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To comprehensively annotate miRNAs and their targets in tea plant, Camellia sinensis, we sequenced small and messenger RNAs of 9 samples of Camellia sinensis var. assamica (YK-10), a diploid elite cultivar widely grown in southwest China. In order to identify targets of miRNAs, we sequenced two degradome sequencing profiles from leaves and roots of YK-10, respectively. By analyzing the small RNA-Seq profiles, we newly identified 137 conserved miRNAs and 23 species specific miRNAs in the genome of YK-10, which significantly improved the annotation of miRNAs in tea plant. Approximately 2000 differently expressed genes were identified when comparing RNA-Seq profiles of any two of the three organs selected in the study. Totally, more than 5000 targets of conserved miRNAs were identified in the two degradome profiles. Furthermore, our results suggest that a few miRNAs play roles in the biosynthesis pathways of theanine, caffeine and flavonoid. These results enhance our understanding of small RNA guided gene regulations in different organs of tea plant.

MicroRNAs are known to regulate almost all developmental processes in plants. These are ubiquitously expressed and most importantly their abundances vary by several orders of magnitude in different tissues and developmental stages. MicroRNA profiles in seedlings and leaves of switchgrass have been reported but not from the inflorescence or tillers. The overall small RNA population in inflorescence differs from the other vegetative organs, and, moreover, miRNAs are important regulators of flowering and flower development, thus inflorescence was chosen. Likewise, emerging tillers were chosen to identify miRNAs that might play a role in tillering, which is an important trait contributing to higher biomass production. The sequencing followed by computational analyses of small RNA libraries generated from inflorescence and emerging tillers revealed the identification of 28 conserved and 4 novel miRNA families. The expression levels of most miRNA families and miRNA variants within a family displayed greater differences between the tissues. Importantly, this study has offered insights into differences in abundances of miRNA families in the inflorescence and tillers. Specifically, the reported miRNA profiles from tillers are a valuable resource to examine which of these miRNAs play important roles in tillering, an important agronomic trait in this bioenergy crop.

Background MicroRNA-mediated gene regulatory networks play a significant role in plant growth and development and environmental stress responses. Results We identified 79 microRNAs (miRNAs) and multiple miRNA variants (isomiRs) belonging to 26 miRNA families in the primary root growth zone of maize seedlings grown at one of three water potentials: well-watered (− 0.02 MPa), mild water deficit stress (− 0.3 MPa), and severe water deficit stress (− 1.6 MPa). The abundances of 3 miRNAs (mild stress) and 34 miRNAs representing 17 families (severe stress) were significantly different in water-deficit stressed relative to well-watered controls (FDR Conclusions We have identified a number of maize miRNAs that respond to specific water deficits applied to the primary root growth zone. We have also identified transcripts that are targets for miRNA regulation in the root growth zone under water-deficit stress. The miR399e,i,j-3p that is known to regulate phosphate uptake in response to nutrient deficiencies responds to water-deficit stress, however, at the seedling stage the seed provides adequate nutrients for root growth thus miR399e,i,j-3p may play a separate role in water-deficit responses. A water-deficit regulated maize transcript, similar to known miR399 target mimics, was identified and we hypothesized that it is another regulatory player, moderating the role of miR399e,i,j-3p, in primary root growth zone water deficit responses.

Agricultural productivity is severely hampered by drought in many parts of the globe. It is well-known that wild plant species can tolerate drought better when compared with their closely related cultivated plant species. Better drought adaptation of wild species over cultivated ones is accounted for their ability to differentially regulate gene expression. miRNAs, known to regulate gene expression at the post-transcriptional level, are admitted to play an important role in plant adaptation to stresses. This study aims at evaluating miRNA dynamics in a drought-tolerant wild Ipomoea campanulata L. and drought-sensitive cultivated Jacquemontia pentantha (Jacq.) of the family Convolvulaceae under ex situ drought. Sequencing profiles revealed that 34 conserved miRNA families were analogous between the two species. Drought altered expression levels of several of these miRNAs in both the species. Drought-tolerant I. campanulata showed upregulation of miR398, miR168, miR858, miR162 and miR408, while miR394 and miR171 were downregulated. Drought-sensitive J. pentantha showed upregulation of miR394, miR156, miR160, miR164, miR167, miR172, miR319, miR395, miR396, miR403 and downregulation of miR157. Basal miRNA levels and their drought mediated regulation were very different between the two species. Differential drought sensitivities of these two plant species can be attributed to these innate variations in miRNA levels and their expression.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate their target mRNA levels by directing cleavage or repressing its translation. Besides its outstanding nutritional and medicinal significances, pineapple serves as a model for studying genome evolution in cereal crops as well as obligate crassulacean acid metabolism (CAM) photosynthesis. Thus, studying miRNAs in pineapple is critical for better understanding their roles in this plant species. Here we carried out computational and experimental analysis of miRNAs and phased small interfering RNAs (phasiRNAs) in pineapple by analyzing small RNA profiles from flowers, fruits and leaves. The analyses have identified 131 conserved miRNAs that could be grouped into 37 families and 16 novel miRNAs. Three TAS3 loci and forty five 21 nucleotide (nt) PHAS loci, and seventy three 24 nt PHAS loci were also identified. The putative targets of the identified miRNAs and phasiRNAs were predicted. The presented results provide a comprehensive view of small regulatory RNAs and their targets in pineapple.

