correspondence of plant mirnas to their mrna targets provides a feature which makes a computational prediction possible. this approach is useful when data mining is performed on the basis of mirna:mrna targets conservation among different species, and was herein applied to the study of globe artichoke and safflower. in this study, mirna identification and analysis were developed through a primary tool, rnahybrid, in order to find all available targets. in a subsequent step we looked for homologous expressed sequence tags, in both species. finally, by means of a bioinformatics pipeline and a relational database, the presence of single targets in homologous ests regions was matched, in both species. finally, some of the identified sequences were validated by means of experimental assays. the overall procedure for mirna, target and homologous regions identification is explained in figure  in silico mirnas identification einverted program from emboss package, was used to find stem loop regions on the complete est datasets of c. cardunculus and c. tinctorius, containing and entries respectively. ests with inverted repeats were analysed to find the occurrence of putative mature mirnas, and their complementary units sequences using mature mirna related to dicotyledon and monocotyledon. the ests with two match were considered only if the sequence length between the matches is equivalent to the orthologous pre-mirnas, stored in mirbase. these ests were inferred as good candidates for mirna precursors. the screening followed by analyses of the secondary structure of the putative pre-mirnas thus allowed identification of mirna candidates within the two considered species. in particular, using these criteria, three ests coding for the homologous to mir were retrieved from the c. cardunculus dataset. these entries showed a bp homologous alignment region with identity > 95% and a less conserved, 3' region. the mirna* strand and mature mirna of the ath-mir homolog were found in the conserved region, in reverse and complementary orientation. when analysed by rnafold, these regions showed a secondary structure comparable to the arabidopsis mir398a/b/c precursor. after a blast search these sequences did not exhibit any significant similarity with known proteins from the uniprot database pointing out that they might not code for proteins, and suggesting that they could code for a putative mir like or homologue in c. cardunculus. in the following est embl:ge embl:el embl:el embl:el using the same procedure we identified orthologous for mir. furthermore, were identified mir167d in embl:ge, mir mir mir mir mir4387c, mir families corresponding to embl:ge embl:ge embl:ge embl:el embl:el embl:ge and embl:ge embl:el in addition we used sequences from ngs study find out that sequence, embl:el represents the precursor of cat-m blastx analysis showed at least three mirnas within coding regions,, corresponding to mir168a and mir in c. tinctorius, and to mir and mir in cynara. the latter was detected within embl:ge that is homologous to the burp domain-containing proteins, identified in many plants, putatively involved in a variety of vegetative and reproductive developmental functions, as well as in responses to environmental stresses. mir identified in embl:ge showed a high similarity level with chaperonin family tcp-1/cpn,. all sequences of precursors of the above mentioned pre-mirnas, fold into a perfect hairpin structure with low free energy levels. experimental identification of mature mirnas an experimental assay was developed for in-vitro isolation of mature mirnas from c. cardunculus. primers were designed for mirnas, based on the alignment of available a. thaliana mature mirnas and est sequences of c. cardunculus and c. tinctorius, considering variable nucleotides at divergent sites. assays were performed on total rna from artichoke leaves. the first strand was synthesized using an oligo-dt primer carrying an adaptor. a 3' primer complementary to the adaptor and a 5' primer corresponding to the mirnas, annealing to their reverse transcribed target, were used for polymerase chain reaction. in six reactions out of ten, mirna-rt-pcr produced the predicted base pair fragments, including the adaptor sequences and the stabilisation tails. cloning and sequencing the pcr products confirmed the identity of four amplified artichoke mirnas, corresponding to mir171b.c, mir833-5p, mir, and mir390b. the latter sequence matched the reported mirna of glycine max that differs from that of a. thaliana in the 19th nucleotide, in which there is an a instead of g. the two mirnas not represented in the sequenced clones, together with the not amplified mirnas, suggested either a lack of expression in the artichoke leaves tested, or a very low abundance preventing isolation or misidentification. three sequenced mirnas appeared highly conserved in plants, due to their occurrence in several species. however, this is the first retrieval within the asteraceae family of mir833-5p, thus far considered specific for arabidopsis only. in spite of the pcr reaction sensitivity, this method allowed the validation of a first set of mirnas from artichoke leaves. to increase their number, a further step is needed to enrich the fractions of small rnas. a wider range of plant developmental stages is also required to allow the identification of further mirnas, involved in other differential regulatory pathways. § degenerated bases are indicated by the following one-letter code: c+t = y; a+c+g = v; a+c+g+t = n. motif cgccgc corresponds to stabilisation tail. * common reverse primers for all mirnas identification of homologous regions between c. cardunculus and c. tinctorius, and the conservation of identical putative targets in both analysed sequences, provided a guide for specific primer design to further assess c. cardunculus mirnas in vivo. this approach allowed the experimental validation of out of selected mirnas, using as template rnas from young c. cardunculus leaves. however, to be able to capture all expressed mirnas and considering that only young leaves were assayed, it appeared necessary to test various phenological stages or plant developmental phases, in order to get a more complete