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1.  High-Throughput Sequencing of RNA Silencing-Associated Small RNAs in Olive (Olea europaea L.) 
PLoS ONE  2011;6(11):e27916.
Small RNAs (sRNAs) of 20 to 25 nucleotides (nt) in length maintain genome integrity and control gene expression in a multitude of developmental and physiological processes. Despite RNA silencing has been primarily studied in model plants, the advent of high-throughput sequencing technologies has enabled profiling of the sRNA component of more than 40 plant species. Here, we used deep sequencing and molecular methods to report the first inventory of sRNAs in olive (Olea europaea L.). sRNA libraries prepared from juvenile and adult shoots revealed that the 24-nt class dominates the sRNA transcriptome and atypically accumulates to levels never seen in other plant species, suggesting an active role of heterochromatin silencing in the maintenance and integrity of its large genome. A total of 18 known miRNA families were identified in the libraries. Also, 5 other sRNAs derived from potential hairpin-like precursors remain as plausible miRNA candidates. RNA blots confirmed miRNA expression and suggested tissue- and/or developmental-specific expression patterns. Target mRNAs of conserved miRNAs were computationally predicted among the olive cDNA collection and experimentally validated through endonucleolytic cleavage assays. Finally, we use expression data to uncover genetic components of the miR156, miR172 and miR390/TAS3-derived trans-acting small interfering RNA (tasiRNA) regulatory nodes, suggesting that these interactive networks controlling developmental transitions are fully operational in olive.
PMCID: PMC3225373  PMID: 22140484
2.  Global small RNA analysis in fast-growing Arabidopsis thaliana with elevated concentrations of ATP and sugars 
BMC Genomics  2014;15:116.
In higher eukaryotes, small RNAs play a role in regulating gene expression. Overexpression (OE) lines of Arabidopsis thaliana purple acid phosphatase 2 (AtPAP2) were shown to grow faster and exhibit higher ATP and sugar contents. Leaf microarray studies showed that many genes involved in microRNAs (miRNAs) and trans-acting siRNAs (tasiRNAs) biogenesis were significantly changed in the fast-growing lines. In this study, the sRNA profiles of the leaf and the root of 20-day-old plants were sequenced and the impacts of high energy status on sRNA expression were analyzed.
9-13 million reads from each library were mapped to genome. miRNAs, tasiRNAs and natural antisense transcripts-generated small interfering RNAs (natsiRNAs) were identified and compared between libraries. In the leaf of OE lines, 15 known miRNAs increased in abundance and 9 miRNAs decreased in abundance, whereas in the root of OE lines, 2 known miRNAs increased in abundance and 9 miRNAs decreased in abundance. miRNAs with increased abundance in the leaf and root samples of both OE lines (miR158b and miR172a/b) were predicted to target mRNAs coding for Dof zinc finger protein and Apetala 2 (AP2) proteins, respectively. Furthermore, a significant change in the miR173-tasiRNAs-PPR/TPR network was observed in the leaves of both OE lines.
In this study, the impact of high energy content on the sRNA profiles of Arabidopsis is reported. While the abundance of many stress-induced miRNAs is unaltered, the abundance of some miRNAs related to plant growth and development (miR172 and miR319) is elevated in the fast-growing lines. An induction of miR173-tasiRNAs-PPR/TPR network was also observed in the OE lines. In contrast, only few cis- and trans-natsiRNAs are altered in the fast-growing lines.
PMCID: PMC3925372  PMID: 24507710
Chloroplasts; Mitochondria; miRNAs; MORF; PPR; tasiRNAs and natsiRNAs
3.  Characterization of the small RNA component of leaves and fruits from four different cucurbit species 
BMC Genomics  2012;13:329.
MicroRNAs (miRNAs) are a class of non-coding small RNAs involved in post-transcriptional regulation of gene expression critical for plant growth and development, stress responses and other diverse biological processes in plants. The Cucurbitaceae or cucurbit family represents some of economically important species, particularly those with edible and medicinal fruits. Genomic tools for the molecular analysis of members of this family are just emerging. Partial draft genome sequence became available recently for cucumber and watermelon facilitating investigation of the small RNA component of the transcriptomes in cucurbits.
We generated four small RNA libraries from bottle gourd (Lagenaria siceraria), Cucurbita moschata, Cucurbita pepo, and, watermelon (Citrullus lanatus var. lanatus) in order to identify conserved and novel lineage specific miRNAs in these cucurbits. Deep sequencing of small RNA libraries from these species resulted in 1,597,263, 532,948, 601,388, and 493,384 unique sRNA reads from bottle gourd, moschata, pepo and watermelon, respectively. Sequence analysis of these four libraries resulted in identification of 21 miRNA families that are highly conserved and 8 miRNA families that are moderately conserved in diverse dicots. We also identified 4 putative novel miRNAs in these plant species. Furthermore, the tasiRNAs were identified and their biogenesis was determined in these cucurbits. Small RNA blot analysis or q-PCR analyses of leaf and fruit tissues of these cucurbits showed differential expression of several conserved miRNAs. Interestingly, the abundance of several miRNAs in leaves and fruits of closely related C. moschata and C. pepo was also distinctly different. Target genes for the most conserved miRNAs are also predicted.
High-throughput sequencing of small RNA libraries from four cucurbit species has provided a glimpse of small RNA component in their transcriptomes. The analysis also showed considerable variation within four cucurbit species with regards to expression of individual miRNAs.
PMCID: PMC3431224  PMID: 22823569
4.  Small RNA transcriptomes of mangroves evolve adaptively in extreme environments 
Scientific Reports  2016;6:27551.
MicroRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) are key players in plant stress responses. Here, we present the sRNA transcriptomes of mangroves Bruguiera gymnorrhiza and Kandelia candel. Comparative computational analyses and target predictions revealed that mangroves exhibit distinct sRNA regulatory networks that differ from those of glycophytes. A total of 32 known and three novel miRNA families were identified. Conserved and mangrove-specific miRNA targets were predicted; the latter were widely involved in stress responses. The known miRNAs showed differential expression between the mangroves and glycophytes, reminiscent of the adaptive stress-responsive changes in Arabidopsis. B. gymnorrhiza possessed highly abundant but less conserved TAS3 trans-acting siRNAs (tasiRNAs) in addition to tasiR-ARFs, with expanded potential targets. Our results indicate that the evolutionary alteration of sRNA expression levels and the rewiring of sRNA-regulatory networks are important mechanisms underlying stress adaptation. We also identified sRNAs that are involved in salt and/or drought tolerance and nutrient homeostasis as possible contributors to mangrove success in stressful environments.
PMCID: PMC4899726  PMID: 27278626
5.  Genome-wide analysis of small RNAs reveals eight fiber elongation-related and 257 novel microRNAs in elongating cotton fiber cells 
BMC Genomics  2013;14:629.
MicroRNAs (miRNAs) and other types of small regulatory RNAs play critical roles in the regulation of gene expression at the post-transcriptional level in plants. Cotton is one of the most economically important crops, but little is known about the roles of miRNAs during cotton fiber elongation.
Here, we combined high-throughput sequencing with computational analysis to identify small RNAs (sRNAs) related to cotton fiber elongation in Gossypium hirsutum L. (G. hirsutum). The sequence analysis confirmed the expression of 79 known miRNA families in elongating fiber cells and identified 257 novel miRNAs, primarily derived from corresponding specific loci in the Gossypium raimondii Ulbr. (G. raimondii) genome. Furthermore, a comparison of the miRNAomes revealed that 46 miRNA families were differentially expressed throughout the elongation period. Importantly, the predicted and experimentally validated targets of eight miRNAs were associated with fiber elongation, with obvious functional relationships with calcium and auxin signal transduction, fatty acid metabolism, anthocyanin synthesis and the xylem tissue differentiation. Moreover, one tasiRNA was also identified, and its target, ARF4, was experimentally validated in vivo.
This study not only facilitated the discovery of 257 novel low-abundance miRNAs in elongating cotton fiber cells but also revealed a potential regulatory network of nine sRNAs important for fiber elongation. The identification and characterization of miRNAs in elongating cotton fiber cells might promote the further study of fiber miRNA regulation mechanisms and provide insight into the importance of miRNAs in cotton.
PMCID: PMC3849097  PMID: 24044642
Cotton; Comparative miRNAome analysis; Fiber cell elongation; High-throughput sequencing; miRNAs; tasiRNA
6.  Endogenous TasiRNAs Mediate Non-Cell Autonomous Effects on Gene Regulation in Arabidopsis thaliana 
PLoS ONE  2009;4(6):e5980.
Different classes of small RNAs (sRNAs) refine the expression of numerous genes in higher eukaryotes by directing protein partners to complementary nucleic acids, where they mediate gene silencing. Plants encode a unique class of sRNAs, called trans-acting small interfering RNAs (tasiRNAs), which post-transcriptionally regulate protein-coding transcripts, as do microRNAs (miRNAs), and both sRNA classes control development through their targets. TasiRNA biogenesis requires multiple components of the siRNA pathway and also miRNAs. But while 21mer siRNAs originating from transgenes can mediate silencing across several cell layers, miRNA action seems spatially restricted to the producing or closely surrounding cells.
Principal Findings
We have previously described the isolation of a genetrap reporter line for TAS3a, the major locus producing AUXIN RESPONS FACTOR (ARF)-regulating tasiRNAs in the Arabidopsis shoot. Its activity is limited to the adaxial (upper) side of leaf primordia, thus spatially isolated from ARF-activities, which are located in the abaxial (lower) side. We show here by in situ hybridization and reporter fusions that the silencing activities of ARF-regulating tasiRNAs are indeed manifested non-cell autonomously to spatially control ARF activities.
Endogenous tasiRNAs are thus mediators of a mobile developmental signal and might provide effective gene silencing at a distance beyond the reach of most miRNAs.
PMCID: PMC2694355  PMID: 19543387
7.  Comparative analysis of non-autonomous effects of tasiRNAs and miRNAs in Arabidopsis thaliana 
Nucleic Acids Research  2010;39(7):2880-2889.
In plants, small interfering RNAs (siRNAs) can trigger a silencing signal that may spread within a tissue to adjacent cells or even systemically to other organs. Movement of the signal is initially limited to a few cells, but in some cases the signal can be amplified and travel over larger distances. How far silencing initiated by other classes of plant small RNAs (sRNAs) than siRNAs can extend has been less clear. Using a system based on the silencing of the CH42 gene, we have tracked the mobility of silencing signals initiated in phloem companion cells by artificial microRNAs (miRNA) and trans-acting siRNA (tasiRNA) that have the same primary sequence. In this system, both the ta-siRNA and the miRNA act at a distance. Non-autonomous effects of the miRNA can be triggered by several different miRNA precursors deployed as backbones. While the tasiRNA also acts non-autonomously, it has a much greater range than the miRNA or hairpin-derived siRNAs directed against CH42, indicating that biogenesis can determine the non-autonomous effects of sRNAs. In agreement with this hypothesis, the silencing signals initiated by different sRNAs differ in their genetic requirements.
PMCID: PMC3074149  PMID: 21134910
8.  Differential expression of microRNAs and other small RNAs in barley between water and drought conditions 
Plant Biotechnology Journal  2014;13(1):2-13.
