Quantification of Circulating Cell-Free DNA in Idiopathic Parkinson's Disease Patients.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders globally and leads to an excessive loss of dopaminergic neurons in the substantia nigra of the brain. Circulating cell-free DNA (ccf-DNA) are double-stranded DNA fragments of different sizes and origins that are released into the serum and cerebrospinal fluid (CSF) due to cell death (i.e., necrosis and apoptosis) or are actively released by viable cells via exocytosis and NETosis. Using droplet digital polymerase chain reaction (ddPCR), we comprehensively analyzed and distinguished circulating cell-free mitochondrial DNA (ccf mtDNA) and circulating cell-free nuclear DNA (ccfDNA) in the serum and CSF of PD and control patients. The quantitative analysis of serum ccf-DNA in PD patients demonstrated a significant increase in ccf mtDNA and ccfDNA compared to that in healthy control patients and a significantly higher copy of ccf mtDNA when compared to ccfDNA. Next, the serum ccf mtDNA levels significantly increased in male PD patients compared to those in healthy male controls. Furthermore, CSF ccf mtDNA in PD patients increased significantly compared to ccfDNA, and ccf mtDNA decreased in PD patients more than it did in healthy controls. These decreases were not statistically significant but were in agreement with previous data. Interestingly, ccf mtDNA increased in healthy control patients in both serum and CSF as compared to ccfDNA. The small sample size of serum and CSF were the main limitations of this study. To the best of our knowledge, this is the first comprehensive study on serum and CSF of PD patients using ddPCR to indicate the distribution of the copy number of ccf mtDNA as well as ccfDNA. If validated, we suggest that ccf mtDNA has greater potential than ccfDNA to lead the development of novel treatments for PD patients.

PEP444c encoded within the MIR444c gene regulates microRNA444c accumulation in barley.

MicroRNAs are small, noncoding RNA molecules that regulate the expression of their target genes. The MIR444 gene family is present exclusively in monocotyledons, and microRNAs444 from this family have been shown to target certain MADS-box transcription factors in rice and barley. We identified three barley MIR444 (MIR444a/b/c) genes and comprehensively characterised their structure and the processing pattern of the primary transcripts (pri-miRNAs444). Pri-microRNAs444 undergo extensive alternative splicing, generating functional and nonfunctional pri-miRNA444 isoforms. We show that barley pri-miRNAs444 contain numerous open reading frames (ORFs) whose transcripts associate with ribosomes. Using specific antibodies, we provide evidence that selected ORFs encoding PEP444a within MIR444a and PEP444c within MIR444c are expressed in barley plants. Moreover, we demonstrate that CRISPR-associated endonuclease 9 (Cas9)-mediated mutagenesis of the PEP444c-encoding sequence results in a decreased level of PEP444 transcript in barley shoots and roots and a 5-fold reduced level of mature microRNA444c in roots. Our observations suggest that PEP444c encoded by the MIR444c gene is involved in microRNA444c biogenesis in barley.

Pi-starvation induced transcriptional changes in barley revealed by a comprehensive RNA-Seq and degradome analyses.

BACKGROUND: Small RNAs (sRNAs) are 20-30 nt regulatory elements which are responsible for plant development regulation and participate in many plant stress responses. Insufficient inorganic phosphate (Pi) concentration triggers plant responses to balance the internal Pi level. RESULTS: In this study, we describe Pi-starvation-responsive small RNAs and transcriptome changes in barley (Hordeum vulgare L.) using Next-Generation Sequencing (NGS) RNA-Seq data derived from three different types of NGS libraries: (i) small RNAs, (ii) degraded RNAs, and (iii) functional mRNAs. We find that differentially and significantly expressed miRNAs (DEMs, Bonferroni adjusted p-value < 0.05) are represented by 15 molecules in shoot and 13 in root; mainly various miR399 and miR827 isomiRs. The remaining small RNAs (i.e., those without perfect match to reference sequences deposited in miRBase) are considered as differentially expressed other sRNAs (DESs, p-value Bonferroni correction < 0.05). In roots, a more abundant and diverse set of other sRNAs (DESs, 1796 unique sequences, 0.13% from the average of the unique small RNA expressed under low-Pi) contributes more to the compensation of low-Pi stress than that in shoots (DESs, 199 unique sequences, 0.01%). More than 80% of differentially expressed other sRNAs are up-regulated in both organs. Additionally, in barley shoots, up-regulation of small RNAs is accompanied by strong induction of two nucleases (S1/P1 endonuclease and 3'-5' exonuclease). This suggests that most small RNAs may be generated upon nucleolytic cleavage to increase the internal Pi pool. Transcriptomic profiling of Pi-starved barley shoots identifies 98 differentially expressed genes (DEGs). A majority of the DEGs possess characteristic Pi-responsive cis-regulatory elements (P1BS and/or PHO element), located mostly in the proximal promoter regions. GO analysis shows that the discovered DEGs primarily alter plant defense, plant stress response, nutrient mobilization, or pathways involved in the gathering and recycling of phosphorus from organic pools. CONCLUSIONS: Our results provide comprehensive data to demonstrate complex responses at the RNA level in barley to maintain Pi homeostasis and indicate that barley adapts to Pi-starvation through elicitation of RNA degradation. Novel P-responsive genes were selected as putative candidates to overcome low-Pi stress in barley plants.

