BACKGROUND
hevea brasiliensis, the sole commercial source of natural rubber, is a tropical perennial species native to the amazon basin.
south-east asian countries supplied 92% of the  <dig> million tons of natural rubber produced in  <dig>  but ever-growing worldwide demand calls for enhanced tree productivity.
this goal is increasingly becoming a crucial challenge for the corresponding research activities in a context of global climate change and redistribution of land for food crops.
natural rubber is a cis- <dig>  polyisoprene polymer biosynthesized in rubber particles located in specialized latex cells.
latex cells are periodically emitted from the cambium and then anastomosed to form latificer mantels  <cit> .
natural rubber is harvested by tapping.
the cytoplasm of laticifers, containing 30-50% of rubber particles, is then expelled by high turgor pressure maintained in the soft bark tissues.
an ethylene generator, 2-chloroethylphosphonic acid , is applied to the tapping panel to stimulate latex production.
ethephon increases the duration of latex flow after tapping and its regeneration between two tappings  <cit> .
tapping panel dryness  is a physiological disease that causes 10-40% annual rubber production losses over the  <dig> years of a rubber cultivation cycle.
this physiological disorder, triggered by oxidative stress, is partially induced by excessive environmental and harvesting stresses  <cit> .
the generation of reactive oxygen species  in latex cells leads to in situ coagulation of rubber particles  <cit> .
research on ros in plants initially focused on their cytotoxicity only, but nowadays ros are also considered as signalling molecules  <cit> .
a ros signalling network emerges and is conserved in all aerobic organisms  <cit> .
the ros-scavenging system acts both to maintain redox homeostasis and to protect cell components from oxidative damage.
biotic and abiotic stress, such as drought, salinity, strong light, temperature, heavy metals, uv radiation, atmospheric contamination, mechanical wounding, nutrient starvation and pathogen attacks are major sources of ros in plants  <cit> .
micrornas  particularly play a key function in responses to abiotic stress .
indeed, small rnas are involved in fine-tuning gene expression in response to physiological, external and developmental stimuli  <cit> .
of these, mirnas have been shown to be a large group of small endogenous rnas that exist in animals, plants and viruses.
since their discovery in caenorrhabditis elegans  <cit>  and in arabidopsis thaliana in  <dig>  <cit> , mirnas have been described as negatively regulating gene expression by targeting specific messenger rnas  for cleavage  <cit>  or translational inhibition  <cit> .
the biogenesis of mirnas has been amply described in plants and animals  <cit> .
briefly, a mir gene is first transcribed to primary mirnas  by rna polymerase ii, the 70- <dig> bp long pri-mirna forms an imperfectly paired hairpin which is processed by rnase iii-like dicer-like i  to generate mir precursors   <cit> .
further cleavage of the pre-mirna by dcli releases a mirna/mirna* duplex.
the duplex is then translocated into the cytoplasm by hasty , the canonical mature mirna of 20- <dig> nucleotides is selectively incorporated into the rna-induced silencing complex  associated with argonaute  <dig> .
in the risc complex, mirnas bind to mrna and inhibit gene expression through perfect or near-perfect complementarity between the mirna and the mrna  <cit> .
in plants, the recent discovery of mir genes generating both canonical mirna  and long mirna  suggests their bifunctionality  <cit> .
the lmirnas derived from mir genes are selectively processed by dcl <dig>  can associate with ago <dig> and guide dna methylation at some of their target loci in trans in rice and moss  <cit> .
the recent and major discovery of the role played by post-transcriptional modifications of mirna precursors highlighted the complexity of mirna biogenesis and the way mirnas act  <cit> .
indeed, adding an oligo-uridine tail to the 3' end of the mirna precursor is a mechanism that controls mirna production in mice  <cit> .
in plants, it has been reported that either a combination of 5' deletion and 3' uridylation of mirna, or rna editing events, alter the targeting and preference of argonaute association  <cit> .
several mirnas have been described as being up-regulated or down-regulated by high salinity, drought and low temperatures  <cit> .
furthermore, the mirna-targeted gene copper zinc superoxide dismutase , which plays a major role in maintaining redox homeostasis, is cleaved by mir <dig> under stress-free conditions in arabidopsis  <cit> .
currently,  <dig>  mature mirnas have been discovered and deposited in the public database, mirbase   <cit> , and  <dig>  mature plant mirnas in the plant microrna database, pmrd http://bioinformatics.cau.edu.cn/pmrd <cit> .
most mirnas have been identified in model species such as arabidopsis, oryza  <cit> , populus  <cit> , physcomitrella  <cit> , vitis  <cit> , whose genomes have been sequenced.
some mirnas have also been identified in glycine max  <cit> , arachis hypogaea  <cit> , solanum lycopersicum  <cit> , brassica napus  <cit> , phaseolus vulgaris  <cit>  and in the euphorbiaceous species ricinus communis  <cit> .
twenty-three mirna families predicted in ricinus communis were experimentally validated in four euphorbiaceous plants   <cit> .
however, the strategy used did not allow exhaustive identification of mirna families in hevea.
the availability of large collections of hevea expressed sequence tags  and next-generation sequencing techniques offers prospects for understanding the post-transcriptional regulation of key functions involved in responses to stress and in latex production in hevea brasiliensis.
this study involved the deep sequencing of small rnas from hevea plants grown under various conditions.
we report here on the identification of  <dig> conserved mirna families, sharing very high homology with that already known in other species.
mirna precursor sequences were identified for nine families.
in addition, ten putative novel mirna families were also identified with their precursors.
a scan of the hevea est databases with mirna sequences revealed their putative targets.
of them, the predicted targets involved in responses to stimuli, the ros-scavenging systems and transcription regulation are presented, and new insights into the control of redox status in hevea are proposed.
