BACKGROUND
the spontaneous 7b- <dig> mutant in tomato  is a photoperiod-dependent male-sterile line, where in long days flowers are male-sterile with stamens that are shrunken and produce non-viable microspores  <cit> .
in short days, flowers are fertile and produce normal stamens and viable pollen.
a proteomic study suggested that microsporogenesis in 7b- <dig> breaks down prior to the meiosis in microspore mother cells , which was associated with altered levels of several important proteins involved in tapetum degeneration and mmcs development  <cit> .
compared to the wt, 7b- <dig> has a higher tolerance to various abiotic stresses, specifically under blue light, is less sensitive to light-induced inhibition  of hypocotyl growth, to blue light-induced stomata opening  <cit> , and has an elevated level of endogenous aba, but less gas, iaa, and cks .
fellner and sawhney,  <cit>  demonstrated that there was a defect in blue light perception in 7b- <dig>  which affected hormonal sensitivity and their endogenous levels.
this information adds on to the fact that the 7b- <dig> is a complex mutation with its primary effect still unknown.
as a stress-tolerant male-sterile mutant, 7b- <dig> is a valuable germplasm for hybrid tomato breeding  <cit> .
recent studies have documented important regulatory functions of srnas in plant growth and development.
there are two main types of srnas based on their biogenesis: small interfering rnas , and micrornas .
sirnas are processed from perfectly double-stranded rna  and comprise different classes; those produced from dsrna synthesized by rna dependent rna polymerase  <dig>    <cit> , by rdr <dig>   <cit> , or by overlapping antisense mrnas   <cit> .
while ta-sirnas target mrnas similarly to mirnas, heterochromatin sirnas cause dna methylation and/or heterochromatin formation that lead to transcriptional gene silencing  <cit> .
ta-sirnas play essential roles in regulating plant development, metabolism and responses to biotic and abiotic stresses  <cit> .
four families of tas genes comprising eight loci have been identified in arabidopsis thaliana, among which mir <dig> targets both tas1a/b/c family and tas <dig> locus, mir <dig> targets tas3a/b/c family, while mir <dig> triggers the production of tas4-derived ta-sirnas .
tas3a-derived tasiarfs, 5´d <dig> and 5´d <dig>  target several arfs, including arf <dig>   <dig> and  <dig>  <cit> , which were proposed to act as suppressors in auxin signaling pathway  <cit> .
recently, a fifth tas family  was identified from tomato, which is triggered by mir <dig>  <cit> .
plant mirnas are typically  <dig> nucleotides  long, which are derived from single-stranded rna transcripts that have the ability to fold into imperfect stem-loop secondary structures.
these hairpins are processed by the ribonuclease iii-like enzyme dicer  into mirna/mirna* duplexes.
one of the strands of the mirna/mirna* duplex  is incorporated into the rna induced silencing complex , which guides the risc to recognize target mrna based on sequence complementarity.
mature mirnas suppress the expression of their target genes by cleavage of the target mrnas or translational repression  <cit> .
many plant mirnas are conserved among species and have been implicated in processes, such as development, signal transduction, abiotic stress tolerance, and resistance to pathogens .
high-throughput sequencing was first used to identify tomato srnas, including mirnas from young green fruits and more recently from several developmental stages of the fruits .
recently, there is a growing interest towards understanding the role of mirnas in regulation of male-sterility in plants.
even though there is no evidence that any mirnas directly cause male-sterility in plants, it was suggested that mirnas and their target genes play important roles in regulation of male-sterility.
wei et al.,  <cit>  identified several conserved and new mirnas, which were differentially expressed during anther development in a male-sterile cotton mutant.
these mirnas targeted hd-zip iii-like, arf <dig>  ap <dig> and acc oxidase  <dig> genes, which are key genes involved in hormone signaling, cell patterning, and anti-oxidant metabolism.
zhang et al.,  <cit>  reported that mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig> and mir <dig> were differentially expressed in sporogenous cell, mmcs and microspores between a male-sterile cotton and its maintainer line.
the predicted targets for these mirnas were involved in cotton growth and development, signal transduction and metabolism pathways.
jiang et al.,  <cit>  identified  <dig> mirnas, which were differentially expressed in pollens of a male-sterile line of brassica campestris and its wild type.
there are increasing evidences showing that the function of mirnas, including mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig> and mir <dig> is crucial during flower development and microsporogenesis .
mir <dig> directs the cleavage of gamyb-related transcripts, which are involved in regulation of anther development and microsporogensis  <cit> .
overexpression of mir <dig> in arabidopsis thaliana resulted in sterile anthers  <cit> .
mir <dig> targets arf6/ <dig>  which regulate ovule and anther development  <cit> .
in arabidopsis thaliana, overexpression of mir <dig> led to male-sterility  <cit> .
mutation in a srna locus in rice which produced a 21-nt srna has led to environment-conditioned male-sterility  <cit> .
srnas in tomato have been mainly studied in fruits development and ripening process, and no report has yet documented their involvement in regulation of male-sterility.
the main goal of our study was to investigate whether srna biogenesis is affected by 7b- <dig> mutation, and if srnas, particularly mirnas, are associated or involved in anther development and regulation of male-sterility in 7b- <dig>  known and new mirnas, ta-sirnas and their targets were identified and their expressions were studied in 7b- <dig> and wt anthers.
targets of mirnas with potential roles in anther development and microsporogenesis were validated using 5′-race and localization of mirnas was further analyzed by in situ hybridization.
RESULTS
deep sequencing of srnas
anthers from the flower buds at three stages of pre-meiosis, meiosis, and post-meiosis  were dissected and pooled as described by sheoran et al.,  <cit> .
two srna libraries were constructed from wt and 7b- <dig> pooled anthers and sequenced, which produced about  <dig> and  <dig> million raw reads, respectively.
after adaptor trimming, reads were mapped to the tomato  genome with no mis-matches allowed .
size class distribution of total and unique srnas and their complexities in each library is presented in fig.
<dig>  the majority of srnas in both 7b- <dig> and wt libraries were 21–24 nt, and formed a bi-modal size distribution typical for plant srnas.
in general, the peak represented by 24-nt size class is greater than the 21-nt class as reported in several plant species  with a few exceptions, as in grapevine, where the 21-nt class was more abundant than the 24-nt class in total read numbers, but not at unique read level  <cit> , and in brassica juncea, where the 21-nt class had the major peak in total and unique reads  <cit> .table  <dig> statistics of srna reads
fig.