Drought is a major constraint to crop production, and microRNAs (miRNAs) play an important role in plant drought tolerance. Analysis of miRNAs and other classes of small RNAs (sRNAs) in barley grown under water and drought conditions reveals that drought selectively regulates expression of miRNAs and other classes of sRNAs. Low-expressed miRNAs and all repeat-associated siRNAs (rasiRNAs) tended towards down-regulation, while tRNA-derived sRNAs (tsRNAs) had the tendency to be up-regulated, under drought. Antisense sRNAs (putative siRNAs) did not have such a tendency under drought. In drought-tolerant transgenic barley overexpressing DREB transcription factor, most of the low-expressed miRNAs were also down-regulated. In contrast, tsRNAs, rasiRNAs and other classes of sRNAs were not consistently expressed between the drought-treated and transgenic plants. The differential expression of miRNAs and siRNAs was further confirmed by Northern hybridization and quantitative real-time PCR (qRT-PCR). Targets of the drought-regulated miRNAs and siRNAs were predicted, identified by degradome libraries and confirmed by qRT-PCR. Their functions are diverse, but most are involved in transcriptional regulation. Our data provide insight into the expression profiles of miRNAs and other sRNAs, and their relationship under drought, thereby helping understand how miRNAs and sRNAs respond to drought stress in cereal crops.
PMCID: PMC4309496  PMID: 24975557
barley; drought; microRNA; small RNA; differential expression
9.  Fast-forward generation of effective artificial small RNAs for enhanced antiviral defense in plants 
RNA & disease (Houston, Tex.)  2016;3(1):e1130.
Artificial small RNAs (sRNAs) are short ≈21-nt non-coding RNAs engineered to inactivate sequence complementary RNAs. In plants, they have been extensively used to silence cellular transcripts in gene function analyses and to target invading RNA viruses to induce resistance. Current artificial sRNA-based antiviral resistance in plants is mainly limited to a single virus, and is jeopardized by the emergence of mutations in the artificial sRNA target site or by the presence of co-infecting viruses. Hence, there is a need to further develop the artificial sRNA approach to generate more broad and durable antiviral resistance in plants. A recently developed toolbox allows for the time and cost-effective large-scale production of artificial sRNA constructs in plants. The toolbox includes the P-SAMS web tool for the automated design of artificial sRNAs, and a new generation of artificial microRNA and synthetic trans-acting small interfering RNA (syn-tasiRNA) vectors for direct cloning and high expression of artificial sRNAs. Here we describe how the simplicity and high-throughput capability of these new technologies should accelerate the study of artificial sRNA-based antiviral resistance in plants. In particular, we discuss the potential of the syn-tasiRNA approach as a promising strategy for developing more effective, durable and broad antiviral resistance in plants.
PMCID: PMC4768481  PMID: 26925463
small RNA; silencing; artificial microRNA; synthetic trans-acting small interfering RNA; plant virus; virus resistance
10.  Analysis of the melon (Cucumis melo) small RNAome by high-throughput pyrosequencing 
BMC Genomics  2011;12:393.
Melon (Cucumis melo L.) is a commercially important fruit crop that is cultivated worldwide. The melon research community has recently benefited from the determination of a complete draft genome sequence and the development of associated genomic tools, which have allowed us to focus on small RNAs (sRNAs). These are short, non-coding RNAs 21-24 nucleotides in length with diverse physiological roles. In plants, they regulate gene expression and heterochromatin assembly, and control protection against virus infection. Much remains to be learned about the role of sRNAs in melon.
We constructed 10 sRNA libraries from two stages of developing ovaries, fruits and photosynthetic cotyledons infected with viruses, and carried out high-throughput pyrosequencing. We catalogued and analysed the melon sRNAs, resulting in the identification of 26 known miRNA families (many conserved with other species), the prediction of 84 melon-specific miRNA candidates, the identification of trans-acting siRNAs, and the identification of chloroplast, mitochondrion and transposon-derived sRNAs. In silico analysis revealed more than 400 potential targets for the conserved and novel miRNAs.
We have discovered and analysed a large number of conserved and melon-specific sRNAs, including miRNAs and their potential target genes. This provides insight into the composition and function of the melon small RNAome, and paves the way towards an understanding of sRNA-mediated processes that regulate melon fruit development and melon-virus interactions.
PMCID: PMC3163571  PMID: 21812964
11.  Sex specific expression and distribution of small RNAs in papaya 
BMC Genomics  2014;15(1):20.
Regulatory function of small non-coding RNAs (sRNA) in response to environmental and developmental cues has been established. Additionally, sRNA, also plays an important role in maintaining the heterochromatin and centromere structures of the chromosome. Papaya, a trioecious species with recently evolved sex chromosomes, has emerged as an excellent model system to study sex determination and sex chromosome evolution in plants. However, role of small RNA in papaya sex determination is yet to be explored.
We analyzed the high throughput sRNAs reads in the Illumina libraries prepared from male, female, and hermaphrodite flowers of papaya. Using the sRNA reads, we identified 29 miRNAs that were not previously reported from papaya. Including this and two previous studies, a total of 90 miRNAs has been identified in papaya. We analyzed the expression of these miRNAs in each sex types. A total of 65 miRNAs, including 31 conserved and 34 novel mirNA, were detected in at least one library. Fourteen of the 65 miRNAs were differentially expressed among different sex types. Most of the miRNA expressed higher in male flowers were related to the auxin signaling pathways, whereas the miRNAs expressed higher in female flowers were the potential regulators of the apical meristem identity genes. Aligning the sRNA reads identified the sRNA hotspots adjacent to the gaps of the X and Y chromosomes. The X and Y chromosomes sRNA hotspots has a 7.8 and 4.4 folds higher expression of sRNA, respectively, relative to the chromosome wide average. Approximately 75% of the reads aligned to the X chromosome hotspot was identical to that of the Y chromosome hotspot.
By analyzing the large-scale sRNA sequences from three sex types, we identified the sRNA hotspots flanking the gaps of papaya X, Y, and Yh chromosome. The sRNAs expression patterns in these regions were reminiscent of the pericentromeric region indicating that the only remaining gap in each of these chromosomes is likely the centromere. We also identified 14 differentially expressed miRNAs in male, female and hermaphrodite flowers of papaya. Our results provide valuable information toward understanding the papaya sex determination.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-20) contains supplementary material, which is available to authorized users.