scenario of the mirna expression patterns. however, this study revealed that some sequences considered specific only for arabidopsis are also present in carduoideae. all analyses carried out in this study highlighted and confirmed the broad conservation and homology of plant mature mirnas. results herein shown strengthen the hypothesis that mirnas play a fundamental role at the cellular level, and that they are nearly always conserved in related organisms. in particular, their conservation suggests a feedback mechanism of genome stabilization and protection, through selective pressures active at the gene expression level. mutations in mirna sequences have in fact a very low probability of being mirrored by complementary changes occurring at their target loci or vice versa, thus setting in place a simple but efficient mechanism of control. however, as for orphan genes, during evolution some structures persist even if their function was lost. since a. thaliana and carduoideae do not share the same evolutionary line, caution is needed when inferring the functionality of the mirna-target links observed. target prediction rnahybrid results showed that and ests in c. tinctorius and c. cardunculus, respectively, have at least one predicted mirna target. the selection parameters used in the analysis were: maximum one loop, one mismatch, and overhangs not longer than nucleotides and mfe cut off value > 70% of the perfect match obtained by rnahybrid, and calculated for each mirna mature sequence. data also showed a pool of ests, for c. scolymus and for c. tinctorius, in which repeated targets occur in the same est. the prediction of multiple targets within the same est in several positions, suggests the possibility of evolutive mechanisms related to co-regulation or amplification. data indicate that, in asteraceae, several types of mirnas could be involved in this mechanism, in a way similar to the multiple and different targets on the same mrnas proposed in arabidopsis, i.e. mir and mir that target tair:at1g tair:at1g and tair:at1g mrnas. blast results and cos identification the comparative approach herein followed allowed the identification of conserved homologous/orthologous sequences in est datasets of c. tinctorius and c. cardunculus, pointing out the unique features, present in this phylogenetic group. a systematic search for aligned regions between c. tinctorius and c. cardunculus ests, through blast analyses, resulted in the identification of homologous sequences. we inferred that about 75% of c. tinctorius and c. cardunculus ests shared at least one homologous region. the aligned regions longer than bp, were considered as significant. they covered about 50% of the complete est dataset and were considered to represent sequences that code for proteins with similar functions in these two species. about c. cardunculus and c. tinctorius est sequences did not show a common region, probably due to incomplete transcriptome sequencing data. conserved target in cos region analyses carried out in cos regions resulted in the identification of mirna targets, conserved in c. cardunculus and c. tinctorius, obtained after aligning of and ests of cynara and carthamus respectively. following a statistical evaluation of the predicted targets, a pool of different mature mirna targets with a signal to noise ratio > and specificity > was identified. the complete est sequences analyses, including target and relative functions, are reported in additional file ests embl:ge and embl:el showed a bp homologous region in which a mir157a/b/c target was present. members of this family are ubiquitous within plants, as they were also found in fabaceae, vitaceae, solanaceae and now in cardueae. blastx analysis showed a significant similarity of these accessions to arabidopsis cyclophillin, an important protein involved in variety of cellular processes in plants, including protein trafficking and maturation, receptor signalling and complex stabilization. a target for mir397a was predicted on a cos region, similar to laccase, a multi-copper containing glycoprotein, member of a multigene family present in plants as well as other organisms. furthermore, mir was predicted in cos region with specificity >; comparison analysis of these ests against the arabidopsis proteome showed significant similarity to copper/zinc superoxide dismutase. in arabidopsis, down-regulation of cu,zn-sod expression involves mir that directs the degradation of its mrna at cu++ low concentrations. three additional mirna families, mir, mir and mir were predicted in arabidopsis and other plants to target transcripts for the cu++ protein plastocyanin and members of the laccase family, as shown by their accumulation in response to low cu++ concentrations, demonstrating a microrna-mediated down regulation. furthermore, the four mirnas above mentioned are known to perform important functions in cu++ homeostasis within the plant cells. moreover, other blastx analyses against the arabidopsis proteome showed similarity of c. cardunculus with a nucleotide binding protein and to another protein with unknown function. in this study, potential targets for mir were found within ests of c. cardunculus, whereas only were conserved in c. tinctorius cos regions. in arabidopsis, mature ath-mir390a is obtained by two precursors. the mature sequence of ath-mir390a is ubiquitous in plants, since it is shared with other species of eudicotyledons, monocotyledons as well as physcomitrella patens. a homologous sequence is also known in coniferophyta with a g→u substitution in position. ath-mir drives the cleavage of the non-protein-coding primary transcripts of trans-acting genes directing the formation of small-interfering ta-sirnas. these mirnas are involved in the cleavage of ago proteins, with rnaseh-like activity cleaving ta-sirnas single-stranded rna transcripts in the region complementary to small rnas. two mir target sites were shown to be necessary in arabidopsis for ta-sirnas precursor rna cleavage, dependent on a specific interaction between ago and mir suggesting a similar putative role of mir in the cardueae. within the c. cardunculus transcriptome, this pool of mirnas conserved in cos regions was considered trustable for subsequent in vivo validation, since their identification was experimentally more probable than the vertical prevision carried out by rnahybrid in each of the two datasets, separately. eight c. cardunculus est sequences were found as possible targets of ath-mir. so far, ath-mir was considered specific in arabidopsis. blastx analysis of these ests showed a significant similarity with an arabidopsis proteasome subunit-like protein. in arabidopsis, mir is predicted to target more than genes of the pentatricopeptide repeat protein family, characterized by tandem repeats of a degenerate amino acid motif. some proteins play a role in post-transcriptional processes within organelles, current evidence suggesting that ppr proteins bind rna as well as other proteins.  