RESULTS
classification of small rnas
a total of  <dig> , <dig> raw reads was generated by solexa sequencing from the small rna library prepared from pooled juvenile and mature plant materials subjected to various types of abiotic stress .
after removing adapters, cleaned reads amounted to  <dig> , <dig> sequences corresponding to  <dig>  unique sequences.
the sequence lengths ranged from  <dig> to  <dig> nucleotides.
although some small rnas were present about a thousand times in our dataset, most were sequenced only a few times.
small rnas consisting of only one or two reads accounted for 32% and 53% of clean sequences, respectively.
annotation of the hevea small rnas was attempted by blastn on the arabidopsis genome.
most of the sequences did not map onto the arabidopsis genome sequence.
of the mapped sequences,  <dig>  were not annotated.
annotated sequences predicted  <dig>  products of mrna degradation,  <dig>  transposable elements,  <dig> rrnas,  <dig> products of pseudogene degradation,  <dig> ncrnas,  <dig> trnas,  <dig> sn/snornas and  <dig> mirnas corresponding to  <dig> families.
further mapping was carried out with all the mirna sequences available in the pmrd database.
in all,  <dig>  sequences matched  to conserved mirnas in other species.
the accessions were classified into  <dig> mirna families .
since most sequences did not map against pmrd, the hevea est database was scanned using the learn pipeline.
this strategy led to the identification of  <dig> additional mirna families, based on the stem-loop structure, which were not described in other species .
the distribution of the  <dig>  unique accessions showed that the most abundant accessions had in descending order  <dig>   <dig>   <dig>   <dig> and  <dig> nucleotides .
accessions with  <dig> and  <dig> nucleotides came from products of mrna degradation and transposable elements according to the mapping results against the arabidopsis genome sequence .
characteristics of conserved and putative novel mirna families in hevea brasiliensis
reads of conserved and putative novel mirnas were distributed according to their nucleotide lengths .
the  <dig> conserved mirna families mostly had  <dig> and  <dig> nucleotides .
for the  <dig> putative novel mirna families, the most represented lengths were  <dig>   <dig> and  <dig> nucleotides .
of the  <dig> conserved mirna families,  <dig> were predominant with more than  <dig>  reads, including  <dig> families that displayed more than  <dig>  reads.
the latter corresponded to hbmir159/ <dig> and hbmir <dig> .
by contrast,  <dig> families were less represented in the dataset with  <dig> families having fewer than  <dig> reads: hbmir <dig>  hbmir <dig>  hbmir <dig>  hbmir <dig>  hbmir <dig>  hbmir <dig>  hbmir <dig>  hbmir <dig>  hbmir <dig>  hbmir <dig>  hbmir <dig>  hbmir <dig>  hbmir <dig>  hbmir <dig>  hbmir <dig>  hbmir <dig>  hbmir <dig> and hbmir <dig> .
a detailed analysis of length distribution within the hevea mirna families revealed some discrepancies .
firstly, the 20- <dig> nucleotide and 23- <dig> nucleotide size classes were detected for both conserved and novel mirna families.
secondly, the most abundant size for mirnas was 21- <dig> nucleotides, except for hbmir <dig> , hbmir <dig> , hbmir <dig> , hbmir <dig> , hbmirn <dig>  and hbmirn <dig> .
thirdly, the putative novel mirnas occurred at low levels with fewer than  <dig> reads, and at extremely low levels for hbmirn <dig> and hbmirn <dig>  with only three sequences .
detection of single-nucleotide mutations in mirna sequences
for all the conserved mirna families, single-nucleotide modifications in the sequences were analysed in detail based on the data presented in  <cit> .
in arabidopsis, seventeen families are concerned by rna editing events and nine families by 5' deletion and 3' uridylation modifications .
in hevea, of the seventeen families, four families displayed single-nucleotide mutations at the sites of post-transcriptional rna modifications described in arabidopsis, namely atmir <dig>  atmir159a, atmir164a, and atmir172a .
by contrast, no - <dig> + uu modification was observed in hevea .
a-u:  <dig> %
sites of post-transcriptional modification identified in arabidopsis  <cit>  were analysed in the hevea small rna dataset.
only sequences present at >  <dig> % are listed
identification of precursor transcripts for conserved and putative novel mirna families from the hevea brasiliensis transcriptome
conserved and putative novel mirna precursors were sought in the hevea clone pb <dig> transcript sequence databases obtained from various organs using the learn pipeline  <cit> .
eight conserved mirna families that mapped against rna sequences displayed a stem-loop structure .
a single mirna precursor transcript was found for the hbmir <dig>  hbmir <dig>  hbmir <dig>  hbmir <dig> and hbmir <dig> families.
two mirna precursor transcripts were identified for the hbmir <dig>  hbmir <dig> and hbmir <dig> families.
in addition, the learn pipeline predicted  <dig> putative novel mirnas with their unique precursors .
mapping the sequences of the small rna dataset on the precursor sequences led to the identification of mirna*for all eight conserved families  and for the five putatively new families hbmirn <dig>  hbmirn <dig>  hbmirn <dig>  hbmirn <dig> and hbmirn <dig>  stem-loop reverse transcription polymerase chain reactions  were successfully performed on seven precursors for the conserved families and on nine for the putatively new families .
the presence of the mirna* sequence in the small rna dataset is indicated.
pictures of the stem-loop structures are presented in additional table  <dig>
the presence of the mirna* sequence in the small rna dataset is indicated .
pictures of the stem-loop structures are presented in additional table  <dig>
comparison of the gene ontology classification of predicted targets for conserved and putative novel mirna families
computational prediction of mirna targets led to the identification of  <dig>  sequences for the  <dig> conserved families and  <dig> for the  <dig> putative novel mirna families.