PMCID: PMC3916515  PMID: 24410969
Carica papaya; Centromere; miRNA; siRNA; Sex chromosome; Sex determination
12.  Deciphering Small Noncoding RNAs during the Transition from Dormant Embryo to Germinated Embryo in Larches (Larix leptolepis) 
PLoS ONE  2013;8(12):e81452.
Small RNAs (sRNAs), as a key component of molecular biology, play essential roles in plant development, hormone signaling, and stress response. However, little is known about the relationships among sRNAs, hormone signaling, and dormancy regulation in gymnosperm embryos. To investigate the roles of sRNAs in embryo dormancy maintenance and release in Larix leptolepis, we deciphered the endogenous “sRNAome” in dormant and germinated embryos. High-throughput sequencing of sRNA libraries showed that dormant embryos exhibited a length bias toward 24-nt while germinated embryos showed a bias toward 21-nt lengths. This might be associated with distinct levels of RNA-dependent RNA polymerase2 (RDR2) and/or RDR6, which is regulated by hormones. Proportions of miRNAs to nonredundant and redundant sRNAs were higher in germinated embryos than in dormant embryos, while the ratio of unknown sRNAs was higher in dormant embryos than in germinated embryos. We identified a total of 160 conserved miRNAs from 38 families, 3 novel miRNAs, and 16 plausible miRNA candidates, of which many were upregulated in germinated embryos relative to dormant embryos. These findings indicate that larches and possibly other gymnosperms have complex mechanisms of gene regulation involving miRNAs and other sRNAs operating transcriptionally and posttranscriptionally during embryo dormancy and germination. We propose that abscisic acid modulates embryo dormancy and germination at least in part through regulation of the expression level of sRNA-biogenesis genes, thus changing the sRNA components.
PMCID: PMC3858266  PMID: 24339932
13.  Physcomitrella patens DCL3 Is Required for 22–24 nt siRNA Accumulation, Suppression of Retrotransposon-Derived Transcripts, and Normal Development 
PLoS Genetics  2008;4(12):e1000314.
Endogenous 24 nt short interfering RNAs (siRNAs), derived mostly from intergenic and repetitive genomic regions, constitute a major class of endogenous small RNAs in flowering plants. Accumulation of Arabidopsis thaliana 24 nt siRNAs requires the Dicer family member DCL3, and clear homologs of DCL3 exist in both flowering and non-flowering plants. However, the absence of a conspicuous 24 nt peak in the total RNA populations of several non-flowering plants has raised the question of whether this class of siRNAs might, in contrast to the ancient 21 nt microRNAs (miRNAs) and 21–22 nt trans-acting siRNAs (tasiRNAs), be an angiosperm-specific innovation. Analysis of non-miRNA, non-tasiRNA hotspots of small RNA production within the genome of the moss Physcomitrella patens revealed multiple loci that consistently produced a mixture of 21–24 nt siRNAs with a peak at 23 nt. These Pp23SR loci were significantly enriched in transposon content, depleted in overlap with annotated genes, and typified by dense concentrations of the 5-methyl cytosine (5 mC) DNA modification. Deep sequencing of small RNAs from two independent Ppdcl3 mutants showed that the P. patens DCL3 homolog is required for the accumulation of 22–24 nt siRNAs, but not 21 nt siRNAs, at Pp23SR loci. The 21 nt component of Pp23SR-derived siRNAs was also unaffected by a mutation in the RNA-dependent RNA polymerase mutant Pprdr6. Transcriptome-wide, Ppdcl3 mutants failed to accumulate 22–24 nt small RNAs from repetitive regions while transcripts from two abundant families of long terminal repeat (LTR) retrotransposon-associated reverse transcriptases were up-regulated. Ppdcl3 mutants also displayed an acceleration of leafy gametophore production, suggesting that repetitive siRNAs may play a role in the development of P. patens. We conclude that intergenic/repeat-derived siRNAs are indeed a broadly conserved, distinct class of small regulatory RNAs within land plants.
Author Summary
Very small RNAs (between ∼21 and ∼30 single-stranded bases) are a ubiquitous component of gene regulation in nearly all eukaryotic organisms. The small RNA repertoire of angiosperms (the flowering plants) is exceptionally diverse and includes conspicuous populations of 21 nt microRNAs, as well a diverse set of 24 nt short, interfering RNAs (siRNAs). The 24 nt siRNAs have well-documented roles in enforcing the silence of parasitic regions of the genome, but are not readily apparent in the small RNA populations of several lineages of ancient, non-flowering plants. We found numerous “hotspots” of small RNA production from the genome of the moss P. patens that produced a mix of 21–24 nt siRNAs. Except for their broad mix of sizes, these hotspots were reminiscent of the 24 nt siRNA loci of angiosperms: they tended to associate with decayed transposons, to avoid annotated genes, and to be densely modified with the epigenetic mark 5-methyl cytosine. Deletion of a P. patens Dicer gene abolished production of 22–24 nt siRNAs both from these loci and transcriptome-wide, especially from repetitive regions. We conclude that both microRNAs and intergenic/repeat-associated siRNAs are ancient small RNA regulators in plants, but that the sizes of the siRNAs themselves have drifted over time.