MicroRNAs are small noncoding RNAs with about 21 nucleotides (nt) that have important functions in both the development and stress responses of plants. Pineapple is an important plant for its agricultural and economical reasons and for its special crassulacean acid metabolism (CAM) photosynthesis system. To better understand the miRNAs and their functions in pineapple, we sequenced three small RNA profiles from flowers, fruits, and leaves, respectively. After analyzing these profiles with bioinformatic methods, 131 conserved miRNAs that could be grouped into 37 families and 16 novel miRNAs were identified. Trans-acting small interfering RNAs (tasiRNAs) are a class of small RNAs that have a different biogenesis pathway but similar functional means by repression of their target genes at different genomic loci. We identified three highly conserved TAS3 loci in pineapple. Recent evidences show that miRNAs also trigger the generations of secondary phased siRNAs (phasiRNAs), from either noncoding genes or coding genes, called as PHAS loci. Our analysis identified 45 PHAS loci encoding 21 nt phasiRNAs and 73 PHAS loci encoding 24 nt phasiRNAs in pineapple. We also predicted the putative targets of the identified miRNAs, tasiRNAs, and phasiRNAs. These results significantly enhance our knowledge of small regulatory RNAs and their potential functions in pineapple.

Plant genomes encode several classes of small regulatory RNAs (sRNAs) that play critical roles in both development and stress responses. Panax notoginseng (Burk.) F.H. Chen (P. notoginseng) is an important traditional Chinese herbal medicinal plant species for its haemostatic effects. Therefore, the root yield of P. notoginseng is a major economically important trait since the roots of P. notoginseng are the parts used to produce medicine. To identify sRNAs that are critical for the root biomass of P. notoginseng, we performed a comprehensive study of miRNA transcriptomes from P. notoginseng roots of different biomasses. We identified 675 conserved miRNAs, of which 180 pre-miRNAs are also identified, and three TAS3 loci in P. notoginseng. By using degradome sequencing, we identified 79 conserved miRNA:target or tasiRNA:target interactions, of which eight were further confirmed with the RLM 5′-RACE experiments. More importantly, our results revealed that a member of miR156 family and one of its SPL target genes have inverse expression levels, which is tightly correlated with greater root biomass contents. These results not only contributes to overall understanding of post-transcriptional gene regulation in roots of P. notoginseng but also could serve as markers for breeding P. notoginseng with greater root yield.

The tobacco hornworm Manduca sexta has served as a model for insect biochemical and physiological research for decades. However, knowledge of the posttranscriptional regulation of gene expression by microRNAs is still rudimentary in this species. Our previous study (Zhang et al., 2012) identified 163 conserved and 13 novel microRNAs in M. sexta, most of which were present at low levels in pupae. To identify additional M. sexta microRNAs and more importantly to examine their possible roles in the expression regulation of immunity-related genes, we constructed four small RNA libraries using fat body and hemocytes from naïve or bacteria-injected larvae and obtained 32.9 million reads of 18-31 nucleotides by Illumina sequencing. Mse-miR-929 and mse-miR-1b (antisense microRNA of mse-miR-1) were predicted in the previous study and now found to be conserved microRNAs in the tissue samples. We also found four novel microRNAs, two of which result from a gene cluster. Mse-miR-281-star, mse-miR-965-star, mse-miR-31-star, and mse-miR-9b-star were present at higher levels than their respective mature strands. Abundance changes of microRNAs were observed after the immune challenge. Based on the quantitative data of mRNA levels in control and induced fat body and hemocytes as well as the results of microRNA target site prediction, we suggest that certain microRNAs and microRNA*s regulate gene expression for pattern recognition, prophenoloxidase activation, cellular responses, antimicrobial peptide synthesis, and conserved intracellular signal transduction (Toll, IMD, JAK-STAT, MAPK-JNK-p38, and small interfering RNA pathways). In summary, this study has enriched our knowledge on M. sexta microRNAs and how some of them may participate in the expression regulation of immunity-related genes.

MicroRNAs (miRNAs) are small non-coding regulatory RNAs that degrade or repress protein synthesis of their messenger RNA targets. This mode of posttranscriptional gene regulation is critical for plant growth and development as well as adaptation to stress conditions. Sacred lotus (Nelumbo nucifera) is a land plant but adapted to the aquatic environment. It is a basal eudicot in the order Proteales, with significant taxonomic importance. Thus identification of miRNAs in sacred lotus could provide information about miRNA evolution, particularly the conservation as well as divergence of miRNAs in dicots. To identify conserved and novel miRNAs in sacred lotus, small RNA libraries from leaves and flowers were sequenced as well as computational strategy was employed. These approaches resulted in identification of 81 miRNAs that can be grouped into 41 conserved/known miRNA families and 52 novel miRNAs forming 49 novel miRNA families. Using 3 mismatches between miRNAs and their mRNA targets as cutoff, we have predicted 137 genes as targets for the conserved and known miRNAs. Overall, this analysis provided a glimpse of miRNA-dependent posttranscriptional gene regulations in sacred lotus.