<dig> size distribution of redundant  and non-redundant  srna reads and their complexity in wt  and 7b- <dig>  anthers
in our study, the 21-nt class had a bigger peak than 24-nt class in total reads in both libraries; however, for the non-redundant distribution, the 24-nt class had the major peak in both libraries.
two key distinguishing features of srna libraries are the size distribution and population complexity as defined by the ratio of unique/total reads.
the lower complexity of the 21-nt class in comparison to the 24-nt class in our study indicated that a relatively small number of unique reads were highly expressed in the 21-nt class, while the 24-nt class high complexity indicated the presence of more unique reads with less redundancy, which is also a typical feature of the 24-nt hcrnas, where often are present in a more chaotic dicing pattern  <cit> .
identification of known and new mirnas
known mirnas from  <dig> known families of mirnas were identified, which were present in both 7b- <dig> and wt libraries .
the composition of mirna families was quite similar in the two libraries, and none showed a clear 7b- <dig> or wt-specific profile.
most of the families had several members, with exception of mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig>  and mir <dig>  which were represented only by one member in both libraries .
after excluding srna reads homologous to known mirnas and other non-coding rnas from rfam v. <dig> database, the remaining 20-22 nt reads were used as input for mirna prediction using the mircat tool  <cit> .
a total of  <dig> putative new mirnas were identified in the two libraries, where  <dig> were present only in wt,  <dig> in 7b- <dig>  and  <dig> in both libraries.
the mirna* sequences were found for two new mirnas, which were present in both libraries and designated as mir#a and mir#b.
these mirnas formed near-perfect hairpin structures .
expression analysis of mirnas
to identify mirnas related to anther development and regulation of male-sterility in 7b- <dig>  expressions of mirnas were compared between 7b- <dig> and wt anther libraries.
the two libraries were normalized using the reads per million  approach  <cit> .
the differential expression analysis was conducted using the offset fold change  approach  <cit>  and two-fold threshold was considered as a cutoff value.
among the known mirnas, mir <dig>  mir <dig>  mir <dig> were up-regulated and mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig>  and mir <dig> were down-regulated in 7b- <dig> anthers .
mir <dig> and mir <dig> had three and two iso-mirs, respectively, while the rest of differentially expressed mirnas were present only by one member.
out of the total of  <dig> putative new mirnas only two potential mirnas, so-called mir#w and mir#m were differentially down-regulated in 7b- <dig>  none of the new mirnas with sequenced mirna* strands were differentially expressed in 7b- <dig> .table  <dig> list of differentially expressed mirnas between wt and 7b- <dig> anthers
7b-1
7b-1
ade is differential expression values which were calculated as log2-fold changes of the expression.
negative and positive values mean up- and down regulation of the expression in 7b- <dig>  respectively.
de value of ± <dig> was considered as a cutoff value for significant changes of the expression
rt-qpcr validation of mirnas expression
expressions of known and new mirnas of interest were further analyzed by rt-qpcr  in 7b- <dig> and wt anthers  and stem.
mir <dig> and mir <dig> were up-regulated at similar extent in all stages of 7b- <dig> anthers.
mir <dig> and mir <dig> were down-regulated in all stages of 7b- <dig> anthers; more strongly at stage  <dig>  mir#m was strongly down-regulated in all stages of 7b- <dig> anthers, while mir#a and mir#b were not differentially expressed and their sequenced mirna* strands were not detected by our rt-qpcr analysis.
the rt-qpcr results were in general agreement with those from sequencing data.
in 7b- <dig> stem, mir <dig>  mir <dig>  mir <dig> and mir#m were all up-regulated, while mir <dig>  mir#a and mir#b were not differentially expressed.
mir#b* was not detected by rt-qpcr.
the results showed a very distinct anther- or stem-specific expression pattern of mir <dig>  mir <dig>  and mir#m, which suggests that they may regulate different biological processes in these tissues.fig.
<dig> rt-qpcr validation of differentially expressed known and new mirnas and mirnas with mirna*.
expression changes are presented as normalized fold changes between 7b- <dig> and wt reference tissue.
positive and negative values indicate up- and down-regulation of the expression, respectively.
two-fold threshold was considered as a cutoff value for significant changes in the expression.
error bars represent standard errors of three biological replicates based on dmnrt
target prediction for known and new mirnas
to better understand the biological roles of mirnas in regulation of 7b- <dig> male-sterility, putative targets of differentially expressed known and new mirnas were identified .
target genes for many of known conserved mirnas have been experimentally validated .
predicted targets for known mirnas in our study were also in agreement with those from the literature, however a number of new putative target genes were also computationally identified, which has yet to be validated.
the mirna targets were categorized into fifteen different biological classes  with the three most frequent being metabolic process, cellular process, and single-organism process.
among the targets worth mentioning  were those, including auxin-responsive factor  <dig> , abc transporters, g protein, f-box and no apical meristem  proteins, b-zip, sbp-box, mad-box and myb transcription factors with potential roles in anther development , cystatin, receptor-like kinase, and bhlh proteins involved in tapetum development/degeneration , kinesin-like protein and gras transcription factor involved in meiosis regulation  <cit> , and wd- <dig> protein with roles in autophagy and apoptosis  <cit> .
among the targets of new mirnas , a gene encoding pectinmethylesterase  inhibitor  was identified as the putative target of mir#m.
pme is a cell wall modifying enzyme, which catalyzes de-esterification of pectin  <cit> .
identification of mirna targets with potential roles in anther development and microsporogenesis regulation in our study, suggested that these mirnas are likely associated or involved in regulation of 7b- <dig> male-sterility.table  <dig> list of the predicted targets of differentially expressed known mirnas
aindicates targets which have identified from multiple loci
aindicates targets which have identified from multiple loci
fig.
<dig> gene ontology of mirna targets.
predicted targets were categorized into different biological classes and numbers in the parenthesis indicate the frequency of members in each category
identification of ta-sirnas and prediction of their targets
tas loci and their associated ta-sirnas were predicted based on the phased 21 nt srnas characteristic of ta-sirna loci  <cit> .
ta-sirnas with abundance below  <dig> reads were excluded from the analysis.
twenty-five ta-sirnas from wt,  <dig> from 7b- <dig> and  <dig> common in both libraries were identified and their target genes were predicted .
as these ta-sirnas had generally low abundances, those identified only in wt or 7b- <dig> could not be considered as wt- or 7b-1-specific and were not further analyzed.
instead, we focused on those which have been found in both libraries; however none were differentially expressed .
ta-sirnas with phased expressions matching to the tomato tas <dig>  were also identified , however tas3-derived 5′d <dig> and 5′d <dig> tasiarfs were not differentially regulated between the two libraries.
the results suggested that ta-sirnas were not likely associated or involved in regulation of male-sterility in 7b- <dig> as they were not differentially regulated.
5′-race validation of mirna and ta-sirna targets
the predicated targets of interest were validated in 7b- <dig> anthers  and stem using 5′-race.
sequence analysis showed  that the 5′ ends for most of the cleaved mrna fragments corresponded to nucleotide complementary to the 10th nucleotide of the corresponding mirna.
mir <dig> and mir <dig> could both cleave myb transcripts  <cit> ; however sequence analysis of the cleavage products showed that myb cleavage in anthers and stem was directed by mir <dig>  not mir <dig>  cleavage products of mir <dig> and mir <dig> can be readily distinguished as they differ in one base.
cleavage products of arf <dig>  target of mir <dig>  were identified from anther, but not stem.
among the predicted targets of mir <dig>  cleavage products of cystatin were identified in both anther and stem libraries, but not mad-box.
cleavage products of pmei, predicted target of mir#m, were also identified in both anther and stem libraries.
cleavage products of arf <dig>   <dig> and  <dig>  targets of d <dig> and d <dig> tasiarfs, were identified from both anther and stem libraries .
these results showed that the above mentioned mirnas, including the newly identified mir#m as well as tasiarfs were functionally active in 7b- <dig> anther and stem, where directed the cleavage of their predicted target transcripts.
furthermore, it provided experimental evidence to support our target predictions.fig.