PMCID: PMC2600652  PMID: 19096705
14.  Identification of novel drought-responsive microRNAs and trans-acting siRNAs from Sorghum bicolor (L.) Moench by high-throughput sequencing analysis 
Small non-coding RNAs (sRNAs) namely microRNAs (miRNAs) and trans-acting small interfering RNAs (tasi-RNAs) play a crucial role in post-transcriptional regulation of gene expression and thus the control plant development and stress responses. In order to identify drought-responsive miRNAs and tasi-RNAs in sorghum, we constructed small RNA libraries from a drought tolerant (M35-1) and susceptible (C43) sorghum genotypes grown under control and drought stress conditions, and sequenced by Illumina Genome Analyzer IIx. Ninety seven conserved and 526 novel miRNAs representing 472 unique miRNA families were identified from sorghum. Ninety-six unique miRNAs were found to be regulated by drought stress, of which 32 were up- and 49 were down-regulated (fold change ≥ 2 or ≤ −2) at least in one genotype, while the remaining 15 miRNAs showed contrasting drought-regulated expression pattern between genotypes. A maximum of 17 and 18 miRNAs was differentially regulated under drought stress condition in the sensitive and tolerant genotypes, respectively. These results suggest that genotype dependent stress responsive regulation of miRNAs may contribute, at least in part, to the differential drought tolerance of sorghum genotypes. We also identified two miR390-directed TAS3 gene homologs and the auxin response factors as tasi-RNA targets. We predicted more than 1300 unique target genes for the novel and conserved miRNAs. These target genes were predicted to be involved in different cellular, metabolic, response to stimulus, biological regulation, and developmental processes. Genome-wide identification of stress-responsive miRNAs, tasi-RNAs and their targets identified in this study will be useful in unraveling the molecular mechanisms underlying drought stress responses and genetic improvement of biomass production and stress tolerance in sorghum.
PMCID: PMC4504434  PMID: 26236318
sorghum; microRNAs; tasiRNA; drought; next-generation sequencing; transcriptome
15.  Asymmetric purine-pyrimidine distribution in cellular small RNA population of papaya 
BMC Genomics  2012;13:682.
The small RNAs (sRNA) are a regulatory class of RNA mainly represented by the 21 and 24-nucleotide size classes. The cellular sRNAs are processed by RNase III family enzyme dicer (Dicer like in plant) from a self-complementary hairpin loop or other type of RNA duplexes. The papaya genome has been sequenced, but its microRNAs and other regulatory RNAs are yet to be analyzed.
We analyzed the genomic features of the papaya sRNA population from three sRNA deep sequencing libraries made from leaves, flowers, and leaves infected with Papaya Ringspot Virus (PRSV). We also used the deep sequencing data to annotate the micro RNA (miRNA) in papaya. We identified 60 miRNAs, 24 of which were conserved in other species, and 36 of which were novel miRNAs specific to papaya. In contrast to the Chargaff’s purine-pyrimidine equilibrium, cellular sRNA was significantly biased towards a purine rich population. Of the two purine bases, higher frequency of adenine was present in 23nt or longer sRNAs, while 22nt or shorter sRNAs were over represented by guanine bases. However, this bias was not observed in the annotated miRNAs in plants. The 21nt species were expressed from fewer loci but expressed at higher levels relative to the 24nt species. The highly expressed 21nt species were clustered in a few isolated locations of the genome. The PRSV infected leaves showed higher accumulation of 21 and 22nt sRNA compared to uninfected leaves. We observed higher accumulation of miRNA* of seven annotated miRNAs in virus-infected tissue, indicating the potential function of miRNA* under stressed conditions.
We have identified 60 miRNAs in papaya. Our study revealed the asymmetric purine-pyrimidine distribution in cellular sRNA population. The 21nt species of sRNAs have higher expression levels than 24nt sRNA. The miRNA* of some miRNAs shows higher accumulation in PRSV infected tissues, suggesting that these strands are not totally functionally redundant. The findings open a new avenue for further investigation of the sRNA silencing pathway in plants.
PMCID: PMC3582581  PMID: 23216749
miRNA; siRNA; Papaya Ringspot Virus (PRSV); Small RNA strand selection; Transgene silencing
16.  Divergent patterns of endogenous small RNA populations from seed and vegetative tissues of Glycine max 
BMC Plant Biology  2012;12:177.
Small non-coding RNAs (smRNAs) are known to have major roles in gene regulation in eukaryotes. In plants, knowledge of the biogenesis and mechanisms of action of smRNA classes including microRNAs (miRNAs), short interfering RNAs (siRNAs), and trans-acting siRNAs (tasiRNAs) has been gained mostly through studies with Arabidopsis. In recent years, high throughput sequencing of smRNA populations has enabled extension of knowledge from model systems to plants with larger, more complex genomes. Soybean (Glycine max) now has many genomics resources available including a complete genome sequence and predicted gene models. Relatively little is known, however, about the full complement of its endogenous smRNAs populations and the silenced genes.
Using Illumina sequencing and computational analysis, we characterized eight smRNA populations from multiple tissues and organs of soybean including developing seed and vegetative tissues. A total of 41 million raw sequence reads collapsed into 135,055 unique reads were mapped to the soybean genome and its predicted cDNA gene models. Bioinformatic analyses were used to distinguish miRNAs and siRNAs and to determine their genomic origins and potential target genes. In addition, we identified two soybean TAS3 gene homologs, the miRNAs that putatively guide cleavage of their transcripts, and the derived tasiRNAs that could target soybean genes annotated as auxin response factors. Tissue-differential expression based on the flux of normalized miRNA and siRNA abundances in the eight smRNA libraries was evident, some of which was confirmed by smRNA blotting. Our global view of these smRNA populations also revealed that the size classes of smRNAs varied amongst different tissues, with the developing seed and seed coat having greater numbers of unique smRNAs of the 24-nt class compared to the vegetative tissues of germinating seedlings. The 24-nt class is known to be derived from repetitive elements including transposons. Detailed analysis of the size classes associated with ribosomal RNAs and transposable element families showed greater diversity of smRNAs in the 22- and 24-nt size classes.
The flux of endogenous smRNAs within multiple stages and tissues of seed development was contrasted with vegetative tissues of soybean, one of the dominant sources of protein and oil in world markets. The smRNAs varied in size class, complexity of origins, and possible targets. Sequencing revealed tissue-preferential expression for certain smRNAs and expression differences among closely related miRNA family members.