Identification of transcription factors that bind to the 5'-UTR of the barley PHO2 gene.

In barley and other higher plants, phosphate homeostasis is maintained by a regulatory network involving the PHO2 (PHOSPHATE2) encoding ubiquitin-conjugating (UBC) E2 enzyme, the PHR1 (PHOSPHATE STARVATION RESPONSE 1) transcription factor (TF), IPS1 (INDUCED BYPHOSPHATESTARVATION1) RNA, and miR399. During phosphate ion (Pi) deprivation, PHR1 positively regulates MIR399 expression, after transcription and processing mature miR399 guides the Ago protein to the 5'-UTR of PHO2 transcripts. Non-coding IPS1 RNA is highly expressed during Pi starvation, and the sequestration of miR399 molecules protects PHO2 mRNA from complete degradation. Here, we reveal new cis- and trans-regulatory elements that are crucial for efficient PHO2 gene expression in barley. We found that the 5'-UTR of PHO2 contains two PHR1 binding sites (P1BSs) and one Pi-responsive PHO element. Using a yeast one-hybrid (Y1H) assay, we identified two candidate proteins that might mediate this transcriptional regulation: a barley PHR1 ortholog and a TF containing an uncharacterized MYB domain. Additional results classified this new potential TF as belonging to the APL (ALTERED PHLOEM DEVELOPMENT) protein family, and we observed its nuclear localization in barley protoplasts. Pi starvation induced the accumulation of barley APL transcripts in both the shoots and roots. Interestingly, the deletion of the P1BS motif from the first intron of the barley 5'-UTR led to a significant increase in the transcription of a downstream ß-glucuronidase (GUS) reporter gene in tobacco leaves. Our work extends the current knowledge about putative cis- and trans-regulatory elements that may affect the expression of the barley PHO2 gene.

mirEX 2.0 - an integrated environment for expression profiling of plant microRNAs.

BACKGROUND: MicroRNAs are the key post-transcriptional regulators of gene expression in development and stress responses. Thus, precisely quantifying the level of each particular microRNA is of utmost importance when studying the biology of any organism. DESCRIPTION: The mirEX 2.0 web portal ( http://www.combio.pl/mirex ) provides a comprehensive platform for the exploration of microRNA expression data based on quantitative Real Time PCR and NGS sequencing experiments, covering various developmental stages, from wild-type to mutant plants. The portal includes mature and pri-miRNA expression levels detected in three plant species (Arabidopsis thaliana, Hordeum vulgare and Pellia endiviifolia), and in A. thaliana miRNA biogenesis pathway mutants. In total, the database contains information about the expression of 461 miRNAs representing 268 families. The data can be explored through the use of advanced web tools, including (i) a graphical query builder system allowing a combination of any given species, developmental stages and tissues, (ii) a modular presentation of the results in the form of thematic windows, and (iii) a number of user-friendly utilities such as a community-building discussion system and extensive tutorial documentation (e.g., tooltips, exemplary videos and presentations). All data contained within the mirEX 2.0 database can be downloaded for use in further applications in a context-based way from the result windows or from a dedicated web page. CONCLUSIONS: The mirEX 2.0 portal provides the plant research community with easily accessible data and powerful tools for application in multi-conditioned analyses of miRNA expression from important plant species in different biological and developmental backgrounds.

The liverwort Pellia endiviifolia shares microtranscriptomic traits that are common to green algae and land plants.