their involvement in biological processes and their molecular functions were attributed using gene ontology .
most of the go terms were represented in the same proportions for predicted targets from conserved and putative novel mirna families.
target genes of the putative novel mirna families all had go terms of conserved mirna families for biological processes and molecular functions.
in biological process terms, the putative novel mirna families had two additional terms .
the number of mirna target genes was smaller for putative novel than for conserved mirnas for some go terms: cellular process , metabolic process  and biological regulation  .
this number was larger for putative novel mirna targets compared to conserved targets for cellular component organization , localization  and response to stimulus  .
as regards the go terms for molecular function, although the distribution for antioxidant activity was the same for the  <dig> classes of mirnas , a decrease in the proportion of transcription regulator activity  and an increase in structural molecule activity  and transporter activity  were observed for the putative novel mirna families .
comparison of predicted target genes for conserved and putative novel mirna families involved in "response to stimulus", and in "antioxidant" and "transcription regulation" activities
as the management of oxidative stress is crucial for rubber productivity, we set out to more effectively understand the mechanisms of redox homeostasis.
we therefore gave priority to predicted mirna-targeted genes involved in the "response to stimulus"  and in "antioxidant" and "transcription regulator" activities  according to the go annotations .
twelve members of the ap2/erf domain-containing transcription factor family were putatively targeted by hbmir <dig>  hbmir <dig>  hbmir <dig>  hbmir <dig>  hbmir <dig>  hbmir <dig>  and hbmir <dig>  two hd-zip iii domain-containing protein members , a transcription factor with a bzip domain , three nac domain-containing proteins , three squamosa promoter binding proteins , four wrky transcription factors , four auxin response factor ; hbmir <dig> and hbmirn5) and five gras family transcription factors  were also potentially targeted.
sequences encoding protein involved in protein degradation, such as cysteine protease  and ubiquitin protein ligase , were identified for genes classified in the "response to stimulus" category.
three sequences related to aba signalling were also identified: an aba-insensitive protein , and an hva22-like protein .
in addition, one sequence encoding ctr <dig>  a negative regulator of the ethylene signalling pathway, was targeted by hbmirn <dig>  mirna biogenesis was also highlighted by the presence of a cap binding protein predicted to be targeted by hbmir <dig>  interestingly, two enzymes involved in natural rubber biosynthesis, hmg-coa reductase and 4-hydroxyl-3-methylbut-2-enyl diphosphate reductase, were predicted to be targeted by hbmir <dig> and hbmir <dig>  respectively.
for the "antioxidant activity" category, several ros-scavenging enzymes were identified.
the chloroplastic and peroxisomal isoforms of cuznsod were putatively targeted by hbmir <dig> and hbmir <dig>  respectively.
another enzyme involved in glutathione biosynthesis, gamma glutamyl cysteine ligase , was predicted to be targeted by hbmirn <dig>  rboh  is an enzyme involved in ros production, which is likely to be targeted by two mirnas .
in addition, targets that are subjected to or are involved in the redox status of proteins, such as genes encoding proteins involved in lignin synthesis, cynnamoyl coa reductase, lignin forming anionic peroxidase precursor and 4-coumarate:coa ligase, were also found to be targeted by hbmir <dig>  hbmir <dig> and hbmir <dig>  respectively.
the cleavage site for three targets has been experimentally validated for a chloroplastic cuznsod, a squamosa promoter binding protein and an arf .
for these targets, the cleavage site is located at the canonical 10th nucleotide.
mirna
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the number of sequences presenting the same cleavage site in indicated below the cleavage site located at the 10th nucleotide
discussion
size distribution of small rnas in hevea may differ from others species
next-generation high-throughput sequencing techniques are powerful tools for mirna identification.
the sequencing and analysis of the hevea small rna library generated more than  <dig> million sequences.
in their vast majority , the sequenced small rnas were only represented by one or two reads.
similarly, 65% of unique mirna sequences were found in arabidopsis by  <dig> sequencing techniques  <cit> .
interestingly, we found that the size distribution of hevea small rnas differed from other species.
the most abundant small rnas had  <dig>   <dig> and  <dig> nucleotides in hevea whereas they have  <dig> and  <dig> nucleotides in other species.
indeed, in arabidopsis thaliana  <cit> , arachis hypogea  <cit>  and oryza sativa  <cit> , the fraction of  <dig> nucleotides has proved to be predominant, accounting for 60%, 45% and 36% of unique small rnas, respectively.
the predominant size has been found to be  <dig> nucleotides in pinus contorta  <cit>  and taxus chiniensis  <cit> .
in hevea, the high percentage of 17- and 19-nucleotide small rnas resulted from the increased amount of rna degradation products and transposable elements in response to abiotic treatments according to the mapping results against the arabidopsis genome , as previously observed in brachypodium  <cit> .
indeed, a change in small rna size distribution has been reported in brachypodium in response to cold treatment.
under normal growth conditions, the 21- and 24-nucleotide classes were the most abundant small rna families, whereas it was the 19-nucleotide class after cold treatment  <cit> .
our dataset was generated from pooled small rnas of various tissues subjected to several types of abiotic stress.
the size distribution may have been differentially affected by each type of abiotic stress and may explain the difference in size distribution in hevea.
long conserved and novel mirnas of 23- <dig> nucleotides consisted of short mirna sequences with an extension.
this lmirna structure suggested that they were generated from the same mir genes.
our observation suggests dual functions for  <dig> canonical mir genes producing 20- <dig> nt and 23- <dig> nt lmirna in hevea, namely hbmir <dig>  hbmir <dig>  hbmir <dig>  hbmir <dig>  hbmir <dig> and hbmirn3- <dig>  across the plant kingdom, lmirna were previously classed as heterochromatic small interfering rnas   <cit> .