Wheat is the most highly cultivated plant species for its grain production throughout the world. Because small RNA-dependent gene regulation is critical for successful completion of plant life cycle including its productivity, identification of not only miRNAs but also confirming their targets in wheat is important. To identify small RNAs including novel miRNAs as well as miRNA targets in wheat, we constructed small RNA and degradome libraries from wheat seedlings. Small RNA analysis resulted in identification of most conserved miRNAs including novel miRNAs that can be grouped into 32 miRNA families. The sequence analysis also led to the characterization of two abundantly expressed rRNA-derived small RNAs. To identify miRNA targets, degradome library was sequenced and the bioinformatic analysis confirmed 53 genes as targets for miRNAs and Tas3-siRNAs. Degradome analysis also confirmed a conserved fine-tuning mechanism of Tas3-siRNA abundance by siRNA-mediated silencing of TAS3 transcripts in diverse plant species. These findings added additional information to the small RNA knowledge-base in wheat.

Background Soybean (Glycine max) production is significantly hampered by frequent droughts in many regions of the world including the United States. Identifying microRNA (miRNA)-controlled posttranscriptional gene regulation under drought will enhance our understanding of molecular basis of drought tolerance in this important cash crop. Indeed, miRNA profiles in soybean exposed to drought were studied but not from the primary root tips, which is not only a main zone of water uptake but also critical for water stress sensing and signaling. Methods Here we report miRNA profiles specifically from well-watered and water-stressed primary root tips (0 to 8 mm from the root apex) of soybean. Small RNA sequencing confirmed the expression of vastly diverse miRNA (303 individual miRNAs) population, and, importantly several conserved miRNAs were abundantly expressed in primary root tips. Results Notably, 12 highly conserved miRNA families were differentially regulated in response to water-deficit; six were upregulated while six others were downregulated at least by one fold (log2) change. Differentially regulated soybean miRNAs are targeting genes include auxin response factors, Cu/Zn Superoxide dismutases, laccases and plantacyanin and several others. Conclusions These results highlighted the importance of miRNAs in primary root tips both under control and water-deficit conditions; under control conditions, miRNAs could be important for cell division, cell elongation and maintenance of the root apical meristem activity including quiescent centre whereas under water stress differentially regulated miRNAs could decrease auxin signaling and oxidative stress as well as other metabolic processes that save energy and water. Electronic supplementary material The online version of this article (doi:10.1186/s12918-016-0374-0) contains supplementary material, which is available to authorized users.

MicroRNA genes (miRNAs) encoding small non-coding RNAs are abundant in plant genomes and play a key role in regulating several biological mechanisms. Five conserved miRNAs, miR156, miR168-1, miR168-2, miR164, and miR166 were selected for analysis from the 21 known plant miRNA families that were recovered from deep sequencing data of small RNA libraries of pumpkin and squash. A total of six novel miRNAs that were not reported before were found to have precursors with reliable fold-back structures and hence considered novel and were designated as cuc_nov_miRNAs. A set of five conserved, six novel miRNAs, and five uncharacterized small RNAs from the deep sequencing data were profiled for their dynamic regulation using qPCR. The miRNAs were evaluated for differential regulation across the tissues among four diverse cucurbit species, including pumpkin and squash (Cucurbita moschata Duch. Ex Poir. and Cucurbita pepo L.), bitter melon (Momordica charantia L.), and Luffa (Loofah) (Luffa acutangula Roxb.). Expression analysis revealed differential regulation of various miRNAs in leaf, stem, and fruit tissues. Importantly, differences in the expression levels were also found in the leaves and fruits of closely related C. moschata and C. pepo. Comparative miRNA profiling and expression analysis in four cucurbits led to identification of conserved miRNAs in cucurbits. Predicted targets for two of the conserved miRNAs suggested miRNAs are involved in regulating similar biological mechanisms in various species of cucurbits.

Summary Switchgrass (Panicum virgatum L.) has been developed into a dedicated herbaceous bioenergy crop. Biomass yield is a major target trait for genetic improvement of switchgrass. microRNAs have emerged as a prominent class of gene regulatory factors that has the potential to improve complex traits such as biomass yield. A miR156b precursor was overexpressed in switchgrass. The effects of miR156 overexpression on SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) genes were revealed by microarray and quantitative RT-PCR analyses. Morphological alterations, biomass yield, saccharification efficiency and forage digestibility of the transgenic plants were characterized. miR156 controls apical dominance and floral transition in switchgrass by suppressing its target SPL genes. Relatively low levels of miR156 overexpression were sufficient to increase biomass yield while producing plants with normal flowering time. Moderate levels of miR156 led to improved biomass but the plants were nonflowering. These two groups of plants produced 58%‐101% more biomass yield compared with the control. However, high miR156 levels resulted in severely stunted growth. The degree of morphological alterations of the transgenic switchgrass depends on miR156 level. Compared with floral transition, a lower miR156 level is required to disrupt apical dominance. The improvement in biomass yield was mainly because of the increase in tiller number. Targeted overexpression of miR156 also improved solubilized sugar yield and forage digestibility, and offered an effective approach for transgene containment.