Liverworts are the most basal group of extant land plants. Nonetheless, the molecular biology of liverworts is poorly understood. Gene expression has been studied in only one species, Marchantia polymorpha. In particular, no microRNA (miRNA) sequences from liverworts have been reported. Here, Illumina-based next-generation sequencing was employed to identify small RNAs, and analyze the transcriptome and the degradome of Pellia endiviifolia. Three hundred and eleven conserved miRNA plant families were identified, and 42 new liverwort-specific miRNAs were discovered. The RNA degradome analysis revealed that target mRNAs of only three miRNAs (miR160, miR166, and miR408) have been conserved between liverworts and other land plants. New targets were identified for the remaining conserved miRNAs. Moreover, the analysis of the degradome permitted the identification of targets for 13 novel liverwort-specific miRNAs. Interestingly, three of the liverwort microRNAs show high similarity to previously reported miRNAs from Chlamydomonas reinhardtii. This is the first observation of miRNAs that exist both in a representative alga and in the liverwort P. endiviifolia but are not present in land plants. The results of the analysis of the P. endivifolia microtranscriptome support the conclusions of previous studies that placed liverworts at the root of the land plant evolutionary tree of life.

Transcriptionally and post-transcriptionally regulated microRNAs in heat stress response in barley.

Heat stress is one of the major abiotic factors that can induce severe plant damage, leading to a decrease in crop plant productivity. Despite barley being a cereal of great economic importance, few data are available concerning its thermotolerance mechanisms. In this work microRNAs (miRNAs) involved in heat stress response in barley were investigated. The level of selected barley mature miRNAs was examined by hybridization. Quantitative real-time PCR (RT-qPCR) was used to monitor the changes in the expression profiles of primary miRNA (pri-miRNA) precursors, as well as novel and conserved target genes during heat stress. The miRNA-mediated cleavage sites in the target transcripts were confirmed by degradome analysis and the 5' RACE (rapid amplification of cDNA ends) approach. Four barley miRNAs (miR160a, 166a, 167h, and 5175a) were found which are heat stress up-regulated at the level of both mature miRNAs and precursor pri-miRNAs. Moreover, the splicing of introns hosting miR160a and miR5175a is also heat induced. The results demonstrate transcriptional and post-transcriptional regulation of heat-responsive miRNAs in barley. The observed induction of miRNA expression is correlated with the down-regulation of the expression level of their experimentally identified new and conservative target genes.

Developmentally regulated expression and complex processing of barley pri-microRNAs.

BACKGROUND: MicroRNAs (miRNAs) regulate gene expression via mRNA cleavage or translation inhibition. In spite of barley being a cereal of great economic importance, very little data is available concerning its miRNA biogenesis. There are 69 barley miRNA and 67 pre-miRNA sequences available in the miRBase (release 19). However, no barley pri-miRNA and MIR gene structures have been shown experimentally. In the present paper, we examine the biogenesis of selected barley miRNAs and the developmental regulation of their pri-miRNA processing to learn more about miRNA maturation in barely. RESULTS: To investigate the organization of barley microRNA genes, nine microRNAs - 156g, 159b, 166n, 168a-5p/168a-3p, 171e, 397b-3p, 1120, and 1126 - were selected. Two of the studied miRNAs originate from one MIR168a-5p/168a-3p gene. The presence of all miRNAs was confirmed using a Northern blot approach. The miRNAs are encoded by genes with diverse organizations, representing mostly independent transcription units with or without introns. The intron-containing miRNA transcripts undergo complex splicing events to generate various spliced isoforms. We identified miRNAs that were encoded within introns of the noncoding genes MIR156g and MIR1126. Interestingly, the intron that encodes miR156g is spliced less efficiently than the intron encoding miR1126 from their specific precursors. miR397b-3p was detected in barley as a most probable functional miRNA, in contrast to rice where it has been identified as a complementary partner miRNA*. In the case of miR168a-5p/168a-3p, we found the generation of stable, mature molecules from both pre-miRNA arms, confirming evolutionary conservation of the stability of both species, as shown in rice and maize. We suggest that miR1120, located within the 3' UTR of a protein-coding gene and described as a functional miRNA in wheat, may represent a siRNA generated from a mariner-like transposable element. CONCLUSIONS: Seven of the eight barley miRNA genes characterized in this study contain introns with their respective transcripts undergoing developmentally specific processing events prior to the dicing out of pre-miRNA species from their pri-miRNA precursors. The observed tendency to maintain the intron encoding miR156g within the transcript, and preferences in splicing the miR1126-harboring intron, may suggest the existence of specific regulation of the levels of intron-derived miRNAs in barley.