PMCID: PMC3534067  PMID: 23031057
17.  Genome wide identification of chilling responsive microRNAs in Prunus persica 
BMC Genomics  2012;13:481.
MicroRNAs (miRNAs) are small RNAs (sRNAs) approximately 21 nucleotides in length that negatively control gene expression by cleaving or inhibiting the translation of target gene transcripts. Within this context, miRNAs and siRNAs are coming to the forefront as molecular mediators of gene regulation in plant responses to annual temperature cycling and cold stress. For this reason, we chose to identify and characterize the conserved and non-conserved miRNA component of peach (Prunus persica (L.) Batsch) focusing our efforts on both the recently released whole genome sequence of peach and sRNA transcriptome sequences from two tissues representing non-dormant leaves and dormant leaf buds. Conserved and non-conserved miRNAs, and their targets were identified. These sRNA resources were used to identify cold-responsive miRNAs whose gene targets co-localize with previously described QTLs for chilling requirement (CR).
Analysis of 21 million peach sRNA reads allowed us to identify 157 and 230 conserved and non-conserved miRNA sequences. Among the non-conserved miRNAs, we identified 205 that seem to be specific to peach. Comparative genome analysis between peach and Arabidopsis showed that conserved miRNA families, with the exception of miR5021, are similar in size. Sixteen of these conserved miRNA families are deeply rooted in land plant phylogeny as they are present in mosses and/or lycophytes. Within the other conserved miRNA families, five families (miR1446, miR473, miR479, miR3629, and miR3627) were reported only in tree species (Populustrichocarpa, Citrus trifolia, and Prunus persica). Expression analysis identified several up-regulated or down-regulated miRNAs in winter buds versus young leaves. A search of the peach proteome allowed the prediction of target genes for most of the conserved miRNAs and a large fraction of non-conserved miRNAs. A fraction of predicted targets in peach have not been previously reported in other species. Several conserved and non-conserved miRNAs and miRNA-regulated genes co-localize with Quantitative Trait Loci (QTLs) for chilling requirement (CR-QTL) and bloom date (BD-QTL).
In this work, we identified a large set of conserved and non-conserved miRNAs and describe their evolutionary footprint in angiosperm lineages. Several of these miRNAs were induced in winter buds and co-localized with QTLs for chilling requirement and bloom date thus making their gene targets potential candidates for mediating plant responses to cold stress. Several peach homologs of genes participating in the regulation of vernalization in Arabidopsis were identified as differentially expressed miRNAs targets, potentially linking these gene activities to cold responses in peach dormant buds. The non-conserved miRNAs may regulate cellular, physiological or developmental processes specific to peach and/or other tree species.
PMCID: PMC3463484  PMID: 22978558
microRNAs; Distribution; Expression; Cold stress; Chilling requirement; Bud development
18.  Differential sRNA Regulation in Leaves and Roots of Sugarcane under Water Depletion 
PLoS ONE  2014;9(4):e93822.
Plants have developed multiple regulatory mechanisms to respond and adapt to stress. Drought stress is one of the major constraints to agricultural productivity worldwide and recent reports have highlighted the importance of plant sRNA in the response and adaptation to water availability. In order to increase our understanding of the roles of sRNA in response to water depletion, cultivars of sugarcane were submitted to treatment of ceasing drip irrigation for 24 hours. Deep sequencing analysis was carried out to identify the sRNA regulated in leaves and roots of sugarcane cultivars with different drought sensitivities. The pool of sRNA selected allowed the analysis of different sRNA classes (miRNA and siRNA). Twenty-eight and 36 families of conserved miRNA were identified in leaf and root libraries, respectively. Dynamic regulation of miRNA was observed and the expression profiles of eight miRNA were verified in leaf samples from three biological replicates by stem-loop qRT-PCR assay using the cultivars: SP90–1638 - sensitive cultivar- and; SP83–2847 and SP83–5073 - tolerant cultivars. Altered miRNA regulation was correlated with changes in mRNA levels of specific targets. Two leaf libraries from individual sugarcane cultivars with contrasting drought-tolerance properties were also analyzed. An enrichment of 22-nt sRNA species was observed in leaf libraries. 22-nt miRNA triggered siRNA production by cleavage of their targets in response to water depletion. A number of genes of the sRNA biogenesis pathway were down-regulated in tolerant genotypes and up-regulated in sensitive in response to water depletion treatment. Our analysis contributes to increase the knowledge on the roles of sRNA in sugarcane submitted to water depletion.
PMCID: PMC3973653  PMID: 24695493
19.  Identification and profiling of miRNAs during herbivory reveals jasmonate-dependent and -independent patterns of accumulation in Nicotiana attenuata 
BMC Plant Biology  2012;12:209.
Plant microRNAs (miRNAs) play key roles in the transcriptional responses to environmental stresses. However, the role of miRNAs in responses to insect herbivory has not been thoroughly explored. To identify herbivory-responsive miRNAs, we identified conserved miRNAs in the ecological model plant Nicotiana attenuata whose interactions with herbivores have been well-characterized in both laboratory and field studies.
We identified 59 miRNAs from 36 families, and two endogenous trans-acting small interfering RNAs (tasiRNA) targeted by miRNAs. We characterized the response of the precursor and mature miRNAs to simulated attack from the specialist herbivore Manduca sexta by quantitative PCR analysis and used ir-aoc RNAi transformants, deficient in jasmonate biosynthesis, to identify jasmonate-dependent and -independent miRNA regulation. Expression analysis revealed that groups of miRNAs and tasiRNAs were specifically regulated by either mechanical wounding or wounding plus oral secretions from M. sexta larvae, and these small RNAs were accumulated in jasmonate-dependent or -independent manners. Moreover, cDNA microarray analysis indicated that the expression patterns of the corresponding target genes were correlated with the accumulation of miRNAs and tasiRNAs.