Sweet Sorghum is largely grown for grain production but also recently emerged as one of the model feedstock plants for biofuel production. In plants, microRNA (miRNA)-guided gene regulation plays a key role in diverse biological processes, thus, their identification in different plant species is essential to understand post-transcriptional gene regulation. To identify miRNAs in Sorghum, we sequenced a small RNA library. Sequence analysis revealed the identity of 29 conserved miRNA families. Importantly, 13 novel miRNAs are identified, seven of which are conserved in closely related monocots. Temporal expression analysis of conserved and novel miRNAs indicated differential expression of several miRNAs. Approximately 125 genes that play diverse roles have been predicted as targets and a few targets were experimentally validated. These results provided insights into miRNA-controlled processes in Sorghum and also laid the foundation for manipulating miRNAs or their targets for improving biomass production and stress tolerance in Sorghum.

Being sessile organisms, plants often have to face challenges posed by environmental stresses. To minimize the cellular damage caused by stress, plants have evolved highly complex but well-coordinated adaptive responses operating at the transcriptional, post-transcriptional, translational and post-translational levels. A thorough understanding of regulation at all levels will provide better tools to improve plant's performance under stress. Dramatic changes in the levels of several hundreds or even thousands of mRNAs/proteins were evident under stress as revealed by high-throughput microarray and proteome analyses and such changes were thought to be dependent on transcriptional (induction or suppression of genes) or post-translational regulation (protein stability and degradation). However, recently discovered 21-24 nt small RNAs (microRNAs [miRNAs] and small-interfering RNAs [siRNAs]), which regulate gene expression at the post-transcriptional level, are also modulated during stress and possibly contribute to the stress-induced changes in profiles of mRNAs or proteins. This review highlights our understanding of the role of small RNAs in plant stress responses.

MicroRNA (miRNA)-guided target RNA expression is vital for a wide variety of biological processes in eukaryotes. Currently, miRBase (version 13) lists 142 and 353 miRNAs from Arabidopsis and rice (Oryza sativa), respectively. The integration of miRNAs in diverse biological networks relies upon the confirmation of their RNA targets. In contrast with the well-characterized miRNA targets that are cleaved in Arabidopsis, only a few such targets have been confirmed in rice. To identify small RNA targets in rice, we applied the 'degradome sequencing' approach, which globally identifies the remnants of small RNA-directed target cleavage by sequencing the 5' ends of uncapped RNAs. One hundred and sixty targets of 53 miRNA families (24 conserved and 29 rice-specific) and five targets of TAS3-small interfering RNAs (siRNAs) were identified. Surprisingly, an additional conserved target for miR398, which has not been reported so far, has been validated. Besides conserved homologous transcripts, 23 non-conserved genes for nine conserved miRNAs and 56 genes for 29 rice-specific miRNAs were also identified as targets. Besides miRNA targets, the rice degradome contained fragments derived from MIRNA precursors. A closer inspection of these fragments revealed a unique pattern distinct from siRNA-producing loci. This attribute can serve as one of the ancillary criteria for separating miRNAs from siRNAs in plants.

Crop yields are significantly reduced by biotic and abiotic stresses throughout the world. To reduce the damage caused by stress factors, plants have evolved sophisticated adaptive responses involving reprogramming gene expression at the transcriptional, post-transcriptional and post-translational levels. A better understanding of such processes will lead to new strategies to improve plant stress tolerance. Recently discovered endogenous small RNAs (microRNAs and small-interfering RNAs) have emerged as important players in plant stress responses. The observation that some of the small RNAs are up- or down-regulated in response to stress implies that these small RNAs have a role in stress tolerance. Stress-induced small RNAs might down-regulate their target genes, which may encode negative regulators of stress responses. Conversely, small RNAs down-regulated in response to stress cause the accumulation of their target mRNAs, which may contribute positively to the adaptation to stress. Here, we review the current status of small RNAs involved in biotic and abiotic stress regulatory networks.

Abiotic stress is one of the primary causes of crop losses worldwide. Much progress has been made in unraveling the complex stress response mechanisms, particularly in the identification of stress responsive protein-coding genes. In addition to protein coding genes, recently discovered microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) have emerged as important players in plant stress responses. Initial clues suggesting that small RNAs are involved in plant stress responses stem from studies showing stress regulation of miRNAs and endogenous siRNAs, as well as from target predictions for some miRNAs. Subsequent studies have demonstrated an important functional role for these small RNAs in abiotic stress responses. This review focuses on recent advances, with emphasis on integration of small RNAs in stress regulatory networks.