Recent Insights into Plant miRNA Biogenesis: Multiple Layers of miRNA Level Regulation.

MicroRNAs are small RNAs, 20-22 nt long, the main role of which is to downregulate gene expression at the level of mRNAs. MiRNAs are fundamental regulators of plant growth and development in response to internal signals as well as in response to abiotic and biotic factors. Therefore, the deficiency or excess of individual miRNAs is detrimental to particular aspects of a plant's life. In consequence, the miRNA levels must be appropriately adjusted. To obtain proper expression of each miRNA, their biogenesis is controlled at multiple regulatory layers. Here, we addressed processes discovered to influence miRNA steady-state levels, such as MIR transcription, co-transcriptional pri-miRNA processing (including splicing, polyadenylation, microprocessor assembly and activity) and miRNA-encoded peptides synthesis. MiRNA stability, RISC formation and miRNA export out of the nucleus and out of the plant cell also define the levels of miRNAs in various plant tissues. Moreover, we show the evolutionary conservation of miRNA biogenesis core proteins across the plant kingdom.

MicroRNA172b-5p/trehalose-6-phosphate synthase module stimulates trehalose synthesis and microRNA172b-3p/AP2-like module accelerates flowering in barley upon drought stress.

MicroRNAs (miRNAs) are major regulators of gene expression during plant development under normal and stress conditions. In this study, we analyzed the expression of 150 conserved miRNAs during drought stress applied to barley ready to flower. The dynamics of miRNAs expression was also observed after rewatering. Target messenger RNA (mRNAs) were experimentally identified for all but two analyzed miRNAs, and 41 of the targets were not reported before. Drought stress applied to barley induced accelerated flowering coordinated by a pair of two differently expressed miRNAs originating from a single precursor: hvu-miR172b-3p and hvu-miR172b-5p. Increased expression of miRNA172b-3p during drought leads to the downregulation of four APETALA2(AP2)-like genes by their mRNA cleavage. In parallel, the downregulation of the miRNA172b-5p level results in an increased level of a newly identified target, trehalose-6-phosphate synthase, a key enzyme in the trehalose biosynthesis pathway. Therefore, drought-treated plants have higher trehalose content, a known osmoprotectant, whose level is rapidly dropping after watering. In addition, trehalose-6-phosphate, an intermediate of the trehalose synthesis pathway, is known to induce flowering. The hvu-miRNA172b-5p/trehalose-6-phosphate synthase and hvu-miRNA172b-3p/AP2-like create a module leading to osmoprotection and accelerated flowering induction during drought.

Excess nitrogen responsive HvMADS27 transcription factor controls barley root architecture by regulating abscisic acid level.

Nitrogen (N) is an important element for plant growth and development. Although several studies have examined plants' response to N deficiency, studies on plants' response to excess N, which is common in fertilizer-based agrosystems, are limited. Therefore, the aim of this study was to examine the response of barley to excess N conditions, specifically the root response. Additionally, genomic mechanism of excess N response in barley was elucidated using transcriptomic technologies. The results of the study showed that barley MADS27 transcription factor was mainly expressed in the roots and its gene contained N-responsive cis-regulatory elements in the promoter region. Additionally, there was a significant decrease in HvMADS27 expression under excess N condition; however, its expression was not significantly affected under low N condition. Phenotypic analysis of the root system of HvMADS27 knockdown and overexpressing barley plants revealed that HvMADS27 regulates barley root architecture under excess N stress. Further analysis of wild-type (WT) and transgenic barley plants (hvmads27 kd and hvmads27 c-Myc OE) revealed that HvMADS27 regulates the expression of HvBG1 ß-glucosidase, which in turn regulates abscisic acid (ABA) level in roots. Overall, the findings of this study showed that HvMADS27 expression is downregulated in barley roots under excess N stress, which induces HvBG1 expression, leading to the release of ABA from ABA-glucose conjugate, and consequent shortening of the roots.

Quantitative Analysis of Plant miRNA Primary Transcripts.

MicroRNAs control plant development and are key regulators of plant responses to biotic and abiotic stresses. Thus, their expression must be carefully controlled since both excess and deficiency of a given microRNA may be deleterious to plant cell. MicroRNA expression regulation can occur at several stages of their biogenesis pathway. One of the most important of these regulatory checkpoints is transcription efficiency. mirEX database is a tool for exploration and visualization of plant pri-miRNA expression profiles. It includes results obtained using high-throughput RT-qPCR platform designed to monitor pri-miRNA expression in different miRNA biogenesis mutants and developmental stages of Arabidopsis, barley, and Pellia plants. A step-by-step instruction for browsing the database and detailed protocol for high-throughput RT-qPCR experiments, including list of primers designed for the amplification of pri-miRNAs, are presented.