We show that a group of miRNAs and tasiRNAs orchestrates the expression of target genes involved in N. attenuata’s responses to herbivore attack.
PMCID: PMC3502350  PMID: 23134682
Anti-herbivore defense; Jasmonate; Manduca sexta; miRNA; Nicotiana attenuata; tasiRNA
20.  Identification and expression profiles of sRNAs and their biogenesis and action-related genes in male and female cones of Pinus tabuliformis 
BMC Genomics  2015;16(1):693.
Small RNA (sRNA) play pivotal roles in reproductive development, and their biogenesis and action mechanisms are well characterised in angiosperm plants; however, corresponding studies in conifers are very limited. To improve our understanding of the roles of sRNA pathways in the reproductive development of conifers, the genes associated with sRNA biogenesis and action pathways were identified and analysed, and sRNA sequencing and parallel analysis of RNA ends (PARE) were performed in male and female cones of the Chinese pine (Pinus tabuliformis).
Based on high-quality reference transcriptomic sequences, 21 high-confidence homologues involved in sRNA biogenesis and action in P. tabuliformis were identified, including two different DCL3 genes and one AGO4 gene. More than 75 % of genes involved in sRNA biogenesis and action have higher expression levels in female than in male cones. Twenty-six microRNA (miRNA) families and 74 targets, including 46 24-nt sRNAs with a 5’ A, which are specifically expressed in male cones or female cones and probably bind to AGO4, were identified.
The sRNA pathways have higher activity in female than in male cones, and the miRNA pathways are the main sRNA pathways in P. tabuliformis. The low level of 24-nt short-interfering RNAs in conifers is not caused by the absence of biogenesis-related genes or AGO-binding proteins, but most likely caused by the low accumulation of these key components. The identification of sRNAs and their targets, as well as genes associated with sRNA biogenesis and action, will provide a good starting point for investigations into the roles of sRNA pathways in cone development in conifers.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1885-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4570457  PMID: 26369937
Pinus tabuliformis Carr; miRNA; siRNA; Parallel analysis of RNA ends (PARE); Male cones; Female cones
21.  Identification of drought-responsive and novel Populus trichocarpa microRNAs by high-throughput sequencing and their targets using degradome analysis 
BMC Genomics  2013;14:233.
MicroRNAs (miRNAs) are endogenous small RNAs (sRNAs) with a wide range of regulatory functions in plant development and stress responses. Although miRNAs associated with plant drought stress tolerance have been studied, the use of high-throughput sequencing can provide a much deeper understanding of miRNAs. Drought is a common stress that limits the growth of plants. To obtain more insight into the role of miRNAs in drought stress, Illumina sequencing of Populus trichocarpa sRNAs was implemented.
Two sRNA libraries were constructed by sequencing data of control and drought stress treatments of poplar leaves. In total, 207 P. trichocarpa conserved miRNAs were detected from the two sRNA libraries. In addition, 274 potential candidate miRNAs were found; among them, 65 candidates with star sequences were chosen as novel miRNAs. The expression of nine conserved miRNA and three novel miRNAs showed notable changes in response to drought stress. This was also confirmed by quantitative real time polymerase chain reaction experiments. To confirm the targets of miRNAs experimentally, two degradome libraries from the two treatments were constructed. According to degradome sequencing results, 53 and 19 genes were identified as targets of conserved and new miRNAs, respectively. Functional analysis of these miRNA targets indicated that they are involved in important activities such as the regulation of transcription factors, the stress response, and lipid metabolism.
We discovered five upregulated miRNAs and seven downregulated miRNAs in response to drought stress. A total of 72 related target genes were detected by degradome sequencing. These findings reveal important information about the regulation mechanism of miRNAs in P. trichocarpa and promote the understanding of miRNA functions during the drought response.
PMCID: PMC3630063  PMID: 23570526
Populus trichocarpa; microRNA; Drought; Target identification
22.  A Comprehensive Expression Profile of MicroRNAs and Other Classes of Non-Coding Small RNAs in Barley Under Phosphorous-Deficient and -Sufficient Conditions 
Phosphorus (P) is essential for plant growth. MicroRNAs (miRNAs) play a key role in phosphate homeostasis. However, little is known about P effect on miRNA expression in barley (Hordeum vulgare L.). In this study, we used Illumina's next-generation sequencing technology to sequence small RNAs (sRNAs) in barley grown under P-deficient and P-sufficient conditions. We identified 221 conserved miRNAs and 12 novel miRNAs, of which 55 were only present in P-deficient treatment while 32 only existed in P-sufficient treatment. Total 47 miRNAs were significantly differentially expressed between the two P treatments (|log2| > 1). We also identified many other classes of sRNAs, including sense and antisense sRNAs, repeat-associated sRNAs, transfer RNA (tRNA)-derived sRNAs and chloroplast-derived sRNAs, and some of which were also significantly differentially expressed between the two P treatments. Of all the sRNAs identified, antisense sRNAs were the most abundant sRNA class in both P treatments. Surprisingly, about one-fourth of sRNAs were derived from the chloroplast genome, and a chloroplast-encoded tRNA-derived sRNA was the most abundant sRNA of all the sRNAs sequenced. Our data provide valuable clues for understanding the properties of sRNAs and new insights into the potential roles of miRNAs and other classes of sRNAs in the control of phosphate homeostasis.
PMCID: PMC3628442  PMID: 23266877
deep sequencing; barley; phosphorus; non-coding small RNAs; miRNAs; differential expression
23.  Genome-wide identification of microRNA and siRNA responsive to endophytic beneficial diazotrophic bacteria in maize 
BMC Genomics  2014;15(1):766.
