Peach miRNAs and their targets with diverse biological significance
In plants, miRNAs are both highly conserved and rapidly evolving, and these features have been characterized in a variety of lineages . While extensive research has been carried out on model plants, less is known about the characterization and functional analysis of miRNAs from plant species with agricultural and horticultural significance such as peach and apple [31, 41, 42]. An earlier study reported computational and experimental identification of eight miRNAs from seven conserved miRNA families in peach , not extensive compared to those identified in other plant species [18–21]. Here we provided a comprehensive analysis of peach miRNAs produced in different tissues and characterized their expression patterns by both sRNA-seq and RNA blot analyses. Most of the identified peach miRNAs were found to be tissue-specific, as previously observed for other plant species [9, 19–21]. Many known miRNAs targets, mostly transcription factors, control diverse physiological processes and genetic programs associated with leaf polarity, lateral root formation, flowering, metabolism and stress responses [36, 39, 43, 44]. In this study, a majority of the targets for peach miRNAs have counterparts previously identified in other species [18–23]. For example, two gene targets identified for miR160 in peach are homologus to those in Arabidopsis encoding two auxin response factors (ARFs) that act as repressors in auxin signaling, while a target for miR167 in peach is homologous to AtARF6 which is an activator in the same pathway . Peach tasiARFs target genes that encode auxin signal repressors as their Arabidopsis counterpart does. Further, miR393 that targets TIR1/AFB2 Auxin Receptor (TAAR) Clade, another class of key players for auxin signaling  was also found to target three TAAR homologs in peach. Evidently, miRNA- and tasiARF-mediated regulation of the auxin signaling pathway previously characterized in Arabidopsis is also conserved in peach. However, some of the identified known miRNAs were found to have additional or novel gene targets in peach (Table 2). For example, miR396 that is known to target a conserved family of growth-regulating factor genes also targeted a rho guanyl-nucleotide exchange factor that is involved with phytochrome signaling. Likewise, miR408, besides targeting the conserved copper ion binding protein, could also target two genes encoding a selenium-binding protein and a D-type cyclin, which regulate plant defense and growth, respectively [46, 47]. These results suggest that some known miRNAs have either acquired new targets or expanded their regulatory functions in peach.
Our work found 47 peach-specific miRNAs, which were differentially regulated in various tissues and/or developmental stages (Figure 2c) and targeted a variety of genes with a wide range of biological functions. For example, miRC6a/b targeted two genes encoding pentatricopeptide (PPR) repeat-containing proteins involved in RNA editing, splicing and translation within mitochondria and chloroplasts [48, 49]. MiRC3 and miRC5 both targeted the same NBS-LRR disease resistance protein . Other peach-specific miRNAs were found to target genes associated with transcription/translation initiation, protein kinases, and esterase/lipase/thioesterase family proteins. MiRC45 was found to target a transcript encoding VIP (VERNALIZATION INDEPENDENCE), a gene that modulates vernalization in Arabidopsis[51, 52]. We identified targets for only 12 of the 47 peach-specific miRNAs, which raises a question of whether these remaining miRNAs are non-functional, or alternatively regulate gene expression by translational repression, not direct cleavage of target mRNAs [53, 54]. Conceivably, further improvement of degradome analysis sensitivity and detailed examination of possible translational repression functions of these miRNAs in peach or transgenic plants would provide information of whether or not these miRNAs are functional in peach.
Differential processing of miRNA precursors in peach
In animals, many miRNAs with distinct spatio-temporal expression patterns are modulated by transcriptional and/or post-transcription regulation . In plants though, miRNA transcripts are thought to be immediately processed into mature miRNAs through the processing machinery (DCL1, HYL1, or SE) inside the nucleus, hence the transcript precursor processing is coupled with the transcription . However, a recent study in maize showed that transcription was not closely associated with transcript precursor processing for two miRNAs in specific tissues. The accumulation of abundant miR166a transcript precursors but not mature miR166a was detected in the tip of the maize shoot apical meristem . Transcript precursors were observed for miR390 by RT-PCR in the L1 but not the L2 layer of the shoot apical meristem while similar levels of mature miR390 was revealed in the same L1 and L2 layers by in situ hybridization . These results suggest that both miR166a and miR390 precursors could be differentially processed in various cell types or tissues, although other possibilities, including miRNA stability and mobility, could not be ruled out [56, 57]. In this study, we detected differential accumulation of mature miRNAs and their transcript precursors for nine peach miRNAs (Additional file 7: Figure S2). By comparing the miRNA precursor processing efficiency among tissues for two chosen miRNAs (miRC1 and miRC26), we found that the processing efficiency of their transcript precursors varied in root, bark, leaf, flower, as well as during fruit development (Figure 3), demonstrating that differential processing modulates miRC1 and miRC26 expression. We also found that there was no correlation between miRNA transcription and precursor processing activities thereby demonstrating the two processes can likely be uncoupled. Our detection of these larger transcripts provided substantial evidence for differential processing, though still not conclusive, suggesting this might be part of the basis for the miRNA expression in specific tissues or developmental stages in peach. Similar observations were found during apple miRNA identification indicating such a regulatory mechanism may be common in fruit crops.