MicroRNAs (miRNAs) are a growing family of small noncoding RNAs that downregulate gene expression in a sequence-specific manner. The identification of the entire set of miRNAs from a model organism is a critical step toward understanding miRNA-guided gene regulation. Rice (Oryza sativa) and Arabidopsis thaliana, two plant model species with fully sequenced genomes, are representatives of monocotyledonous and dicotyledonous flowering plants, respectively. Thus far, experimental identification of miRNAs in plants has been confined to Arabidopsis. Computational analysis based on conservation with known miRNAs from Arabidopsis has predicted 20 families of miRNAs in rice. To identify miRNAs that are difficult to predict in silico or not conserved in Arabidopsis, we generated three cDNA libraries of small RNAs from rice shoot, root, and inflorescence tissues. We identified 35 miRNAs, of which 14 are new, and these define 13 new families. Thirteen of the new miRNAs are not conserved in Arabidopsis. Four of the new miRNAs are conserved in related monocot species but not in Arabidopsis, which suggests that these may have evolved after the divergence of monocots and dicots. The remaining nine new miRNAs appear to be absent in the known sequences of other plant species. Most of the rice miRNAs are expressed ubiquitously in all tissues examined, whereas a few display tissue-specific expression. We predicted 46 genes as targets of the new rice miRNAs: 16 of these predicted targets encode transcription factors, and other target genes appear to play roles in diverse physiological processes. Four target genes have been experimentally verified by detection of miRNA-mediated mRNA cleavage. Our identification of new miRNAs in rice suggests that these miRNAs may have evolved independently in rice or been lost in other species.

MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are small noncoding RNAs that have recently emerged as important regulators of mRNA degradation, translational repression, and chromatin modification. In Arabidopsis thaliana, 43 miRNAs comprising 15 families have been reported thus far. In an attempt to identify novel and abiotic stress regulated miRNAs and siRNAs, we constructed a library of small RNAs from Arabidopsis seedlings exposed to dehydration, salinity, or cold stress or to the plant stress hormone abscisic acid. Sequencing of the library and subsequent analysis revealed 26 new miRNAs from 34 loci, forming 15 new families. Two of the new miRNAs from three loci are members of previously reported miR171 and miR319 families. Some of the miRNAs are preferentially expressed in specific tissues, and several are either upregulated or downregulated by abiotic stresses. Ten of the miRNAs are highly conserved in other plant species. Fifty-one potential targets with diverse function were predicted for the newly identified miRNAs based on sequence complementarity. In addition to miRNAs, we identified 102 other novel endogenous small RNAs in Arabidopsis. These findings suggest that a large number of miRNAs and other small regulatory RNAs are encoded by the Arabidopsis genome and that some of them may play important roles in plant responses to environmental stresses as well as in development and genome maintenance.

Background MiRNAs and phasiRNAs are negative regulators of gene expression. These small RNAs have been extensively studied in plant model species but only 10 mature microRNAs are present in miRBase version 21, the most used miRNA database, and no phasiRNAs have been identified for the model legume Phaseolus vulgaris. Thanks to the recent availability of the first version of the common bean genome, degradome data and small RNA libraries, we are able to present here a catalog of the microRNAs and phasiRNAs for this organism and, particularly, we suggest new protagonists in the symbiotic nodulation events. Results We identified a set of 185 mature miRNAs, including 121 previously unpublished sequences, encoded by 307 precursors and distributed in 98 families. Degradome data allowed us to identify a total of 181 targets for these miRNAs. We reveal two regulatory networks involving conserved miRNAs: those known to play crucial roles in the establishment of nodules, and novel miRNAs present only in common bean, suggesting a specific role for these sequences. In addition, we identified 125 loci that potentially produce phased small RNAs, with 47 of them having all the characteristics of being triggered by a total of 31 miRNAs, including 14 new miRNAs identified in this study. Conclusions We provide here a set of new small RNAs that contribute to the broader knowledge of the sRNAome of Phaseolus vulgaris. Thanks to the identification of the miRNA targets from degradome analysis and the construction of regulatory networks between the mature microRNAs, we present here the probable functional regulation associated with the sRNAome and, particularly, in N2-fixing symbiotic nodules. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1639-5) contains supplementary material, which is available to authorized users.

DICER-like 1 (DCL1) is a major player in microRNA (miRNA) biogenesis and accordingly, its few known loss-of-function mutants are either lethal or display arrested development. Consequently, generation of dcl1 mutants by reverse genetics and functional analysis of DCL1 in late-developing organs are challenging. Here, these challenges were resolved through the unique use of trans-activated RNA interference. Global, as well as organ-specific tomato DCL1 (SlDCL1) silencing was induced by crossing the generated responder line (OP:SlDCL1IR) with the appropriate driver line. Constitutive trans-activation knocked down SlDCL1 levels by ~95%, resulting in severe abnormalities including post-germination growth arrest accompanied by decreased miRNA and 21-nucleotide small RNA levels, but prominently elevated levels of 22-nucleotide small RNAs. The increase in the 22-nucleotide small RNAs was correlated with specific up-regulation of SlDCL2b and SlDCL2d, which are probably involved in their biogenesis. Leaf- and flower-specific OP:SlDCL1IR trans-activation inhibited blade outgrowth, induced premature bud senescence and produced pale petals, respectively, emphasizing the importance of SlDCL1-dependent small RNAs in these processes. Together, these results establish OP:SlDCL1IR as an efficient tool for analysing processes regulated by SlDCL1-mediated gene regulation in tomato.