in arabidopsis, the hc-sirna derived from the mirna-generating site is dependent on dcl <dig>  rdr <dig> and poliv associated with ago <dig> and direct dna methylation for some target sites  <cit> .
as regards the higher number of reads for the  <dig> nucleotide compared with the 20- <dig> nucleotide mirnas, the  <dig> mirna families producing 20- <dig> nt and 23- <dig> nt mirnas might mostly regulate their targets through direct dna methylation in response to abiotic stress.
this may confirm the results of a recent study showing the existence of site-specific abiotic stress-induced dna methylation for five genes in hevea  <cit> .
ancient and recently differentiated hevea brasiliensis mirnas provide additional information for evolutionary studies
the  <dig> hevea brasiliensis conserved mirna families were more or less similar to those of other species.
in hevea, five mirna families  have been found in more than forty plant species  <cit> , and nine correspond to ancient mirna already present in the common ancestor of embryophytes , two in the common ancestor of tracheophytes  and nine in the common ancestor of spermaphytes   <cit> .
interestingly, the so-called monocotyledon-specific hbmir <dig> family was identified in this study  <cit> .
this suggests that hbmir <dig> appeared before the speciation between monocotyledons and dicotyledons.
three woody species-specific mirna families were also found in hevea brasiliensis: hbmir <dig> , hbmir <dig>  and hbmir <dig>  and were probably recruited independently by woody species during their evolution.
the  <dig> novel mirna families identified in this work are likely to be more recent, after hevea diversification, as they have not been identified up to now in other euphorbiaceous plants.
furthermore, this information complements previous studies on euphorbiaceous species  <cit>  in which only  <dig> conserved mirnas were identified.
our data could be used to study the evolution of mir genes.
we showed that 70% of the putatively new mir genes were able to produce lmirna of 23- <dig> nucleotides, compared to 29% for conserved mir genes.
this corresponds to what has been observed in arabidopsis, where it was previously suggested that young mir genes consistently produced sirnas, while ancient mir genes produced predominantly canonical mirnas, showing that the evolution of mir genes is associated with changes in the use of dcl, resulting in specific mirna size classes  <cit> .
in addition, several so-called plant species-specific mirnas were also found in hevea, such as hbmir <dig> initially found only in pinus taeda  <cit> , hbmir <dig>  hbmir <dig>  hbmir <dig> and hbmir <dig> found in populus euphratica  <cit>  and hbmir <dig> in populus trichocarpa  <cit> .
our data suggest that those mirnas should no longer be considered as species-specific mirna.
our study also revealed some conserved and divergent mechanisms at post-transcriptional level.
indeed, four families of mirna were subjected to rna editing events at the sites previously observed in arabidopsis.
however, for the thirteen families left, rna editing was not observed under our experimental conditions.
under our experimental conditions, the - <dig> + uu modification was not observed in hevea for the nine families selected from ebhardt et al.
<cit> .
once the hevea genome sequence is available, it will be possible to conduct an exhaustive analysis of the discrepancies between members of a multigenic family and rna editing events.
predicted target/mirna pairs conserved between plant species and putative new ones in hevea brasiliensis
in comparison with other plants, several regulation mechanisms seem to be conserved in hevea brasiliensis, such as the regulation of hd-zip iii protein by hbmir <dig>  <cit> , squamosa promoter binding protein by hbmir <dig>  <cit> , nac domain protein by hbmir <dig>  <cit> , apetala2-like protein by hbmir <dig>  <cit> , ccaat-binding transcription factor by hbmir <dig> and cysteine proteinase by hbmir <dig>  <cit> .
these ancient mirnas regulate ancestral transcription factors that coordinate highly conserved functions, such as organ polarity and separation, cell division, or hormonal control  <cit> .
we predicted potentially new mirna/target pairs in hevea.
new target/mirna pairs were found for transcription factors, such as, for example, the nac domain protein by hbmir <dig> and hbmirn <dig>  the gras domain protein by hbmir <dig>  hbmir <dig> and hbmirn <dig>  wrky by hbmir <dig> and hbmir <dig>  and the apetala2-like transcription factor by hbmir <dig>  hbmir <dig>  hbmir <dig>  hbmir <dig> and hbmir <dig>  .
in addition, we also detected new pairs involved in protein degradation , in response to hormones  and in the regulation of mirna .
further analyses are needed to validate the inhibition of these targets at transcriptional or translational level and to unravel specific regulation in hevea brasiliensis regarding the response to stress in latex cells, and latex production.
conserved and divergent regulation of redox homeostasis in hevea brasiliensis
several enzymes involved in ros production or detoxification were found in our analysis.
two ros-producing enzymes, rboh, are likely to be targeted by hbmir <dig> and hbmir <dig>  they are key regulators in the ros signalling network as they integrate different signal transduction pathways, such as calcium, protein phosphorylation and lipid signalling with ros production  <cit> .
careful attention was also paid to targets classified in the go term "antioxidant activity".
for conserved mirnas, two cuznsod chloroplastic and peroxisomal isoforms were predicted to be targeted by hbmir <dig> and hbmir <dig> respectively.
surprisingly, the cuznsod cytosolic isoform was not predicted by the learn pipeline and, even manually, no correct alignment was possible with hbmir <dig>  so far, validation by 5' race pcr has failed under our experimental conditions.
by contrast, previous studies in arabidopsis showed that hbmir <dig> targeted both chloroplastic and cytosolic isoforms  <cit> .
some mirna families have been reported to be h2o2-responsive in rice, such as osmir <dig>  osmir <dig>  osmir <dig>  osmir <dig>  osmir <dig> and osmir <dig>  <cit> .
in rice, osmir <dig> targets laccase genes involved in lignin biosynthesis and those targets were also found for hbmir <dig> in hevea.