The brome mosaic virus-based recombination vector triggers a limited gene silencing response depending on the orientation of the inserted sequence.

In some RNA viruses (e.g. in brome mosaic virus, BMV), the same factor (intra- or intermolecular hybridization between viral RNA molecules) is capable of inducing two different processes: RNA silencing and RNA recombination. To determine whether there is some interplay between these two phenomena, we have examined if the BMV-based recombination vector containing a plant-genome-derived sequence can function as a gene-silencing vector. Surprisingly, we found that neither dsRNA forming during the replication of the BMV-based vector nor highly structured regions of its genome were effective RNAi triggers. Only mutants carrying a sequence complementary to the target mRNA functioned as gene silencing vectors and were steadily maintained in the infected plant. The constructs containing a sense sequence or inverted repeats did not induce gene silencing but instead were eliminated from the plant cells.

Barley microRNAs as metabolic sensors for soil nitrogen availability.

Barley (Hordeum vulgare) is one of the most important crops in the world, ranking 4th in the worldwide production. Crop breeders are facing increasing environmental obstacles in the field, such as drought, salinity but also toxic over fertilization which not only impacts quality of the grain but also an yield. One of the most prevalent mechanisms of gene expression regulation in plants is microRNA-mediated silencing of target genes. We identified 13 barley microRNAs and 2 microRNAs* that are nitrogen excess responsive. Four microRNAs respond only in root, eight microRNAs only in shoot and one displays broad response in roots and shoots. We demonstrate that 2 microRNAs* are induced in barley shoot by nitrogen excess. For all microRNAs we identified putative target genes and confirmed microRNA-guided cleavage sites for ten out of thirteen mRNAs. None of the identified microRNAs or their target genes is known as nitrogen excess responsive. Analysis of expression pattern of thirteen target mRNAs and their cognate microRNAs showed expected correlations of their levels. The plant microRNAs analyzed are also known to respond to nitrogen deprivation and exhibit the opposite expression pattern when nitrogen excess/deficiency conditions are compared. Thus, they can be regarded as metabolic sensors of the regulation of nitrogen homeostasis in plants.

A Functional Network of Novel Barley MicroRNAs and Their Targets in Response to Drought.

The regulation of mRNA (messenger RNA) levels by microRNA-mediated activity is especially important in plant responses to environmental stresses. In this work, we report six novel barley microRNAs, including two processed from the same precursor that are severely downregulated under drought conditions. For all analyzed microRNAs, we found target genes that were upregulated under drought conditions and that were known to be involved in a plethora of processes from disease resistance to chromatin-protein complex formation and the regulation of transcription in mitochondria. Targets for novel barley microRNAs were confirmed through degradome data analysis and RT-qPCR using primers flanking microRNA-recognition site. Our results show a broad transcriptional response of barley to water deficiency conditions through microRNA-mediated gene regulation and facilitate further research on drought tolerance in crops.

Plant PHR Transcription Factors: Put on A Map.

The phosphate starvation response (PHR) protein family exhibits the MYB and coiled-coil domains. In plants, within the either 5' untranslated regions (UTRs) or promoter regions of phosphate starvation-induced (PSI) genes are characteristic cis-regulatory elements, namely PHR1 binding sequence (P1BS). The most widely studied PHR protein family members, such as AtPHR1 in Arabidopsis thaliana (L.) and OsPHR2 in Oryza sativa (L.), may activate the gene expression of a broad range of PSI genes by binding to such elements in a phosphate (Pi) dependent manner. In Pi signaling, PHR transcription factors (TFs) can be selectively activated or deactivated by other proteins to execute the final step of signal transduction. Several new proteins have been associated with the AtPHR1/OsPHR2 signaling cascade in the last few years. While the PHR TF transcriptional role has been studied intensively, here we highlight the recent findings of upstream molecular components and other signaling pathways that may interfere with the PHR final mode of action in plants. Detailed information about transcriptional regulation of the AtPHR1 gene itself and its upstream molecular events has been reviewed.

Polyamines - A New Metabolic Switch: Crosstalk With Networks Involving Senescence, Crop Improvement, and Mammalian Cancer Therapy.