Endogenous small RNAs can be grouped into several distinct classes of 21-nt-long microRNAs (miRNAs), short interfering RNAs (siRNAs), trans-acting siRNAs (tasiRNAs), and 24-nt long heterochromatic siRNAs. miRNAs are increasingly being recognized as significant effectors of gene regulation in a wide range of organisms. These molecules are typically ∼21-nt long and are amenable for cloning by streamlined protocols. Here we detail the methodology for cloning small RNAs in rice to identify novel miRNAs and other important small RNAs. Briefly, small RNA molecules are size fractionated, attached to adaptors, and subsequently converted into cDNA and PCR amplified. Current high-throughput sequencing technologies allow sequencing of the PCR products directly.

Small RNAs (microRNAs and other classes of endogenous small interfering RNAs) play important roles in a wide variety of biological processes. However, integration of small RNAs in diverse biological networks relies on the confirmation of their RNA targets. In plants, miRNAs negatively regulate mRNA targets by guiding a cleavage in the complementary site that leaves a 3' polyadenylated RNA possessing monophosphate at its 5' end. This chapter describes a detailed step-by-step protocol for cloning such sliced 3' products in order to identify small RNA targets. Using this protocol, we have identified more than 150 small RNA targets in rice; some are conserved and others are non-conserved targets for rice small RNAs.

Background The regulatory role of small RNAs (sRNAs) in various biological processes is an active area of investigation; however, there has been limited information available on the role of sRNAs in plant-insect interactions. This study was designed to identify sRNAs in cotton-melon aphid (Aphis gossypii) during the Vat-mediated resistance interaction with melon (Cucumis melo). Methodology/principal findings The role of miRNAs was investigated in response to aphid herbivory, during both resistant and susceptible interactions. sRNA libraries made from A. gossypii tissues feeding on Vat⁺ and Vat⁻ plants revealed an unexpected abundance of 27 nt long sRNA sequences in the aphids feeding on Vat⁺ plants. Eighty-one conserved microRNAs (miRNAs), twelve aphid-specific miRNAs, and nine novel candidate miRNAs were also identified. Plant miRNAs found in the aphid libraries were most likely ingested during phloem feeding. The presence of novel miRNAs was verified by qPCR experiments in both resistant Vat⁺ and susceptible Vat⁻ interactions. The comparative analyses revealed that novel miRNAs were differentially regulated during the resistant and susceptible interactions. Gene targets predicted for the miRNAs identified in this study by in silico analyses revealed their involvement in morphogenesis and anatomical structure determination, signal transduction pathways, cell differentiation and catabolic processes. Conclusion/significance In this study, conserved and novel miRNAs were reported in A. gossypii. Deep sequencing data showed differences in the abundance of miRNAs and piRNA-like sequences in A. gossypii. Quantitative RT-PCR revealed that A. gossypii miRNAs were differentially regulated during resistant and susceptible interactions. Aphids can also ingest plant miRNAs during phloem feeding that are stable in the insect.

MicroRNA (miRNA) are small noncoding molecules approximately 22 nucleotides in length that mediate translational repression of target messenger RNA (mRNA) transcripts. miRNA play important roles in cellular differentiation and proliferation, regulation of cellular metabolism, and are associated with enhanced tumorigenesis observed with many cancers. These small RNA molecules function by binding with imperfect complementarity to sequences located in the 3′ untranslated region (UTR) of target mRNA. MiRNA are expressed from both intergenic or intronic regions of the genome in an RNA polymerase II-dependent manner and their expression is subject to both feed-forward and negative feedback regulatory loops. Nascent transcripts known as primary miRNA (pri-mRNA) undergo nuclear processing to generate ~ 60–80 nt precursor miRNA (pre-miRNA) containing imperfect stem-loop structures. Pre-miRNA are subsequently exported out of the nucleus whereupon they are further processed by the RNase III enzyme Dicer resulting in the formation of a ~ 20–24 nt miRNA duplex. Thermodynamic stability of each strand determines which strand of the miRNA duplex will be retained within the RNA-induced silencing complex (RISC) to function as the mature miRNA. This mature miRNA then guides the RISC to sequences within the 3′UTR of target mRNAs to repress protein translation. Due to the roles that miRNA have in regulating cellular proliferation and metabolism, they could potentially interfere with recombinant protein expression in cell-based systems.

Due to their immobile nature, plants are constantly challenged by various abiotic stress factors such as drought, salinity, heavy metals, cold, heat, and many others which negatively impact plant growth and development as well as reproduction. Plants perceive such stress signals and respond by reprogramming their gene expression, which enables them to adapt to stress conditions. This altered gene expression includes transcriptional regulation of the stress-responsive genes, which is the major mode of gene regulation, but post-transcriptional gene regulation also plays a profound role in gene regulation during stress. Recently identified microRNAs (miRNAs) are post-transcriptional gene regulatory molecules that silence target gene expression by promoting degradation and/or inhibiting protein production of the target transcript. Several recent studies have firmly established that the miRNA levels are dynamically regulated in plants exposed to stress, which suggests that miRNAs are an integral part of plant stress regulatory networks. Here, we summarize these recent developments.