Small RNA (sRNA) has been described as a regulator of gene expression. In order to understand the role of maize sRNA (Zea mays – hybrid UENF 506-8) during association with endophytic nitrogen-fixing bacteria, we analyzed the sRNA regulated by its association with two diazotrophic bacteria, Herbaspirillum seropedicae and Azospirillum brasilense.
Deep sequencing analysis was done with RNA extracted from plants inoculated with H. seropedicae, allowing the identification of miRNA and siRNA. A total of 25 conserved miRNA families and 15 novel miRNAs were identified. A dynamic regulation in response to inoculation was also observed. A hypothetical model involving copper-miRNA is proposed, emphasizing the fact that the up-regulation of miR397, miR398, miR408 and miR528, which is followed by inhibition of their targets, can facilitate association with diazotrophic bacteria. Similar expression patterns were observed in samples inoculated with A. brasilense. Moreover, novel miRNA and siRNA were classified in the Transposable Elements (TE) database, and an enrichment of siRNA aligned with TE was observed in the inoculated samples. In addition, an increase in 24-nt siRNA mapping to genes was observed, which was correlated with an increase in methylation of the coding regions and a subsequent reduction in transcription.
Our results show that maize has RNA-based silencing mechanisms that can trigger specific responses when plants interact with beneficial endophytic diazotrophic bacteria. Our findings suggest important roles for sRNA regulation in maize, and probably in other plants, during association with diazotrophic bacteria, emphasizing the up-regulation of Cu-miRNA.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-766) contains supplementary material, which is available to authorized users.
PMCID: PMC4168055  PMID: 25194793
Herbaspirillum seropedicae; miRNA; siRNA; Azospirillum brasilense; Epigenetics
24.  DRB2 Is Required for MicroRNA Biogenesis in Arabidopsis thaliana 
PLoS ONE  2012;7(4):e35933.
The Arabidopsis thaliana (Arabidopsis) DOUBLE-STRANDED RNA BINDING (DRB) protein family consists of five members, DRB1 to DRB5. The biogenesis of two developmentally important small RNA (sRNA) species, the microRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) by DICER-LIKE (DCL) endonucleases requires the assistance of DRB1 and DRB4 respectively. The importance of miRNA-directed target gene expression in plant development is exemplified by the phenotypic consequence of loss of DRB1 activity (drb1 plants).
Principal Findings
Here we report that the developmental phenotype of the drb235 triple mutant plant is the result of deregulated miRNA biogenesis in the shoot apical meristem (SAM) region. The expression of DRB2, DRB3 and DRB5 in wild-type seedlings is restricted to the SAM region. Small RNA sequencing of the corresponding tissue of drb235 plants revealed altered miRNA accumulation. Approximately half of the miRNAs detected remained at levels equivalent to those of wild-type plants. However, the accumulation of the remaining miRNAs was either elevated or reduced in the triple mutant. Examination of different single and multiple drb mutants revealed a clear association between the loss of DRB2 activity and altered accumulation for both the elevated and reduced miRNA classes. Furthermore, we show that the constitutive over-expression of DRB2 outside of its wild-type expression domain can compensate for the loss of DRB1 activity in drb1 plants.
Our results suggest that in the SAM region, DRB2 is both antagonistic and synergistic to the role of DRB1 in miRNA biogenesis, adding an additional layer of gene regulatory complexity in this developmentally important tissue.
PMCID: PMC3335824  PMID: 22545148
25.  The Complexity of Posttranscriptional Small RNA Regulatory Networks Revealed by In Silico Analysis of Gossypium arboreum L. Leaf, Flower and Boll Small Regulatory RNAs 
PLoS ONE  2015;10(6):e0127468.
MicroRNAs (miRNAs) and secondary small interfering RNAs (principally phased siRNAs or trans-acting siRNAs) are two distinct subfamilies of small RNAs (sRNAs) that are emerging as key regulators of posttranscriptional gene expression in plants. Both miRNAs and secondary-siRNAs (sec-siRNAs) are processed from longer RNA precursors by DICER-LIKE proteins (DCLs). Gossypium arboreum L., also known as tree cotton or Asian cotton, is a diploid, possibly ancestral relative of tetraploid Gossypium hirsutum L., the predominant type of commercially grown cotton worldwide known as upland cotton. To understand the biological significance of these gene regulators in G. arboreum, a bioinformatics analysis was performed on G. arboreum small RNAs produced from G. arboreum leaf, flower, and boll tissues. Consequently, 263 miRNAs derived from 353 precursors, including 155 conserved miRNAs (cs-miRNAs) and 108 novel lineage-specific miRNAs (ls-miRNAs). Along with miRNAs, 2,033 miRNA variants (isomiRNAs) were identified as well. Those isomiRNAs with variation at the 3’-miRNA end were expressed at the highest levels, compared to other types of variants. In addition, 755 pha-siRNAs derived 319 pha-siRNA gene transcripts (PGTs) were identified, and the potential pha-siRNA initiators were predicted. Also, 2,251 non-phased siRNAs were found as well, of which 1,088 appeared to be produced by so-called cis- or trans-cleavage of the PGTs observed at positions differing from pha-siRNAs. Of those sRNAs, 148 miRNAs/isomiRNAs and 274 phased/non-phased siRNAs were differentially expressed in one or more pairs of tissues examined. Target analysis revealed that target genes for both miRNAs and pha-siRNAs are involved a broad range of metabolic and enzymatic activities. We demonstrate that secondary siRNA production could result from initial cleavage of precursors by both miRNAs or isomiRNAs, and that subsequently produced phased and unphased siRNAs could result that also serve as triggers of a second round of both cis- and trans-cleavage of additional siRNAs, leading to the formation of complex sRNA regulatory networks mediating posttranscriptional gene silencing. Results from this study extended our knowledge on G. arboreum sRNAs and their biological importance, which would facilitate future studies on regulatory mechanism of tissue development in cotton and other plant species.
PMCID: PMC4466472  PMID: 26070200

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