<dig> 5′- race validation of mirna target genes in 7b- <dig> anther and stem.
gene transcripts are in 5′-3′ and mirnas in 3′-5′directions.
the arrows indicate the cleavage sites of target mrna and numbers above them indicate frequency  of sequences found at the exact mirnas cleavage sites.
watson-crick pairing , g-u wobble pairing , and other mismatches  are indicated
rt-qpcr analysis of mirna and ta-sirna targets
figure  <dig> shows rt-qpcr analysis of mirna targets of interest and tasiarfs in 7b- <dig> anthers and stem.
gamybl <dig> was down-regulated in anthers of all stages and more strongly in stem.
arf <dig> was up-regulated in anthers of all stages and stem.
cystatin and pmei were both up-regulated in anthers of all stages, but down-regulated in stem.
tasiarfs  were not differentially expressed in anthers and stem.
arf2/3/ <dig> were not differentially expressed in anthers, but up-regulated in stem .
analysis showed a consistent negative correlation between mirnas expression and accumulation level of their targets, except for mir390-tas3-tasiarfs , and mir167-arf <dig> .
in addition to mirna and their targets, expression of two cell wall modifying enzymes, cysteine protease and polygalacturonase, were also analyzed in 7b- <dig> anthers and stem .
cysteine protease was down-regulated in anthers, more strongly at stage  <dig>  while up-regulated in stem.
polygalacturonase was strongly down-regulated in anthers at stage  <dig>  but not differentially regulated at stage  <dig>  nor in stem.fig.
<dig> rt-qpcr validation of mirna target genes and tasiarfs in 7b- <dig> anthers and stem.
expression changes are presented as normalized fold changes between 7b- <dig> and wt reference tissue.
positive and negative values indicate up- and down-regulation of the expression, respectively.
two-fold threshold was considered as a cutoff value for significant changes in the expression.
error bars represent standard errors of three biological replicates based on dmnrt
in situ hybridization
figure  <dig> shows in situ localization of mir <dig>  gamybl <dig>  pmei and cystatin in wt and 7b- <dig> anthers in the late meiotic  and in binucleate microspores stages as for the cases of pmei and cystatin.
mir <dig>  gamybl <dig> and cystatin were all predominantly expressed in tapetum, tetrads and free microspores in wt and more strongly in 7b- <dig> as the case of mir <dig> and cystatin.
panels l and m of the figure show wt and 7b- <dig> anthers, respectively at binucleate microspores stage, where tapetum was degenerated in wt, but not degenerated and vacuolated in 7b- <dig>  cystatin was strongly expressed in vacuolated tapetal cells in 7b- <dig> .
pmei was strongly expressed in tapetum, tetrads and free microspores in 7b- <dig> anthers, but its expression was mainly restricted to tapetum at binucleate microspores stage .
as mentioned earlier, in some of the 7b- <dig> anthers, microspores were not separated and remained attached after meiosis.
pmei was strongly expressed in arrested binucleate microspores in 7b- <dig> .
no detectable hybridization signal was observed for the murine mir122a probe, which served as a negative control.fig.
<dig> in situ localization of mir <dig>  gamybl <dig>  pmei and cystatin.
a and b localization of mir <dig> in wt and 7b- <dig> anthers, respectively.
c and d
gamybl <dig> in wt and 7b- <dig> anthers, respectively.
e and f
pmei in wt anthers at late meiotic and binucleate microspores stages, respectively.
g, h, and i
pmei in 7b- <dig> anthers at late meiotic stage, free binucleate microspores, and arrested binucleate microspores, respectively.
j and k
cystatin in wt and 7b- <dig> anthers, respectively.
l and m
cystatin in wt and 7b- <dig> anthers at binucleate microspores stage, respectively.
n negative control, where a murine mir122a-specific probe was used.
arrows indicate the localization sites
discussion
the main goal of our study was to investigate the srna profiles, particularly mirnas, between the 7b- <dig> mutant and wt anthers and to identify differentially expressed mirnas and their targets with potential roles in anther development and regulation of male-sterility in 7b- <dig>  analysis showed that the overall size distribution and population complexity of srnas were quite similar between the 7b- <dig> and wt anther libraries.
composition of srnas could indicate the roles and activity level of different categories of srnas in a particular tissue or species or associated biogenetic machineries.
in our study, the 21-nt class had a larger peak than the 24-nt class in total reads in both libraries; however, in non-redundant format, the 24-nt class had the major peak in both libraries.
the 24-nt class was also found to be the major peak in anthers of a male-sterile cotton  <cit> .
higher abundance of the 21-nt class in total reads in anthers of 7b- <dig> and wt in our study, could suggest a more active roles for this class of srnas and an intensified contribution from mirna biogenesis pathways that produce the more precisely defined srna species at those stages compared to those of other crops having the 24-nt class with higher redundancy.
even though there is no direct evidence that any mirnas are causative genes for male-sterility in plants, we hypothesized that differential expression of mirnas could be associated with the regulation of male-sterility in 7b- <dig>  thirty-two families of known mirnas were identified in both 7b- <dig> and wt anther libraries.
three and seven mirna families were up and down-regulated, respectively in 7b- <dig> anthers.
out of the  <dig> putative new mirnas only two, so-called mir#w and mir#m, were differentially expressed in 7b- <dig> anther.
expressions of mirnas of interest were further analyzed at different stages of 7b- <dig> anthers and stem.
in general, changes of the expression were strongest in anthers at stage  <dig>  where the mmcs were about to undergo the meiosis.
mir <dig>  mir <dig>  and mir#m had distinctively different expression patterns in anthers and stem, which strongly suggested that these mirnas regulate different biological processes in these tissues.
the predicted targets of differentially expressed mirnas were categorized into fifteen different biological classes, with metabolic process, cellular process, and single-organism process being the three most frequent classes.
among these mirnas, mir <dig>  mir <dig>  mir <dig>  mir <dig> and mir#m were those of particular interest as their targets had potential roles in anther development, microsporogenesis and production of ta-sirnas as the case of mir <dig>  a schematic presentation of mirna-target pairs and their role in regulation of male-sterility in 7b- <dig> mutant is illustrated in fig.
<dig>  5′-race validation of these mirna targets in 7b- <dig> anther and stem showed that they were active and directed the cleavage of their targets in these tissues, except for mir <dig> in 7b- <dig> stem.fig.
<dig> schematic presentation of mirna-target pairs and their role in regulation of anther development and male-sterility in 7b- <dig> mutant.
arrows below mirnas and mirna targets show up- and down-regulation of the expression.
“●” indicates no change in the expression.
pictures of 7b- <dig> anthers are adopted from sheoran et al.,  <cit>
mir <dig> targets several gamybs  <cit> , among them atmyb <dig>  atmyb <dig> act redundantly in regulation of anther development  <cit> .
overexpression of mir <dig> disrupted anther development and led to male-sterility in arabidopsis thaliana  <cit> .