Polyamines (PAs) are low molecular weight organic cations comprising biogenic amines that play multiple roles in plant growth and senescence. PA metabolism was found to play a central role in metabolic and genetic reprogramming during dark-induced barley leaf senescence (DILS). Robust PA catabolism can impact the rate of senescence progression in plants. We opine that deciphering senescence-dependent polyamine-mediated multidirectional metabolic crosstalks is important to understand regulation and involvement of PAs in plant death and re-mobilization of nutrients during senescence. This will involve optimizing the use of PA biosynthesis inhibitors, robust transgenic approaches to modulate PA biosynthetic and catabolic genes, and developing novel germplasm enriched in pro- and anti-senescence traits to ensure sustained crop productivity. PA-mediated delay of senescence can extend the photosynthesis capacity, thereby increasing grain starch content in malting grains such as barley. On the other hand, accelerating the onset of senescence can lead to increases in mineral and nitrogen content in grains for animal feed. Unraveling the "polyamine metabolic switch" and delineating the roles of PAs in senescence should further our knowledge about autophagy mechanisms involved in plant senescence as well as mammalian systems. It is noteworthy that inhibitors of PA biosynthesis block cell viability in animal model systems (cell tumor lines) to control some cancers, in this instance, proliferative cancer cells were led toward cell death. Likewise, PA conjugates work as signal carriers for slow release of regulatory molecule nitric oxide in the targeted cells. Taken together, these and other outcomes provide examples for developing novel therapeutics for human health wellness as well as developing plant resistance/tolerance to stress stimuli.

miRNA Detection by Stem-Loop RT-qPCR in Studying microRNA Biogenesis and microRNA Responsiveness to Abiotic Stresses.

This chapter is devoted to a PCR-based method for analyzing the expression level of mature miRNAs which utilizes the TaqMan® technology. Stem-loop RT-qPCR requires preparation of separate cDNA templates for each analyzed miRNA as reverse transcription occurs in the presence of a miRNA-specific stem-loop reverse primer. In quantitative analysis, SYBR® Green is not used but the more sensitive TaqMan® probe that on 5' end contains a covalently attached fluorophore and on 3' quencher. When quencher and fluorophore are spatially separated due to nucleolytic DNA polymerase activity, the signal is released and quantified. This section provides a detailed and comprehensive protocol allowing for the successful analysis of mature miRNA levels in analyzed sample. Reverse transcription combined with classic real-time PCR as well as ddPCR™ (Droplet Digital™ PCR) will be presented.

The miRNAome dynamics during developmental and metabolic reprogramming of tomato root infected with potato cyst nematode.

Cyst-forming plant-parasitic nematodes are pests threatening many crops. By means of their secretions cyst nematodes induce the developmental and metabolic reprogramming of host cells that lead to the formation of a syncytium, which is the sole food source for growing nematodes. The in depth micro RNA (miRNA) dynamics in the syncytia induced by Globodera rostochiensis in tomato roots was studied. The miRNAomes were obtained from syncytia covering the early and intermediate developmental stages, and were the subject of differential expression analysis. The expression of 1235 miRNAs was monitored. The fold change (log2FC) ranged from -7.36 to 8.38, indicating that this transcriptome fraction was very variable. Moreover, we showed that the DE (differentially expressed) miRNAs do not fully overlap between the selected time points, suggesting infection stage specific regulation by miRNA. The correctness of RNA-seq expression profiling was confirmed by qRT-PCR (quantitative Real Time Polymerase Chain Reaction) for seven miRNA species. Down- and up-regulated miRNA species, including their isomiRs, were further used to identify their potential targets. Among them there are a large number of transcription factors linked to different aspects of plant development belonging to gene families, such as APETALA2 (AP2), SQUAMOSA (MADS-box), MYB, GRAS, and AUXIN RESPONSE FACTOR (ARF). The substantial portion of potential target genes belong to the NB-LRR and RLK (RECEPTOR-LIKE KINASE) families, indicating the involvement of miRNA mediated regulation in defense responses. We also collected the evidence for target cleavage in the case of 29 miRNAs using one of three alternative methods: 5' RACE (5' Rapid Amplification of cDNA Ends), a search of tasiRNA within our datasets, and the meta-analysis of tomato degradomes in the GEO (Gene Expression Omnibus) database. Eight target transcripts showed a negative correlation with their respective miRNAs at two or three time points. These results indicate a large regulatory potential for miRNAs in tuning the development and defense responses.