CONCLUSIONS
a library of small rnas was deeply sequenced to identify conserved and novel mirnas in hevea brasiliensis.
combined with the hevea transcript database, this led to the identification of  <dig> mirna families and  <dig> novel mirnas.
our study revealed new insights for evolutionary studies on woody plants and the potentially specific regulation of redox homeostasis in hevea.
all the putative targets of interest for hevea stress biology and latex production will be experimentally validated and the target genes and their corresponding mirnas will undergo co-expression analysis.

 hevea brasiliensis, the sole commercial source of natural rubber, is a tropical perennial species native to the amazon basin. south-east asian countries supplied 92% of the million tons of natural rubber produced in but ever-growing worldwide demand calls for enhanced tree productivity. this goal is increasingly becoming a crucial challenge for the corresponding research activities in a context of global climate change and redistribution of land for food crops. natural rubber is a cis- polyisoprene polymer biosynthesized in rubber particles located in specialized latex cells. latex cells are periodically emitted from the cambium and then anastomosed to form latificer mantels. natural rubber is harvested by tapping. the cytoplasm of laticifers, containing 30-50% of rubber particles, is then expelled by high turgor pressure maintained in the soft bark tissues. an ethylene generator, 2-chloroethylphosphonic acid, is applied to the tapping panel to stimulate latex production. ethephon increases the duration of latex flow after tapping and its regeneration between two tappings. tapping panel dryness is a physiological disease that causes 10-40% annual rubber production losses over the years of a rubber cultivation cycle. this physiological disorder, triggered by oxidative stress, is partially induced by excessive environmental and harvesting stresses. the generation of reactive oxygen species in latex cells leads to in situ coagulation of rubber particles. research on ros in plants initially focused on their cytotoxicity only, but nowadays ros are also considered as signalling molecules. a ros signalling network emerges and is conserved in all aerobic organisms. the ros-scavenging system acts both to maintain redox homeostasis and to protect cell components from oxidative damage. biotic and abiotic stress, such as drought, salinity, strong light, temperature, heavy metals, uv radiation, atmospheric contamination, mechanical wounding, nutrient starvation and pathogen attacks are major sources of ros in plants. micrornas particularly play a key function in responses to abiotic stress. indeed, small rnas are involved in fine-tuning gene expression in response to physiological, external and developmental stimuli. of these, mirnas have been shown to be a large group of small endogenous rnas that exist in animals, plants and viruses. since their discovery in caenorrhabditis elegans and in arabidopsis thaliana in, mirnas have been described as negatively regulating gene expression by targeting specific messenger rnas for cleavage or translational inhibition. the biogenesis of mirnas has been amply described in plants and animals. briefly, a mir gene is first transcribed to primary mirnas by rna polymerase ii, the 70- bp long pri-mirna forms an imperfectly paired hairpin which is processed by rnase iii-like dicer-like i to generate mir precursors. further cleavage of the pre-mirna by dcli releases a mirna/mirna* duplex. the duplex is then translocated into the cytoplasm by hasty, the canonical mature mirna of 20- nucleotides is selectively incorporated into the rna-induced silencing complex associated with argonaute. in the risc complex, mirnas bind to mrna and inhibit gene expression through perfect or near-perfect complementarity between the mirna and the mrna. in plants, the recent discovery of mir genes generating both canonical mirna and long mirna suggests their bifunctionality. the lmirnas derived from mir genes are selectively processed by dcl can associate with ago and guide dna methylation at some of their target loci in trans in rice and moss. the recent and major discovery of the role played by post-transcriptional modifications of mirna precursors highlighted the complexity of mirna biogenesis and the way mirnas act. indeed, adding an oligo-uridine tail to the 3' end of the mirna precursor is a mechanism that controls mirna production in mice. in plants, it has been reported that either a combination of 5' deletion and 3' uridylation of mirna, or rna editing events, alter the targeting and preference of argonaute association. several mirnas have been described as being up-regulated or down-regulated by high salinity, drought and low temperatures. furthermore, the mirna-targeted gene copper zinc superoxide dismutase, which plays a major role in maintaining redox homeostasis, is cleaved by mir under stress-free conditions in arabidopsis. currently, mature mirnas have been discovered and deposited in the public database, mirbase, and mature plant mirnas in the plant microrna database, pmrd http://bioinformatics.cau.edu.cn/pmrd. most mirnas have been identified in model species such as arabidopsis, oryza, populus, physcomitrella, vitis, whose genomes have been sequenced. some mirnas have also been identified in glycine max, arachis hypogaea, solanum lycopersicum, brassica napus, phaseolus vulgaris and in the euphorbiaceous species ricinus communis. twenty-three mirna families predicted in ricinus communis were experimentally validated in four euphorbiaceous plants. however, the strategy used did not allow exhaustive identification of mirna families in hevea. the availability of large collections of hevea expressed sequence tags and next-generation sequencing techniques offers prospects for understanding the post-transcriptional regulation of key functions involved in responses to stress and in latex production in hevea brasiliensis. this study involved the deep sequencing of small rnas from hevea plants grown under various conditions. we report here on the identification of conserved mirna families, sharing very high homology with that already known in other species. mirna precursor sequences were identified for nine families. in addition, ten putative novel mirna families were also identified with their precursors. a scan of the hevea est databases with mirna sequences revealed their putative targets. of them, the predicted targets involved in responses to stimuli, the ros-scavenging systems and transcription regulation are presented, and new insights into the control of redox status in hevea are proposed.  