5′-race analysis in our study showed that mir <dig> directed the cleavage of gamybl <dig> transcripts out of the  <dig> predicted myb targets in anther and stem.
osgamybl <dig> and  <dig> are expressed in rice anthers and regulated by mir <dig>  however functions of these gene are not characterized  <cit> .
legamybl <dig> plays a role in seed development in tomato  <cit> , but it is not functionally characterized with respect to anther development.
analysis showed that the expression of gamybl <dig> was negatively correlated with the mir <dig> level in 7b- <dig> during anther development and in stem.
in situ hybridization analysis in 7b- <dig> anthers also showed that mir <dig> and gamybl <dig> were both expressed mainly in tapetum, tetrads and microspores.
mir <dig> and gamybl <dig> were down- and up-regulated, respectively in ga-treated 7b- <dig> anthers .
these observations indicated that mir159-gamybl <dig> cleavage cascade and ga level in flower buds are tightly linked to the regulation of anther development and male-sterility in 7b- <dig>
most of the elongated mutants have been reported as ga-overproducers ; however elongated stem of 7b- <dig> had lower levels of gas compared to wt in long days  <cit> .
several mybs have been identified, which positively regulate stem elongation in light via interaction with phytochromes and/or regulation of light-responsive genes  <cit> .
down-regulation of gamybl <dig> in 7b- <dig> stem is likely associated with the elongated stem phenotype, however functional analysis are needed to understand the actual function of this gene.
mir <dig> cleaves arf6/ <dig> transcripts  <cit> ; however we only identified arf <dig> in our target prediction, which was also validated by 5′-race in 7b- <dig> anther.
tomato overexpressing mir <dig> and arf6-arf <dig> double-null mutant of arabidopsis thaliana both had flowers with severe defects associated with female and male-sterilities, respectively  <cit> .
therefore, proper regulation of mir167-arf6/ <dig> is required for normal female and male organ development.
rt-qpcr analysis showed strong down-regulation of mir <dig> in 7b- <dig> anthers, which was negatively correlated with regulation of arf <dig> in anthers.
although differential regulation of mir167-arf <dig> cleavage cascade in 7b- <dig> anthers could be linked and due to 7b- <dig> mutation, its actual function with respect to anther development and male-sterility in 7b- <dig> remains to be further characterized.
in 7b- <dig> stem, neither the arf <dig> cleavage products were found nor the arf <dig> expression was correlated with mir <dig> level.
tomato overexpressing mir <dig> had lower levels of arf6/ <dig> transcripts and a shorter hypocotyl  <cit> , while other researchers reported that light-grown arabidopsis thaliana arf8- <dig> mutant had elongated hypocotyl  <cit> .
although up-regulation of mir <dig> and arf <dig> in 7b- <dig> stem could be independently associated and/or affected by 7b- <dig> mutation, understanding their functions with respect to anther development and male-sterility in 7b- <dig> requires further functional analysis.
to the best of our knowledge no reports have yet documented ta-sirnas in regulation of male-sterility in plants.
ta-sirnas including tas3-derived tasiarfs were not differentially expressed between 7b- <dig> and wt; however mir <dig> was up-regulated in 7b- <dig> anther and stem.
this suggested that although mir <dig> expression was affected by 7b- <dig> mutation, ta-sirna biogenesis and tasiarfs-guided regulation of arf2/3/ <dig> were not affected by 7b- <dig> mutation nor related to anther development in 7b- <dig>  nevertheless, 5′-race analysis showed that tasiarfs are active in 7b- <dig> anther and stem, where directed the cleavage of arf2/3/ <dig> transcripts.
target genes of arf2/3/ <dig> are largely unknown, thus the mechanisms linking these arfs to a context-specific role in regulation of 7b- <dig> stem growth if any are poorly understood.
our cytological studies showed  that anther development in 7b- <dig>  was not synchronized in contrast to the wt.
we observed that mmcs did undergo meiosis and formed tetrads despite the previous report suggested a breakdown of meiosis in mmcs  <cit> .
more importantly, we found that in some anther lobes, the newly formed microspores were not separated, while in others they formed free microspores.
in qrt <dig> and qrt <dig> mutants of arabidopsis thaliana, microspores failed to separate from tetrads as pectin was not degraded in the primary cell wall  <cit> .
pmei  was up-regulated in 7b- <dig> anthers, where the expression was mainly localized in tapetum, tetrads and microspores.
pmei was strongly expressed in the arrested binucleate microspores, compared to the free binucleate microspores, where a basal expression was detected.
pmes and pmeis are key regulators of pollen cell wall and pollen tube development .
up-regulation of pmei in 7b- <dig> anthers may have suppressed the pme activity and impaired the subsequent enzymatic degradation of pectin in the primary cell walls around tetrads.
in qrt <dig>  mutation in a gene encoding a polygalacturonase  impaired the degradation of pectin in the primary cell wall, similar to qrt <dig> and qrt <dig> mutants  <cit> .
polygalacturonase was also down-regulated in 7b- <dig> anthers in our study.
based on our cytological and transcriptional data, we suggest a potential association between the regulation of mir#m-pmei and polygalacturonase and anther development in 7b- <dig> as inhibition of pme and polygalacturonase activities could have disrupted pectin degradation and proper separation of tetrads and/or tapetum degeneration.
however, it could not be the cause of male-sterility in 7b- <dig> as some of the anthers were still able to produce mature pollens.
arabidopsis thaliana overexpressing pme showed reduced cell elongation in hypocotyl  <cit> .
al-qsous et al.,  <cit>  suggested that higher level of pme transcript in elongated hypocotyl of flax was associated with cell wall stiffening.
overexpression of a polygalacturonase gene, pgx <dig>  enhanced hypocotyl elongation in etiolated arabidopsis thaliana  <cit> .
understating the function of pme and polygalacturonase in regulation of stem elongation in 7b- <dig> if they have indeed altered the pectin level and how they are connected to the cell expansion requires further analysis.
while conserved mirnas regulate expression of the genes involved in basic developmental processes, the non-conserved or new mirnas may be involved in the development of traits that are specific for certain species.
new mirnas are being continually identified from different species, including tomato  <cit> .
in addition to mir#m, we identified a number of new mirnas, two with sequenced mirnas*, which could form a near prefect hairpin structures.
sheoran et al.,  <cit>  identified a number of differentially expressed proteins between 7b- <dig> and wt anthers, where cystatin showed the strongest up-regulation of all in 7b- <dig> anthers.
the cystatin inhibitory activity was also higher in 7b- <dig> anthers relative to wt  <cit> .
in plants, cysteine proteases act as key regulators of programmed cell death in tapetal cells and pollen development, and suppression of their expression has often resulted in delay or failure in tapetum degeneration, pollen abortion and male-sterility .
mir <dig> directed the cleavage of cystatin in 7b- <dig> anthers and stem.
cystatin was up-regulated and cysteine protease was strongly down-regulated as a result in 7b- <dig> anthers, with a pattern closely correlated to the tapetum degeneration during anther development.
these observations provided a strong evidence to suggest that suppression of cysteine protease could have caused a delay or failure in tapetum degeneration, thereby affecting microsporogenesis and anther development in 7b- <dig>  the length of stem in 7b- <dig> is tightly correlated with the length of epidermal cells  <cit> .
minic et al.,  <cit>  identified several cysteine proteases in developing stems of arabidopsis thaliana, suggested to be involved in cell expansion and secondary wall formation.
higher expression of cysteine protease in our study could be associated with the higher cell expansion rate and longer epidermis cells in 7b- <dig> stem.
overall in our study, we found that mirna-mediated regulation of gene expression was perturbed in the 7b- <dig> mutant line.
the findings strongly supported that mir159-gamybl <dig>  mir396-cystatin and mir#m-pmei cleavage cascades were tightly connected to the regulation of microsporogenesis and anther development in 7b- <dig>  stem elongation in 7b- <dig> may be regulated via a complex web of molecular components and interaction between mirnas and hormones, which requires further functional studies to be understood.
a number of new mirnas were also identified for the first time from tomato, which provides new opportunities to study the unexplored functions of these mirnas in tomato.
our data could be used as a benchmark for future studies of the molecular mechanisms of male-sterility in other crops.