Although the mechanism underlying differential processing of miRNA precursors in plants remains unknown, it has been characterized in animal cells where miRNA biogenesis is initiated by processing the pri-miRNA transcript into miRNA precursors (pre-miRNA) by the microprocessor complex containing the dsRNA-binding protein DGCR8 and the RNase III enzyme Drosha as well as multiple accessory proteins [55, 58]. The processed pre-miRNAs are exported to the cytoplasm and cleaved into mature 22-nt miRNAs by Dicer proteins. Two groups of protein factors that utilize distinct strategies to modulate differential processing of miRNA precursors have been identified and characterized. Group one factors (SMADs and p53) interact with the microprocessor complex and accessory factors (EWSR1, p68 and p72) to activate or repress the processing of pri-miRNAs [59–62]. Group two factors (Lin28, hnRNPs, KSRP and TRBP) recognize and bind to the terminal loop of specific miRNA precursors to facilitate the processing or degradation of the bound miRNA precursors in specific tissues or cell types [63–65]. Conceivably, various factors expressed in specific tissues or developmental stages could directly or indirectly interact in the same manner to modulate the processing of specific miRNA precursors in peach. Our findings here implied a prevailing differential stabilization of miRNA precursors, most likely to be dependent on the tissue-specific action of DCLs, HYLs, HENs and other RNA-binding proteins.
The conserved trans-acting siRNA pathway in peach
To date, four TAS gene families have been characterized in Arabidopsis, of which the miR390-TAS3 and miR828-TAS4 pathways are conserved in plants [3, 4]. Here we identified both TAS3 and TAS4 peach orthologs, together with their corresponding trigger miRNAs (Figure 4a,b). We also found similar siRNA biogenesis patterns in the cleaved TAS3 and TAS4 transcripts. The tasiRNA species are conserved as evidenced by the identification of homologous tasiARFs and TAS4-siRNA(−81) and their targets either confirmed by degradome analysis (Figure 4c) and/or in silico prediction. Together, these data indicate that both miR390-TAS3 and miR828-TAS4 biogenesis pathways and functions are at least partially conserved in peach. Since auxin signaling and modulation is essential for diverse biological processes in peach, especially for fruit development and ripening [66, 67], miR390-TAS3 biogenesis-derived tasiARFs in specific tissues could orchestrate auxin signaling that could be directly relevant to fruit growth and development. In Arabidopsis
TAS4-siRNA(−81) has been shown to target genes (AtMYB75
AtMYB90 and AtMYB113) that positively regulate anthocyanin production [9, 38]. The induction of AtMYB75 along with anthocyanin accumulation activates miR828, TAS4 and TAS4-siRNA(−81) . This feedback regulatory loop is proposed to maintain proper anthocyanin levels in plant tissues under nutrient stress condition . This role of TAS4-siRNA(−81) could also be important in peach, where anthocyanin production is directly related to fruit color, which can be highly variable and is considered as an important fruit quality trait [29, 68]. While direct gene targets for TAS4-siRNA(−81) have not been confirmed by the degradome analysis, in silico prediction showed that TAS4-siRNA(−81) can target at least two peach MYBs highly homologous to AtMYB113. Thus, miR828, TAS4
TAS4-siRNA(−81) and the targeted MYBs could form a similar feedback regulatory circle that control anthocyanin accumulation and possibly fruit coloration during peach fruit ripening, which is further supported by the observation of detectable miR828 expression in the mature but not the young fruit (Figure 2b).
MiRNA-mediated MYB regulatory networks in peach and their biological relevance
While MYBs are known to be targeted by miRNAs in other species [9, 21, 38], the target number is limited . In peach, at least 49 MYBs can be potentially targeted by miR159, miR828 and miR858 (Figure 5a). This expansion may have to do with the specialized developmental programs that potentially are regulated through specific MYBs, including but not limited to the lignin synthesis and deposition that orchestrates the stone hardening process and the flavonoid synthesis that is important to fruit color, nutritive properties and disease resistance [29, 69–71]. The lignin and flavonoid biosynthesis pathways are biochemically competitive, drawing from the same phenylpropanoid precursors. Thus, they need to be tightly coordinated during peach fruit development to enable efficient phenylpropanoid metabolism in a tissue specific fashion . While we were only able to detect miR828 expression during fruit development (Figure 2b), we cannot rule out the potential roles of miR858 and miR159 since they could be highly cell- or tissue-, or stage-specific during fruit development, and their expression window period might be missed in this study. Still, the potential regulatory roles of miR858, miR159 and miR828 in lignin, cell wall and flavonoid metabolism and synthesis pathways provides evidence for a significant role of sRNA in coordinating fruit development.
The finding that miR858 shares five MYB targets with miR828 and two with miR159, respectively, and three miR828-targeted MYBs undergo siRNA biogenesis supports the notion of the evolution of a miRNA- and siRNA-mediated silencing reinforcement regulatory mechanism in peach. The co-targeting of the same MYBs by two miRNAs is expected to strengthen their silencing function while miRNA cleavage followed by siRNA biogenesis reinforces the same silencing effect. Although these are two unrelated biological events, they achieve the same goal, executing a strong responsive regulatory function; hence the miRNA-mediated co-targeting could also enable the targeted MYBs to be under refined spatio-temporal regulation. Thus the observed distinct expression patterns of miR828 and miR858 among various tissues and fruit developmental stages would modulate the co-targeted MYB expression in an exquisite spatio-temporal manner, to precisely regulate the co-ordination of lignification in stone and mesocarp- and ectocarp-specific fruit coloring during peach fruit development and ripening.