classification of small rnas a total of, raw reads was generated by solexa sequencing from the small rna library prepared from pooled juvenile and mature plant materials subjected to various types of abiotic stress. after removing adapters, cleaned reads amounted to, sequences corresponding to unique sequences. the sequence lengths ranged from to nucleotides. although some small rnas were present about a thousand times in our dataset, most were sequenced only a few times. small rnas consisting of only one or two reads accounted for 32% and 53% of clean sequences, respectively. annotation of the hevea small rnas was attempted by blastn on the arabidopsis genome. most of the sequences did not map onto the arabidopsis genome sequence. of the mapped sequences, were not annotated. annotated sequences predicted products of mrna degradation, transposable elements, rrnas, products of pseudogene degradation, ncrnas, trnas, sn/snornas and mirnas corresponding to families. further mapping was carried out with all the mirna sequences available in the pmrd database. in all, sequences matched to conserved mirnas in other species. the accessions were classified into mirna families. since most sequences did not map against pmrd, the hevea est database was scanned using the learn pipeline. this strategy led to the identification of additional mirna families, based on the stem-loop structure, which were not described in other species. the distribution of the unique accessions showed that the most abundant accessions had in descending order and nucleotides. accessions with and nucleotides came from products of mrna degradation and transposable elements according to the mapping results against the arabidopsis genome sequence. characteristics of conserved and putative novel mirna families in hevea brasiliensis reads of conserved and putative novel mirnas were distributed according to their nucleotide lengths. the conserved mirna families mostly had and nucleotides. for the putative novel mirna families, the most represented lengths were and nucleotides. of the conserved mirna families, were predominant with more than reads, including families that displayed more than reads. the latter corresponded to hbmir159/ and hbmir. by contrast, families were less represented in the dataset with families having fewer than reads: hbmir hbmir hbmir hbmir hbmir hbmir hbmir hbmir hbmir hbmir hbmir hbmir hbmir hbmir hbmir hbmir hbmir and hbmir. a detailed analysis of length distribution within the hevea mirna families revealed some discrepancies. firstly, the 20- nucleotide and 23- nucleotide size classes were detected for both conserved and novel mirna families. secondly, the most abundant size for mirnas was 21- nucleotides, except for hbmir, hbmir, hbmir, hbmir, hbmirn and hbmirn. thirdly, the putative novel mirnas occurred at low levels with fewer than reads, and at extremely low levels for hbmirn and hbmirn with only three sequences. detection of single-nucleotide mutations in mirna sequences for all the conserved mirna families, single-nucleotide modifications in the sequences were analysed in detail based on the data presented in. in arabidopsis, seventeen families are concerned by rna editing events and nine families by 5' deletion and 3' uridylation modifications. in hevea, of the seventeen families, four families displayed single-nucleotide mutations at the sites of post-transcriptional rna modifications described in arabidopsis, namely atmir atmir159a, atmir164a, and atmir172a. by contrast, no - + uu modification was observed in hevea. a-u: % sites of post-transcriptional modification identified in arabidopsis were analysed in the hevea small rna dataset. only sequences present at > % are listed identification of precursor transcripts for conserved and putative novel mirna families from the hevea brasiliensis transcriptome conserved and putative novel mirna precursors were sought in the hevea clone pb transcript sequence databases obtained from various organs using the learn pipeline. eight conserved mirna families that mapped against rna sequences displayed a stem-loop structure. a single mirna precursor transcript was found for the hbmir hbmir hbmir hbmir and hbmir families. two mirna precursor transcripts were identified for the hbmir hbmir and hbmir families. in addition, the learn pipeline predicted putative novel mirnas with their unique precursors. mapping the sequences of the small rna dataset on the precursor sequences led to the identification of mirna*for all eight conserved families and for the five putatively new families hbmirn hbmirn hbmirn hbmirn and hbmirn stem-loop reverse transcription polymerase chain reactions were successfully performed on seven precursors for the conserved families and on nine for the putatively new families. the presence of the mirna* sequence in the small rna dataset is indicated. pictures of the stem-loop structures are presented in additional table the presence of the mirna* sequence in the small rna dataset is indicated. pictures of the stem-loop structures are presented in additional table comparison of the gene ontology classification of predicted targets for conserved and putative novel mirna families computational prediction of mirna targets led to the identification of sequences for the conserved families and for the putative novel mirna families. their involvement in biological processes and their molecular functions were attributed using gene ontology. most of the go terms were represented in the same proportions for predicted targets from conserved and putative novel mirna families. target genes of the putative novel mirna families all had go terms of conserved mirna families for biological processes and molecular functions. in biological process terms, the putative novel mirna families had two additional terms. the number of mirna target genes was smaller for putative novel than for conserved mirnas for some go terms: cellular process, metabolic process and biological regulation. this number was larger for putative novel mirna targets compared to conserved targets for cellular component organization, localization and response to stimulus. as regards the go terms for molecular function, although the distribution for antioxidant activity was the same for the classes of mirnas, a decrease in the proportion of transcription regulator activity and an increase in structural molecule activity and transporter activity were observed for the putative novel mirna families. comparison of predicted target genes for conserved and putative novel mirna families involved in "response to stimulus", and in "antioxidant" and "transcription regulation" activities as the management of oxidative stress is crucial for rubber productivity, we set out to more effectively understand the mechanisms of redox homeostasis. we therefore gave priority to predicted mirna-targeted genes involved in the "response to stimulus" and in "antioxidant" and "transcription regulator" activities according to the go annotations. twelve members of the ap2/erf domain-containing transcription factor family were putatively targeted by hbmir hbmir hbmir hbmir hbmir hbmir and hbmir two hd-zip iii domain-containing protein members, a transcription factor with a bzip domain, three nac domain-containing proteins, three squamosa promoter binding proteins, four wrky transcription factors, four