CONCLUSIONS
using srna sequencing, we studied mirna profiles during anther development between 7b- <dig> mutant and wt.
comparison of expression of mirnas and their targets between 7b- <dig> and wt and in situ localization analysis suggested potential involvement of several mirna-target pairs in regulation of anther development and male-sterility in 7b- <dig>  in addition a number of new mirnas were identified and validated for the first time from tomato.
availability of supporting data
the sequences could be found in ncbi database under accession number gse <dig>

 the spontaneous 7b- mutant in tomato is a photoperiod-dependent male-sterile line, where in long days flowers are male-sterile with stamens that are shrunken and produce non-viable microspores. in short days, flowers are fertile and produce normal stamens and viable pollen. a proteomic study suggested that microsporogenesis in 7b- breaks down prior to the meiosis in microspore mother cells, which was associated with altered levels of several important proteins involved in tapetum degeneration and mmcs development. compared to the wt, 7b- has a higher tolerance to various abiotic stresses, specifically under blue light, is less sensitive to light-induced inhibition of hypocotyl growth, to blue light-induced stomata opening, and has an elevated level of endogenous aba, but less gas, iaa, and cks. fellner and sawhney, demonstrated that there was a defect in blue light perception in 7b- which affected hormonal sensitivity and their endogenous levels. this information adds on to the fact that the 7b- is a complex mutation with its primary effect still unknown. as a stress-tolerant male-sterile mutant, 7b- is a valuable germplasm for hybrid tomato breeding. recent studies have documented important regulatory functions of srnas in plant growth and development. there are two main types of srnas based on their biogenesis: small interfering rnas, and micrornas. sirnas are processed from perfectly double-stranded rna and comprise different classes; those produced from dsrna synthesized by rna dependent rna polymerase, by rdr, or by overlapping antisense mrnas. while ta-sirnas target mrnas similarly to mirnas, heterochromatin sirnas cause dna methylation and/or heterochromatin formation that lead to transcriptional gene silencing. ta-sirnas play essential roles in regulating plant development, metabolism and responses to biotic and abiotic stresses. four families of tas genes comprising eight loci have been identified in arabidopsis thaliana, among which mir targets both tas1a/b/c family and tas locus, mir targets tas3a/b/c family, while mir triggers the production of tas4-derived ta-sirnas. tas3a-derived tasiarfs, 5´d and 5´d target several arfs, including arf and, which were proposed to act as suppressors in auxin signaling pathway. recently, a fifth tas family was identified from tomato, which is triggered by mir. plant mirnas are typically nucleotides long, which are derived from single-stranded rna transcripts that have the ability to fold into imperfect stem-loop secondary structures. these hairpins are processed by the ribonuclease iii-like enzyme dicer into mirna/mirna* duplexes. one of the strands of the mirna/mirna* duplex is incorporated into the rna induced silencing complex, which guides the risc to recognize target mrna based on sequence complementarity. mature mirnas suppress the expression of their target genes by cleavage of the target mrnas or translational repression. many plant mirnas are conserved among species and have been implicated in processes, such as development, signal transduction, abiotic stress tolerance, and resistance to pathogens. high-throughput sequencing was first used to identify tomato srnas, including mirnas from young green fruits and more recently from several developmental stages of the fruits. recently, there is a growing interest towards understanding the role of mirnas in regulation of male-sterility in plants. even though there is no evidence that any mirnas directly cause male-sterility in plants, it was suggested that mirnas and their target genes play important roles in regulation of male-sterility. wei et al., identified several conserved and new mirnas, which were differentially expressed during anther development in a male-sterile cotton mutant. these mirnas targeted hd-zip iii-like, arf ap and acc oxidase genes, which are key genes involved in hormone signaling, cell patterning, and anti-oxidant metabolism. zhang et al., reported that mir mir mir mir mir and mir were differentially expressed in sporogenous cell, mmcs and microspores between a male-sterile cotton and its maintainer line. the predicted targets for these mirnas were involved in cotton growth and development, signal transduction and metabolism pathways. jiang et al., identified mirnas, which were differentially expressed in pollens of a male-sterile line of brassica campestris and its wild type. there are increasing evidences showing that the function of mirnas, including mir mir mir mir mir and mir is crucial during flower development and microsporogenesis. mir directs the cleavage of gamyb-related transcripts, which are involved in regulation of anther development and microsporogensis. overexpression of mir in arabidopsis thaliana resulted in sterile anthers. mir targets arf6/ which regulate ovule and anther development. in arabidopsis thaliana, overexpression of mir led to male-sterility. mutation in a srna locus in rice which produced a 21-nt srna has led to environment-conditioned male-sterility. srnas in tomato have been mainly studied in fruits development and ripening process, and no report has yet documented their involvement in regulation of male-sterility. the main goal of our study was to investigate whether srna biogenesis is affected by 7b- mutation, and if srnas, particularly mirnas, are associated or involved in anther development and regulation of male-sterility in 7b- known and new mirnas, ta-sirnas and their targets were identified and their expressions were studied in 7b- and wt anthers. targets of mirnas with potential roles in anther development and microsporogenesis were validated using 5′-race and localization of mirnas was further analyzed by in situ hybridization.  deep sequencing of srnas anthers from the flower buds at three stages of pre-meiosis, meiosis, and post-meiosis were dissected and pooled as described by sheoran et al.,. two srna libraries were constructed from wt and 7b- pooled anthers and sequenced, which produced about and million raw reads, respectively. after adaptor trimming, reads were mapped to the tomato genome with no mis-matches allowed. size class distribution of total and unique srnas and their complexities in each library is presented in fig.  the majority of srnas in both 7b- and wt libraries were 21–24 nt, and formed a bi-modal size distribution typical for plant srnas. in general, the peak represented by 24-nt size class is greater than the 21-nt class as reported in several plant species with a few exceptions, as in grapevine, where the 21-nt class was more abundant than the 24-nt class in total read numbers, but not at unique read level, and in brassica juncea, where the 21-nt class had the major peak in total and unique reads.table statistics of srna reads fig. size distribution of redundant and non-redundant srna reads and their complexity in wt and 7b- anthers in our study, the 21-nt class had a bigger peak than 24-nt class in total reads in both libraries; however, for the non-redundant distribution, the 24-nt class had the major peak in both libraries. two key distinguishing features of srna libraries are the size distribution and population complexity as defined by the ratio of unique/total reads. the lower complexity of the 21-nt class in comparison to the 24-nt class in our study indicated that a relatively small number of unique reads were highly expressed in the 21-nt class, while the 24-nt class high complexity indicated the presence of more unique reads with less redundancy, which is also a typical feature of the 24-nt hcrnas, where often are present in a more chaotic dicing pattern. identification of known and new mirnas known mirnas from known families of mirnas were identified, which were present in both 7b- and wt libraries. the composition of mirna families was quite similar in the two libraries, and none showed a clear 7b- or wt-specific profile. most of the families had several members, with exception of mir mir mir mir mir mir mir mir mir and mir which were represented only by one member in both libraries. after excluding srna reads homologous to known mirnas and other non-coding rnas from rfam v. database, the remaining 20-22 nt reads were used as input for mirna prediction using the mircat tool. a total of putative new mirnas were