In plants, microRNAs (miRNAs) regulate their mRNA targets by precisely guiding cleavages between the 10th and 11th nucleotides in the complementary regions. High-throughput sequencing-based methods, such as PARE or degradome profiling coupled with a computational analysis of the sequencing data, have recently been developed for identifying miRNA targets on a genome-wide scale. The existing algorithms limit the number of mismatches between a miRNA and its targets and strictly do not allow a mismatch or G:U Wobble pair at the position 10 or 11. However, evidences from recent studies suggest that cleavable targets with more mismatches exist indicating that a relaxed criterion can find additional miRNA targets. In order to identify targets including the ones with weak complementarities from degradome data, we developed a computational method called SeqTar that allows more mismatches and critically mismatch or G:U pair at the position 10 or 11. Precisely, two statistics were introduced in SeqTar, one to measure the alignment between miRNA and its target and the other to quantify the abundance of reads at the center of the miRNA complementary site. By applying SeqTar to publicly available degradome data sets from Arabidopsis and rice, we identified a substantial number of novel targets for conserved and non-conserved miRNAs in addition to the reported ones. Furthermore, using RLM 5'-RACE assay, we experimentally verified 12 of the novel miRNA targets (6 each in Arabidopsis and rice), of which some have more than 4 mismatches and have mismatches or G:U pairs at the position 10 or 11 in the miRNA complementary sites. Thus, SeqTar is an effective method for identifying miRNA targets in plants using degradome data sets.

The discovery of microRNAs (miRNAs) as gene regulators has led to a paradigm shift in the understanding of post-transcriptional gene regulation in plants and animals. miRNAs have emerged as master regulators of plant growth and development. Evidence suggesting that miRNAs play a role in plant stress responses arises from the discovery that miR398 targets genes with known roles in stress tolerance. In addition, the expression profiles of most miRNAs that are implicated in plant growth and development are significantly altered during stress. These later findings imply that attenuated plant growth and development under stress may be under the control of stress-responsive miRNAs. Here we review recent progress in the understanding of miRNA-mediated plant stress tolerance.

Cold stress adversely affects plant growth and development and thus limits crop productivity. Diverse plant species tolerate cold stress to a varying degree, which depends on reprogramming gene expression to modify their physiology, metabolism, and growth. Cold signal in plants is transmitted to activate CBF-dependent (C-repeat/drought-responsive element binding factor-dependent) and CBF-independent transcriptional pathway, of which CBF-dependent pathway activates CBF regulon. CBF transcription factor genes are induced by the constitutively expressed ICE1 (inducer of CBF expression 1) by binding to the CBF promoter. ICE1-CBF cold response pathway is conserved in diverse plant species. Transgenic analysis in different plant species revealed that cold tolerance can be significantly enhanced by genetic engineering CBF pathway. Posttranscriptional regulation at pre-mRNA processing and export from nucleus plays a role in cold acclimation. Small noncoding RNAs, namely micro-RNAs (miRNAs) and small interfering RNAs (siRNAs), are emerging as key players of posttranscriptional gene silencing. Cold stress-regulated miRNAs have been identified in Arabidopsis and rice. In this chapter, recent advances on cold stress signaling and tolerance are highlighted.

In plants, small RNA (microRNAs and other endogenous small RNAs)-guided target gene expression is vital for a wide variety of biological processes including adaptation to stress conditions. Identification of stress-regulated microRNAs or other classes of endogenous small RNAs advances our understanding of post-transcriptional gene regulation important for plant stress tolerance. This chapter describes a detailed step-by-step protocol for cloning of small RNAs. Following 5' and 3' adapter ligation to the purified small RNAs, cDNA will be synthesized using reverse transcription, which will be further amplified using a polymerase chain reaction. The resulting small DNA fragments can be either cloned and sequenced using traditional sequencing method or subjected to direct high-throughput pyrosequencing or sequencing-by-synthesis technology.

Background MicroRNAs (miRNAs) are small ~22-nt regulatory RNAs that can silence target genes, by blocking their protein production or degrading the mRNAs. Pig is an important animal in the agriculture industry because of its utility in the meat production. Besides, pig has tremendous biomedical importance as a model organism because of its closer proximity to humans than the mouse model. Several hundreds of miRNAs have been identified from mammals, humans, mice and rats, but little is known about the miRNA component in the pig genome. Here, we adopted an experimental approach to identify conserved and unique miRNAs and characterize their expression patterns in diverse tissues of pig. Results By sequencing a small RNA library generated using pooled RNA from the pig heart, liver and thymus; we identified a total of 120 conserved miRNA homologs in pig. Expression analysis of conserved miRNAs in 14 different tissue types revealed heart-specific expression of miR-499 and miR-208 and liver-specific expression of miR-122. Additionally, miR-1 and miR-133 in the heart, miR-181a and miR-142-3p in the thymus, miR-194 in the liver, and miR-143 in the stomach showed the highest levels of expression. miR-22, miR-26b, miR-29c and miR-30c showed ubiquitous expression in diverse tissues. The expression patterns of pig-specific miRNAs also varied among the tissues examined. Conclusion Identification of 120 miRNAs and determination of the spatial expression patterns of a sub-set of these in the pig is a valuable resource for molecular biologists, breeders, and biomedical investigators interested in post-transcriptional gene regulation in pig and in related mammals, including humans.