the confirmed role played by mirnas in both physiological and pathological processes make them an interesting object of study. herein we followed a comparative transcriptome in silico approach that let us mining for putative mirnas and their possible targets in globe artichoke and safflower. using this procedure we identified mirnas in both species and after statistical analysis conserved targets, found on the cos regions and belonging to mirna families, with a signal to noise ratio > and ≥ specificity, allowing for their in vivo functional analyses. the identification of ests codifying for mirna precursors in asteraceae family, showed that the rate of precursors retrieve is estimated at %, alike to the real rate found in arabidopsis, highlighting the validity of the developed method; moreover catm a novel mirna predicted from sufflower, let us to retrieve a est that codified the entire precursor. the complementarity between mirnas, targets and sequence conservation in cynara and carthamus helped us predict regulated genes, as well. the bioinformatic approach identified ests whose sequences were maintained in both species as most probable targets. the occurrence and mining of cos regions from both species validated the analysed targets, suggesting that many true positives were also possibly missed. in fact, any real target identified in one species was ignored if the counterpart sequence was not found in the other species, a problem occurring when the ests in public databases do not represent the whole transcriptome. the presence of multiple targets within a gene agrees with the concept of cooperatively acting mirnas. finally, a possible outcome of our approach would be the development of bioinformatic tools detecting multi-species cos regions using publicly available ests, altogether with an evaluation of their level of conservation. in fact, recent data concerning mirnas identified in safflower confirmed the validity of the in-silico approach herein applied, since 52% of the mirna groups identified in this study were also present in the deep sequencing data produced from different organs of this plant. 
 out of ests from safflower and of ests from artichoke showed at least one predicted mirna target. and ests of safflower and artichoke organized in cos shared different mirna targets, considered functionally conserved, and statistically significant when compared with random sequences. most of the mirna targets appeared highly/moderately conserved, highlighting an important and conserved function. after statistical analysis targets, found on the cos regions and belonging to mirna families, have a signal to noise ratio > with ≥ specificity. the putative mirnas identified belong to dicotyledon plants and to families only in monocotyledon. plant mirnas show imperfect but extensive complementarity to mrna targets, making their computational prediction possible, useful when data mining is applied on different species. in this study we introduce a stringent parameter for the comparative sequence analysis, represented by the identification of the same target in the cos region. micrornas are small rnas providing an rna-based system of gene regulation highly conserved in plants and animals. mature mirnas and targets were predicted within est sequences of safflower and artichoke.
 micrornas are small rnas providing an rna-based system of gene regulation highly conserved in plants and animals. in plants, mirnas control mrna degradation or restrain translation, affecting development and responses to stresses. plant mirnas show imperfect but extensive complementarity to mrna targets, making their computational prediction possible, useful when data mining is applied on different species. in this study we used a comparative approach to identify both mirnas and their targets, in artichoke and safflower.  two complete expressed sequence tags datasets from artichoke and safflower, were analysed with a bioinformatic pipeline and in vitro experiments, identifying potential mirnas. for each est, using rnahybrid program and non redundant mirna mature sequences, available in mirbase as reference, we searched matching putative targets. out of ests from safflower and of ests from artichoke showed at least one predicted mirna target. blast analysis showed that 75% of all ests shared at least a common homologous region and about 50% of these displayed bp or longer aligned sequences as conserved homologous/orthologous regions. and ests of safflower and artichoke organized in cos shared different mirna targets, considered functionally conserved, and statistically significant when compared with random sequences. four highly significant mirnas selected from in silico data were experimentally validated in globe artichoke leaves.  mature mirnas and targets were predicted within est sequences of safflower and artichoke. most of the mirna targets appeared highly/moderately conserved, highlighting an important and conserved function. in this study we introduce a stringent parameter for the comparative sequence analysis, represented by the identification of the same target in the cos region. after statistical analysis targets, found on the cos regions and belonging to mirna families, have a signal to noise ratio > with ≥ specificity. the putative mirnas identified belong to dicotyledon plants and to families only in monocotyledon. 20- june eighth annual meeting of the italian society of bioinformatics pisa, italy 