identified in the two libraries, where were present only in wt, in 7b- and in both libraries. the mirna* sequences were found for two new mirnas, which were present in both libraries and designated as mir#a and mir#b. these mirnas formed near-perfect hairpin structures. expression analysis of mirnas to identify mirnas related to anther development and regulation of male-sterility in 7b- expressions of mirnas were compared between 7b- and wt anther libraries. the two libraries were normalized using the reads per million approach. the differential expression analysis was conducted using the offset fold change approach and two-fold threshold was considered as a cutoff value. among the known mirnas, mir mir mir were up-regulated and mir mir mir mir mir mir and mir were down-regulated in 7b- anthers. mir and mir had three and two iso-mirs, respectively, while the rest of differentially expressed mirnas were present only by one member. out of the total of putative new mirnas only two potential mirnas, so-called mir#w and mir#m were differentially down-regulated in 7b- none of the new mirnas with sequenced mirna* strands were differentially expressed in 7b-.table list of differentially expressed mirnas between wt and 7b- anthers 7b-1 7b-1 ade is differential expression values which were calculated as log2-fold changes of the expression. negative and positive values mean up- and down regulation of the expression in 7b- respectively. de value of ± was considered as a cutoff value for significant changes of the expression rt-qpcr validation of mirnas expression expressions of known and new mirnas of interest were further analyzed by rt-qpcr in 7b- and wt anthers and stem. mir and mir were up-regulated at similar extent in all stages of 7b- anthers. mir and mir were down-regulated in all stages of 7b- anthers; more strongly at stage mir#m was strongly down-regulated in all stages of 7b- anthers, while mir#a and mir#b were not differentially expressed and their sequenced mirna* strands were not detected by our rt-qpcr analysis. the rt-qpcr results were in general agreement with those from sequencing data. in 7b- stem, mir mir mir and mir#m were all up-regulated, while mir mir#a and mir#b were not differentially expressed. mir#b* was not detected by rt-qpcr. the results showed a very distinct anther- or stem-specific expression pattern of mir mir and mir#m, which suggests that they may regulate different biological processes in these tissues.fig. rt-qpcr validation of differentially expressed known and new mirnas and mirnas with mirna*. expression changes are presented as normalized fold changes between 7b- and wt reference tissue. positive and negative values indicate up- and down-regulation of the expression, respectively. two-fold threshold was considered as a cutoff value for significant changes in the expression. error bars represent standard errors of three biological replicates based on dmnrt target prediction for known and new mirnas to better understand the biological roles of mirnas in regulation of 7b- male-sterility, putative targets of differentially expressed known and new mirnas were identified. target genes for many of known conserved mirnas have been experimentally validated. predicted targets for known mirnas in our study were also in agreement with those from the literature, however a number of new putative target genes were also computationally identified, which has yet to be validated. the mirna targets were categorized into fifteen different biological classes with the three most frequent being metabolic process, cellular process, and single-organism process. among the targets worth mentioning were those, including auxin-responsive factor, abc transporters, g protein, f-box and no apical meristem proteins, b-zip, sbp-box, mad-box and myb transcription factors with potential roles in anther development, cystatin, receptor-like kinase, and bhlh proteins involved in tapetum development/degeneration, kinesin-like protein and gras transcription factor involved in meiosis regulation, and wd- protein with roles in autophagy and apoptosis. among the targets of new mirnas, a gene encoding pectinmethylesterase inhibitor was identified as the putative target of mir#m. pme is a cell wall modifying enzyme, which catalyzes de-esterification of pectin. identification of mirna targets with potential roles in anther development and microsporogenesis regulation in our study, suggested that these mirnas are likely associated or involved in regulation of 7b- male-sterility.table list of the predicted targets of differentially expressed known mirnas aindicates targets which have identified from multiple loci aindicates targets which have identified from multiple loci fig. gene ontology of mirna targets. predicted targets were categorized into different biological classes and numbers in the parenthesis indicate the frequency of members in each category identification of ta-sirnas and prediction of their targets tas loci and their associated ta-sirnas were predicted based on the phased 21 nt srnas characteristic of ta-sirna loci. ta-sirnas with abundance below reads were excluded from the analysis. twenty-five ta-sirnas from wt, from 7b- and common in both libraries were identified and their target genes were predicted. as these ta-sirnas had generally low abundances, those identified only in wt or 7b- could not be considered as wt- or 7b-1-specific and were not further analyzed. instead, we focused on those which have been found in both libraries; however none were differentially expressed. ta-sirnas with phased expressions matching to the tomato tas were also identified, however tas3-derived 5′d and 5′d tasiarfs were not differentially regulated between the two libraries. the results suggested that ta-sirnas were not likely associated or involved in regulation of male-sterility in 7b- as they were not differentially regulated. 5′-race validation of mirna and ta-sirna targets the predicated targets of interest were validated in 7b- anthers and stem using 5′-race. sequence analysis showed that the 5′ ends for most of the cleaved mrna fragments corresponded to nucleotide complementary to the 10th nucleotide of the corresponding mirna. mir and mir could both cleave myb transcripts; however sequence analysis of the cleavage products showed that myb cleavage in anthers and stem was directed by mir not mir cleavage products of mir and mir can be readily distinguished as they differ in one base. cleavage products of arf target of mir were identified from anther, but not stem. among the predicted targets of mir cleavage products of cystatin were identified in both anther and stem libraries, but not mad-box. cleavage products of pmei, predicted target of mir#m, were also identified in both anther and stem libraries. cleavage products of arf and targets of d and d tasiarfs, were identified from both anther and stem libraries. these results showed that the above mentioned mirnas, including the newly identified mir#m as well as tasiarfs were functionally active in 7b- anther and stem, where directed the cleavage of their predicted target transcripts. furthermore, it provided experimental evidence to support our target predictions.fig. 5′- race validation of mirna target genes in 7b- anther and stem. gene transcripts are in 5′-3′ and mirnas in 3′-5′directions. the arrows indicate the cleavage sites of target mrna and numbers above them indicate frequency of sequences found at the exact mirnas cleavage sites. watson-crick pairing, g-u wobble pairing, and other mismatches are indicated rt-qpcr analysis of mirna and ta-sirna targets figure  shows rt-qpcr analysis of mirna targets of interest and tasiarfs in 7b- anthers and stem. gamybl was down-regulated in anthers of all stages and more strongly in stem. arf was up-regulated in anthers of all stages and stem. cystatin and pmei were both up-regulated in anthers of all stages, but down-regulated in stem. tasiarfs were not differentially expressed in anthers and stem. arf2/3/ were not differentially expressed in anthers, but up-regulated in stem. analysis showed a consistent negative correlation between mirnas expression and accumulation level of their targets, except for mir390-tas3-tasiarfs, and mir167-arf. in addition to mirna and their targets, expression of two cell wall modifying enzymes, cysteine protease and polygalacturonase, were also analyzed in 7b- anthers and stem. cysteine protease was down-regulated in anthers, more strongly at stage while up-regulated in stem. polygalacturonase was strongly down-regulated in anthers at stage but not differentially regulated at stage nor in stem.fig. rt-qpcr validation of mirna target genes and tasiarfs in 7b- anthers and stem. expression changes are presented as normalized fold changes between 7b- and wt reference