Background Small RNA-guided gene silencing at the transcriptional and post-transcriptional levels has emerged as an important mode of gene regulation in plants and animals. Thus far, conventional sequencing of small RNA libraries from rice led to the identification of most of the conserved miRNAs. Deep sequencing of small RNA libraries is an effective approach to uncover rare and lineage- and/or species-specific microRNAs (miRNAs) in any organism. Results In order to identify new miRNAs and possibly abiotic-stress regulated small RNAs in rice, three small RNA libraries were constructed from control rice seedlings and seedlings exposed to drought or salt stress, and then subjected to pyrosequencing. A total of 58,781, 43,003 and 80,990 unique genome-matching small RNAs were obtained from the control, drought and salt stress libraries, respectively. Sequence analysis confirmed the expression of most of the conserved miRNAs in rice. Importantly, 23 new miRNAs mostly each derived from a unique locus in rice genome were identified. Six of the new miRNAs are conserved in other monocots. Additionally, we identified 40 candidate miRNAs. Allowing not more than 3 mis-matches between a miRNA and its target mRNA, we predicted 20 targets for 9 of the new miRNAs. Conclusion Deep sequencing proved to be an effective strategy that allowed the discovery of 23 low-abundance new miRNAs and 40 candidate miRNAs in rice.

Background MicroRNAs (miRNAs) are recently discovered small non-coding RNAs that play pivotal roles in gene expression, specifically at the post-transcriptional level in plants and animals. Identification of miRNAs in large number of diverse plant species is important to understand the evolution of miRNAs and miRNA-targeted gene regulations. Now-a-days, publicly available databases play a central role in the in-silico biology. Because, at least ~21 miRNA families are conserved in higher plants, a homology based search using these databases can help identify orthologs or paralogs in plants. Results We searched all publicly available nucleotide databases of genome survey sequences (GSS), high-throughput genomics sequences (HTGS), expressed sequenced tags (ESTs) and nonredundant (NR) nucleotides and identified 682 miRNAs in 155 diverse plant species. We found more than 15 conserved miRNA families in 11 plant species, 10 to14 families in 10 plant species and 5 to 9 families in 29 plant species. Nineteen conserved miRNA families were identified in important model legumes such as Medicago, Lotus and soybean. Five miRNA families – miR319, miR156/157, miR169, miR165/166 and miR394 – were found in 51, 45, 41, 40 and 40 diverse plant species, respectively. miR403 homologs were found in 16 dicots, whereas miR437 and miR444 homologs, as well as the miR396d/e variant of the miR396 family, were found only in monocots, thus providing large-scale authenticity for the dicot- and monocot-specific miRNAs. Furthermore, we provide computational and/or experimental evidence for the conservation of 6 newly found Arabidopsis miRNA homologs (miR158, miR391, miR824, miR825, miR827 and miR840) and 2 small RNAs (small-85 and small-87) in Brassica spp. Conclusion Using all publicly available nucleotide databases, 682 miRNAs were identified in 155 diverse plant species. By combining the expression analysis with the computational approach, we found that 6 miRNAs and 2 small RNAs that have been identified only in Arabidopsis thus far, are also conserved in Brassica spp. These findings will be useful for tracing the evolution of small RNAs by examining their expression in common ancestors of the Arabidopsis-Brassica lineage.

A small RNA library was used to identify 58 miRNAs from 43 miRNA families from wheat (Triticum aestivum L.), and 46 potential targets were predicted., Background MicroRNAs (miRNAs) are a class of small, non-coding regulatory RNAs that regulate gene expression by guiding target mRNA cleavage or translational inhibition. So far, identification of miRNAs has been limited to a few model plant species, such as Arabidopsis, rice and Populus, whose genomes have been sequenced. Wheat is one of the most important cereal crops worldwide. To date, only a few conserved miRNAs have been predicted in wheat and the computational identification of wheat miRNAs requires the genome sequence, which is unknown. Results To identify novel as well as conserved miRNAs in wheat (Triticum aestivum L.), we constructed a small RNA library. High throughput sequencing of the library and subsequent analysis revealed the identification of 58 miRNAs, comprising 43 miRNA families. Of these, 35 miRNAs belong to 20 conserved miRNA families. The remaining 23 miRNAs are novel and form 23 miRNA families in wheat; more importantly, 4 of these new miRNAs (miR506, miR510, miR514 and miR516) appear to be monocot-specific. Northern blot analysis indicated that some of the new miRNAs are preferentially expressed in certain tissues. Based on sequence homology, we predicted 46 potential targets. Thus, we have identified a large number of monocot-specific and wheat-specific miRNAs. These results indicate that both conserved and wheat-specific miRNAs play important roles in wheat growth and development, stress responses and other physiological processes. Conclusion This study led to the discovery of 58 wheat miRNAs comprising 43 miRNA families; 20 of these families are conserved and 23 are novel in wheat. It provides a first large scale cloning and characterization of wheat miRNAs and their predicted targets.