auxin response factor; hbmir and hbmirn5) and five gras family transcription factors were also potentially targeted. sequences encoding protein involved in protein degradation, such as cysteine protease and ubiquitin protein ligase, were identified for genes classified in the "response to stimulus" category. three sequences related to aba signalling were also identified: an aba-insensitive protein, and an hva22-like protein. in addition, one sequence encoding ctr a negative regulator of the ethylene signalling pathway, was targeted by hbmirn mirna biogenesis was also highlighted by the presence of a cap binding protein predicted to be targeted by hbmir interestingly, two enzymes involved in natural rubber biosynthesis, hmg-coa reductase and 4-hydroxyl-3-methylbut-2-enyl diphosphate reductase, were predicted to be targeted by hbmir and hbmir respectively. for the "antioxidant activity" category, several ros-scavenging enzymes were identified. the chloroplastic and peroxisomal isoforms of cuznsod were putatively targeted by hbmir and hbmir respectively. another enzyme involved in glutathione biosynthesis, gamma glutamyl cysteine ligase, was predicted to be targeted by hbmirn rboh is an enzyme involved in ros production, which is likely to be targeted by two mirnas. in addition, targets that are subjected to or are involved in the redox status of proteins, such as genes encoding proteins involved in lignin synthesis, cynnamoyl coa reductase, lignin forming anionic peroxidase precursor and 4-coumarate:coa ligase, were also found to be targeted by hbmir hbmir and hbmir respectively. the cleavage site for three targets has been experimentally validated for a chloroplastic cuznsod, a squamosa promoter binding protein and an arf. for these targets, the cleavage site is located at the canonical 10th nucleotide. mirna:::::::::::::::::::::: guugugcucucucucuucuguca ▲::::::.:::::::::::::: uggcaugcagggagccaggca ▲.:::::::.::::::.:::::.: gucaugcgggugaccugggaaacaua ▲ the number of sequences presenting the same cleavage site in indicated below the cleavage site located at the 10th nucleotide discussion size distribution of small rnas in hevea may differ from others species next-generation high-throughput sequencing techniques are powerful tools for mirna identification. the sequencing and analysis of the hevea small rna library generated more than million sequences. in their vast majority, the sequenced small rnas were only represented by one or two reads. similarly, 65% of unique mirna sequences were found in arabidopsis by sequencing techniques. interestingly, we found that the size distribution of hevea small rnas differed from other species. the most abundant small rnas had and nucleotides in hevea whereas they have and nucleotides in other species. indeed, in arabidopsis thaliana, arachis hypogea and oryza sativa, the fraction of nucleotides has proved to be predominant, accounting for 60%, 45% and 36% of unique small rnas, respectively. the predominant size has been found to be nucleotides in pinus contorta and taxus chiniensis. in hevea, the high percentage of 17- and 19-nucleotide small rnas resulted from the increased amount of rna degradation products and transposable elements in response to abiotic treatments according to the mapping results against the arabidopsis genome, as previously observed in brachypodium. indeed, a change in small rna size distribution has been reported in brachypodium in response to cold treatment. under normal growth conditions, the 21- and 24-nucleotide classes were the most abundant small rna families, whereas it was the 19-nucleotide class after cold treatment. our dataset was generated from pooled small rnas of various tissues subjected to several types of abiotic stress. the size distribution may have been differentially affected by each type of abiotic stress and may explain the difference in size distribution in hevea. long conserved and novel mirnas of 23- nucleotides consisted of short mirna sequences with an extension. this lmirna structure suggested that they were generated from the same mir genes. our observation suggests dual functions for canonical mir genes producing 20- nt and 23- nt lmirna in hevea, namely hbmir hbmir hbmir hbmir hbmir and hbmirn3- across the plant kingdom, lmirna were previously classed as heterochromatic small interfering rnas. in arabidopsis, the hc-sirna derived from the mirna-generating site is dependent on dcl rdr and poliv associated with ago and direct dna methylation for some target sites. as regards the higher number of reads for the nucleotide compared with the 20- nucleotide mirnas, the mirna families producing 20- nt and 23- nt mirnas might mostly regulate their targets through direct dna methylation in response to abiotic stress. this may confirm the results of a recent study showing the existence of site-specific abiotic stress-induced dna methylation for five genes in hevea. ancient and recently differentiated hevea brasiliensis mirnas provide additional information for evolutionary studies the hevea brasiliensis conserved mirna families were more or less similar to those of other species. in hevea, five mirna families have been found in more than forty plant species, and nine correspond to ancient mirna already present in the common ancestor of embryophytes, two in the common ancestor of tracheophytes and nine in the common ancestor of spermaphytes. interestingly, the so-called monocotyledon-specific hbmir family was identified in this study. this suggests that hbmir appeared before the speciation between monocotyledons and dicotyledons. three woody species-specific mirna families were also found in hevea brasiliensis: hbmir, hbmir and hbmir and were probably recruited independently by woody species during their evolution. the novel mirna families identified in this work are likely to be more recent, after hevea diversification, as they have not been identified up to now in other euphorbiaceous plants. furthermore, this information complements previous studies on euphorbiaceous species in which only conserved mirnas were identified. our data could be used to study the evolution of mir genes. we showed that 70% of the putatively new mir genes were able to produce lmirna of 23- nucleotides, compared to 29% for conserved mir genes. this corresponds to what has been observed in arabidopsis, where it was previously suggested that young mir genes consistently produced sirnas, while ancient mir genes produced predominantly canonical mirnas, showing that the evolution of mir genes is associated with changes in the use of dcl, resulting in specific mirna size classes. in addition, several so-called plant species-specific mirnas were also found in hevea, such as hbmir initially found only in pinus taeda, hbmir hbmir hbmir and hbmir found in populus euphratica and hbmir in populus trichocarpa. our data suggest that those mirnas should no longer be considered as species-specific mirna. our study also revealed some conserved and divergent mechanisms at post-transcriptional