tissue. positive and negative values indicate up- and down-regulation of the expression, respectively. two-fold threshold was considered as a cutoff value for significant changes in the expression. error bars represent standard errors of three biological replicates based on dmnrt in situ hybridization figure  shows in situ localization of mir gamybl pmei and cystatin in wt and 7b- anthers in the late meiotic and in binucleate microspores stages as for the cases of pmei and cystatin. mir gamybl and cystatin were all predominantly expressed in tapetum, tetrads and free microspores in wt and more strongly in 7b- as the case of mir and cystatin. panels l and m of the figure show wt and 7b- anthers, respectively at binucleate microspores stage, where tapetum was degenerated in wt, but not degenerated and vacuolated in 7b- cystatin was strongly expressed in vacuolated tapetal cells in 7b-. pmei was strongly expressed in tapetum, tetrads and free microspores in 7b- anthers, but its expression was mainly restricted to tapetum at binucleate microspores stage. as mentioned earlier, in some of the 7b- anthers, microspores were not separated and remained attached after meiosis. pmei was strongly expressed in arrested binucleate microspores in 7b-. no detectable hybridization signal was observed for the murine mir122a probe, which served as a negative control.fig. in situ localization of mir gamybl pmei and cystatin. a and b localization of mir in wt and 7b- anthers, respectively. c and d gamybl in wt and 7b- anthers, respectively. e and f pmei in wt anthers at late meiotic and binucleate microspores stages, respectively. g, h, and i pmei in 7b- anthers at late meiotic stage, free binucleate microspores, and arrested binucleate microspores, respectively. j and k cystatin in wt and 7b- anthers, respectively. l and m cystatin in wt and 7b- anthers at binucleate microspores stage, respectively. n negative control, where a murine mir122a-specific probe was used. arrows indicate the localization sites discussion the main goal of our study was to investigate the srna profiles, particularly mirnas, between the 7b- mutant and wt anthers and to identify differentially expressed mirnas and their targets with potential roles in anther development and regulation of male-sterility in 7b- analysis showed that the overall size distribution and population complexity of srnas were quite similar between the 7b- and wt anther libraries. composition of srnas could indicate the roles and activity level of different categories of srnas in a particular tissue or species or associated biogenetic machineries. in our study, the 21-nt class had a larger peak than the 24-nt class in total reads in both libraries; however, in non-redundant format, the 24-nt class had the major peak in both libraries. the 24-nt class was also found to be the major peak in anthers of a male-sterile cotton. higher abundance of the 21-nt class in total reads in anthers of 7b- and wt in our study, could suggest a more active roles for this class of srnas and an intensified contribution from mirna biogenesis pathways that produce the more precisely defined srna species at those stages compared to those of other crops having the 24-nt class with higher redundancy. even though there is no direct evidence that any mirnas are causative genes for male-sterility in plants, we hypothesized that differential expression of mirnas could be associated with the regulation of male-sterility in 7b- thirty-two families of known mirnas were identified in both 7b- and wt anther libraries. three and seven mirna families were up and down-regulated, respectively in 7b- anthers. out of the putative new mirnas only two, so-called mir#w and mir#m, were differentially expressed in 7b- anther. expressions of mirnas of interest were further analyzed at different stages of 7b- anthers and stem. in general, changes of the expression were strongest in anthers at stage where the mmcs were about to undergo the meiosis. mir mir and mir#m had distinctively different expression patterns in anthers and stem, which strongly suggested that these mirnas regulate different biological processes in these tissues. the predicted targets of differentially expressed mirnas were categorized into fifteen different biological classes, with metabolic process, cellular process, and single-organism process being the three most frequent classes. among these mirnas, mir mir mir mir and mir#m were those of particular interest as their targets had potential roles in anther development, microsporogenesis and production of ta-sirnas as the case of mir a schematic presentation of mirna-target pairs and their role in regulation of male-sterility in 7b- mutant is illustrated in fig.  5′-race validation of these mirna targets in 7b- anther and stem showed that they were active and directed the cleavage of their targets in these tissues, except for mir in 7b- stem.fig. schematic presentation of mirna-target pairs and their role in regulation of anther development and male-sterility in 7b- mutant. arrows below mirnas and mirna targets show up- and down-regulation of the expression. “●” indicates no change in the expression. pictures of 7b- anthers are adopted from sheoran et al., mir targets several gamybs, among them atmyb atmyb act redundantly in regulation of anther development. overexpression of mir disrupted anther development and led to male-sterility in arabidopsis thaliana. 5′-race analysis in our study showed that mir directed the cleavage of gamybl transcripts out of the predicted myb targets in anther and stem. osgamybl and are expressed in rice anthers and regulated by mir however functions of these gene are not characterized. legamybl plays a role in seed development in tomato, but it is not functionally characterized with respect to anther development. analysis showed that the expression of gamybl was negatively correlated with the mir level in 7b- during anther development and in stem. in situ hybridization analysis in 7b- anthers also showed that mir and gamybl were both expressed mainly in tapetum, tetrads and microspores. mir and gamybl were down- and up-regulated, respectively in ga-treated 7b- anthers. these observations indicated that mir159-gamybl cleavage cascade and ga level in flower buds are tightly linked to the regulation of anther development and male-sterility in 7b- most of the elongated mutants have been reported as ga-overproducers; however elongated stem of 7b- had lower levels of gas compared to wt in long days. several mybs have been identified, which positively regulate stem elongation in light via interaction with phytochromes and/or regulation of light-responsive genes. down-regulation of gamybl in 7b- stem is likely associated with the elongated stem phenotype, however functional analysis are needed to understand the actual function of this gene. mir cleaves arf6/ transcripts; however we only identified arf in our target prediction, which was also validated by 5′-race in 7b- anther. tomato overexpressing mir and arf6-arf double-null mutant of arabidopsis thaliana both had flowers with severe defects associated with female and male-sterilities, respectively. therefore, proper regulation of mir167-arf6/ is required for normal female and male organ development. rt-qpcr analysis showed strong down-regulation of mir in 7b- anthers, which was negatively correlated with regulation of arf in anthers. although differential regulation of mir167-arf cleavage cascade in 7b- anthers could be linked and due to 7b- mutation, its actual function with respect to anther development and male-sterility in 7b- remains to be further characterized. in 7b- stem, neither the arf cleavage products were found nor the arf expression was correlated with mir level. tomato overexpressing mir had lower levels of arf6/ transcripts and a shorter hypocotyl, while other researchers reported that light-grown arabidopsis thaliana arf8- mutant had elongated hypocotyl. although up-regulation of mir and arf in 7b- stem could be independently associated and/or affected by 7b- mutation, understanding their functions with respect to anther development and male-sterility in 7b- requires further functional analysis. to the best of our knowledge no reports have yet documented ta-sirnas in regulation of male-sterility in plants. ta-sirnas including tas3-derived tasiarfs were not differentially expressed between 7b- and wt; however mir was up-regulated in 7b- anther and stem. this suggested that although mir expression was affected by 7b- mutation, ta-sirna biogenesis and tasiarfs-guided regulation of arf2/3/ were not affected by 7b- mutation nor related to anther development in 7b- nevertheless, 5′-race analysis showed that tasiarfs are active in 7b- anther and stem, where directed the cleavage of arf2/3/ transcripts. target genes of arf2/3/ are largely unknown, thus the mechanisms linking these arfs to a context-specific role in regulation of 7b- stem growth if any are poorly understood. our cytological studies showed that anther development in 7b- was not synchronized in contrast to the wt. we observed that mmcs did undergo meiosis and formed tetrads despite the previous report suggested a breakdown of meiosis in mmcs. more importantly, we found that in some anther lobes, the newly formed microspores were not separated, while in others they formed free microspores. in qrt and qrt mutants of arabidopsis thaliana, microspores failed to separate from tetrads as pectin was not degraded in the primary cell wall. pmei was up-regulated in 7b- anthers, where the expression was mainly localized in tapetum, tetrads and microspores. pmei was strongly expressed in the arrested binucleate microspores, compared to the free binucleate microspores, where a basal expression was detected. pmes and pmeis are key regulators of pollen cell wall and pollen tube development. up-regulation of pmei in 7b- anthers may have suppressed the pme activity and impaired the subsequent enzymatic degradation of pectin in the primary cell walls around tetrads. in qrt mutation in a gene encoding a polygalacturonase impaired the degradation of pectin in the primary cell wall, similar to qrt and qrt mutants. polygalacturonase was also down-regulated in 7b- anthers in our study. based on our cytological and transcriptional data, we suggest a potential association between the regulation of mir#m-pmei and polygalacturonase and anther development in 7b- as inhibition of pme and polygalacturonase activities could have disrupted pectin degradation and proper separation of tetrads and/or tapetum degeneration. however, it could not be the cause of male-sterility in 7b- as some of the anthers were still able to produce mature pollens. arabidopsis thaliana overexpressing pme showed reduced cell elongation in hypocotyl. al-qsous et al., suggested that higher level of pme transcript in elongated hypocotyl of flax was associated with cell wall stiffening. overexpression of a polygalacturonase gene, pgx enhanced hypocotyl elongation in etiolated arabidopsis thaliana. understating the function of pme and polygalacturonase in regulation of stem elongation in 7b- if they have indeed altered the pectin level and how they are connected to the cell expansion requires further analysis. while conserved mirnas regulate expression of the genes involved in basic developmental processes, the non-conserved or new mirnas may be involved in the development of traits that are specific for certain species. new mirnas are being continually identified from different species, including tomato. in addition to mir#m, we identified a number of new mirnas, two with sequenced mirnas*, which could form a near prefect hairpin structures. sheoran et al., identified a number of differentially expressed proteins between 7b- and wt anthers, where cystatin showed the strongest up-regulation of all in 7b- anthers. the cystatin inhibitory activity was also higher in 7b- anthers relative to wt. in plants, cysteine proteases act as key regulators of programmed cell death in tapetal cells and pollen development, and suppression of their expression has often resulted in delay or failure in tapetum degeneration, pollen abortion and male-sterility. mir directed the cleavage of cystatin in 7b- anthers and stem. cystatin was up-regulated and cysteine protease was strongly down-regulated as a result in 7b- anthers, with a pattern closely correlated to the tapetum degeneration during anther development. these observations provided a strong evidence to suggest that suppression of cysteine protease could have caused a delay or failure in tapetum degeneration, thereby affecting microsporogenesis and anther development in 7b- the length of stem in 7b- is tightly correlated with the length of epidermal cells. minic et al., identified several cysteine proteases in developing stems of arabidopsis thaliana, suggested to be involved in cell expansion and secondary wall formation. higher expression of cysteine protease in our study could be associated with the higher cell expansion rate and longer epidermis cells in 7b- stem. overall in our study, we found that mirna-mediated regulation of gene expression was perturbed in the 7b- mutant line. the findings strongly supported that mir159-gamybl mir396-cystatin and mir#m-pmei cleavage cascades were tightly connected to the regulation of microsporogenesis and anther development in 7b- stem elongation in 7b- may be regulated via a complex web of molecular components and interaction between mirnas and hormones, which requires further functional studies to be understood. a number of new mirnas were also identified for the first time from tomato, which provides new opportunities to study the unexplored functions of these mirnas in tomato. our data could be used as a benchmark for future studies of the molecular mechanisms of male-sterility in other crops.  using srna sequencing, we studied mirna profiles during anther development between 7b- mutant and wt. comparison of expression of mirnas and their targets between 7b- and wt and in situ localization analysis suggested potential involvement of several mirna-target pairs in regulation of anther development and male-sterility in 7b- in addition a number of new mirnas were identified and validated for the first time from tomato. availability of supporting data the sequences could be found in ncbi database under accession number gse 
two srna libraries from 7b- and wild type anthers were sequenced and thirty two families of known mirnas and new mirnas were identified in both libraries. mir mir mir were up-regulated and mir mir mir mir mir mir mir and two new mirnas, mir#w and mir#m were down-regulated in 7b- anthers. overall, we identified mirnas with potential roles in anther development and regulation of male-sterility in 7b- a number of new mirnas were also identified from tomato for the first time. qpcr analysis showed differential expression of mirna/target pairs of interest in anthers and stem of 7b- suggesting that they may regulate different biological processes in these tissues. small rnas in tomato have been mainly characterized in fruit development and ripening, but none have been studied with respect to flower development and regulation of male-sterility. our data could be used as a benchmark for future studies of the molecular mechanisms of male-sterility in other crops. using srna sequencing, we identified mirnas that are potentially involved in anther development and regulation of male-sterility in 7b- mutant. mirna targets with potential roles in anther development were validated using 5′-race. expression level of most mirna/target pairs showed negative correlation, except for few. in situ hybridization showed predominant expression of mir gamybl pmei and cystatin in tapetum, tetrads and microspores.
 the 7b- tomato line is a photoperiod-sensitive male-sterile mutant, with potential application in hybrid seed production. small rnas in tomato have been mainly characterized in fruit development and ripening, but none have been studied with respect to flower development and regulation of male-sterility. using srna sequencing, we identified mirnas that are potentially involved in anther development and regulation of male-sterility in 7b- mutant.  two srna libraries from 7b- and wild type anthers were sequenced and thirty two families of known mirnas and new mirnas were identified in both libraries. mir mir mir were up-regulated and mir mir mir mir mir mir mir and two new mirnas, mir#w and mir#m were down-regulated in 7b- anthers. ta-sirnas were not differentially expressed and likely not associated with 7b- male-sterility. mirna targets with potential roles in anther development were validated using 5′-race. qpcr analysis showed differential expression of mirna/target pairs of interest in anthers and stem of 7b- suggesting that they may regulate different biological processes in these tissues. expression level of most mirna/target pairs showed negative correlation, except for few. in situ hybridization showed predominant expression of mir gamybl pmei and cystatin in tapetum, tetrads and microspores.  overall, we identified mirnas with potential roles in anther development and regulation of male-sterility in 7b- a number of new mirnas were also identified from tomato for the first time. our data could be used as a benchmark for future studies of the molecular mechanisms of male-sterility in other crops. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords 7b- mutationsolanum lycopersicummale-sterilityabiotic stressanother developmentmeiosisissue-copyright-statement© the author 2015 