level. indeed, four families of mirna were subjected to rna editing events at the sites previously observed in arabidopsis. however, for the thirteen families left, rna editing was not observed under our experimental conditions. under our experimental conditions, the - + uu modification was not observed in hevea for the nine families selected from ebhardt et al.. once the hevea genome sequence is available, it will be possible to conduct an exhaustive analysis of the discrepancies between members of a multigenic family and rna editing events. predicted target/mirna pairs conserved between plant species and putative new ones in hevea brasiliensis in comparison with other plants, several regulation mechanisms seem to be conserved in hevea brasiliensis, such as the regulation of hd-zip iii protein by hbmir, squamosa promoter binding protein by hbmir, nac domain protein by hbmir, apetala2-like protein by hbmir, ccaat-binding transcription factor by hbmir and cysteine proteinase by hbmir. these ancient mirnas regulate ancestral transcription factors that coordinate highly conserved functions, such as organ polarity and separation, cell division, or hormonal control. we predicted potentially new mirna/target pairs in hevea. new target/mirna pairs were found for transcription factors, such as, for example, the nac domain protein by hbmir and hbmirn the gras domain protein by hbmir hbmir and hbmirn wrky by hbmir and hbmir and the apetala2-like transcription factor by hbmir hbmir hbmir hbmir and hbmir. in addition, we also detected new pairs involved in protein degradation, in response to hormones and in the regulation of mirna. further analyses are needed to validate the inhibition of these targets at transcriptional or translational level and to unravel specific regulation in hevea brasiliensis regarding the response to stress in latex cells, and latex production. conserved and divergent regulation of redox homeostasis in hevea brasiliensis several enzymes involved in ros production or detoxification were found in our analysis. two ros-producing enzymes, rboh, are likely to be targeted by hbmir and hbmir they are key regulators in the ros signalling network as they integrate different signal transduction pathways, such as calcium, protein phosphorylation and lipid signalling with ros production. careful attention was also paid to targets classified in the go term "antioxidant activity". for conserved mirnas, two cuznsod chloroplastic and peroxisomal isoforms were predicted to be targeted by hbmir and hbmir respectively. surprisingly, the cuznsod cytosolic isoform was not predicted by the learn pipeline and, even manually, no correct alignment was possible with hbmir so far, validation by 5' race pcr has failed under our experimental conditions. by contrast, previous studies in arabidopsis showed that hbmir targeted both chloroplastic and cytosolic isoforms. some mirna families have been reported to be h2o2-responsive in rice, such as osmir osmir osmir osmir osmir and osmir. in rice, osmir targets laccase genes involved in lignin biosynthesis and those targets were also found for hbmir in hevea.  a library of small rnas was deeply sequenced to identify conserved and novel mirnas in hevea brasiliensis. combined with the hevea transcript database, this led to the identification of mirna families and novel mirnas. our study revealed new insights for evolutionary studies on woody plants and the potentially specific regulation of redox homeostasis in hevea. all the putative targets of interest for hevea stress biology and latex production will be experimentally validated and the target genes and their corresponding mirnas will undergo co-expression analysis. 
our study provided additional information for evolutionary studies and revealed potentially specific regulation of the control of redox status in hevea. the results showed the most abundant size for mirnas to be nucleotides, except for seven families. by combining the learn pipeline, data from the plant microrna database and hevea est sequences, we identified conserved mirna families already characterized in other plant species, and putatively novel mirna families. in hevea brasiliensis, environmental and harvesting stresses are known to affect natural rubber production. the two mirna class sizes were detected for both conserved and putative novel mirna families, suggesting their functional duality. plants respond to external stimuli through fine regulation of gene expression partially ensured by small rnas. deep sequencing of small rnas was carried out on plantlets subjected to severe abiotic stress using the solexa technique. deep sequencing of small rnas combined with transcriptomic data is a powerful tool for identifying conserved and novel mirnas when the complete genome is not yet available. they negatively regulate gene expression by targeting the cleavage or translational inhibition of target messenger rnas. gene expressionmirnamir genenext-generation sequencingrubber treetranscriptiontranscriptomeabiotic stressmirna editing several mir genes produced both 20- nucleotides and 23- nucleotides. mirna targets were computationally predicted and analysed.
 plants respond to external stimuli through fine regulation of gene expression partially ensured by small rnas. of these, micrornas play a crucial role. they negatively regulate gene expression by targeting the cleavage or translational inhibition of target messenger rnas. in hevea brasiliensis, environmental and harvesting stresses are known to affect natural rubber production. this study set out to identify abiotic stress-related mirnas in hevea using next-generation sequencing and bioinformatic analysis.  deep sequencing of small rnas was carried out on plantlets subjected to severe abiotic stress using the solexa technique. by combining the learn pipeline, data from the plant microrna database and hevea est sequences, we identified conserved mirna families already characterized in other plant species, and putatively novel mirna families. the results showed the most abundant size for mirnas to be nucleotides, except for seven families. several mir genes produced both 20- nucleotides and 23- nucleotides. the two mirna class sizes were detected for both conserved and putative novel mirna families, suggesting their functional duality. the est databases were scanned with conserved and novel mirna sequences. mirna targets were computationally predicted and analysed. the predicted targets involved in "responses to stimuli" and to "antioxidant" and "transcription activities" are presented.  deep sequencing of small rnas combined with transcriptomic data is a powerful tool for identifying conserved and novel mirnas when the complete genome is not yet available. our study provided additional information for evolutionary studies and revealed potentially specific regulation of the control of redox status in hevea. gene expressionmirnamir genenext-generation sequencingrubber treetranscriptiontranscriptomeabiotic stressmirna editing 