Exploring the Metatranscriptome of Bacterial Communities of Two Moss Species Thriving in Different Environments-Terrestrial and Aquatic.

Mosses host diverse bacterial communities essential for their fitness, nutrient acquisition, stress tolerance, and pathogen defense. Understanding the microbiome's taxonomic composition is the first step, but unraveling their functional capabilities is crucial for grasping their ecological significance. Metagenomics characterizes microbial communities by composition, while metatranscriptomics explores gene expression, providing insights into microbiome functionality beyond the structure. Here, we present for the first time a metatranscriptomic study of two moss species, Hypnum cupressiforme (Hedw.) and Platyhypnidium riparioides (Hedw.) Dixon., renowned as key biomonitors of atmospheric and water pollution. Our investigation extends beyond taxonomic profiling and offers a profound exploration of moss bacterial communities. Pseudomonadota and Actinobacteria are the dominant bacterial phyla in both moss species, but their proportions differ. In H. cupressiforme, Actinobacteria make up 62.45% and Pseudomonadota 32.48%, while in P. riparioides, Actinobacteria account for only 25.67% and Pseudomonadota 69.08%. This phylum-level contrast is reflected in genus-level differences. Our study also shows the expression of most genes related to nitrogen cycling across both microbiomes. Additionally, functional annotation highlights disparities in pathway prevalence, including carbon dioxide fixation, photosynthesis, and fatty acid biosynthesis, among others. These findings hint at potential metabolic distinctions between microbial communities associated with different moss species, influenced by their specific genotypes and habitats. The integration of metatranscriptomic data holds promise for enhancing our understanding of bryophyte-microbe partnerships, opening avenues for novel applications in conservation, bioremediation, and sustainable agriculture.

Ailanthus altissima Seed Oil-A Valuable Source of Lipid-Soluble Components with DNA Protective and Antiproliferative Activities.

The present study is focused on the chemical and lipid composition of seed oil of the European ornamental and invasive wood plant Ailanthus altissima (Simaroubaceae). Total lipids, proteins, carbohydrates, ash, and moisture in the seeds were determined. A high yield of glyceride oil (30.7%) was found, as well as a high content of fibers (29.6%) and proteins (18.7%). Physicochemical properties of the oil define it as semi-dry (129.4 g I2/100 g Iodine value) with oxidative stability, refractive index, saponification value, and relative density similar to widely used oils with nutritional value and health benefits. The composition of the seed oil was determined chromatographically. Unsaturated fatty acids (95.3%) predominated in the seed oil, of which linoleic acid (48.6%) and oleic acid (44.8%) were the major ones. The main lipid-soluble bioactive components were ß-sitosterol (72.6%), γ-tocopherol (74.6%), phosphatidylinositol (29.5%), and phosphatidic acids (25.7%). The proven in vitro DNA-protective ability of seed oil is reported for the first time. The seed oil exhibited a weak antiproliferative effect on HT-29 and PC3 tumor cell lines and showed no cytotoxicity on the BALB/c 3T3 cell line. In brief, the present study reveals that A. altissima seed oil can be used as a healthy food.

Exploring the Genomic Landscape of Bacillus paranthracis PUMB_17 as a Proficient Phosphatidylcholine-Specific Phospholipase C Producer.

Phospholipases find versatile applications across industries, including detergent production, food modification, pharmaceuticals (especially in drug delivery systems), and cell signaling research. In this study, we present a strain of Bacillus paranthracis for the first time, demonstrating significant potential in the production of phosphatidylcholine-specific phospholipase C (PC-PLC). The investigation thoroughly examines the B. paranthracis PUMB_17 strain, focusing on the activity of PC-PLC and its purification process. Notably, the PUMB_17 strain displays extracellular PC-PLC production with high specific activity during the late exponential growth phase. To unravel the genetic makeup of PUMB_17, we employed nanopore-based whole-genome sequencing and subsequently conducted a detailed genome annotation. The genome comprises a solitary circular chromosome spanning 5,250,970 bp, featuring a guanine-cytosine ratio of 35.49. Additionally, two plasmids of sizes 64,250 bp and 5845 bp were identified. The annotation analysis reveals the presence of 5328 genes, encompassing 5186 protein-coding sequences, and 142 RNA genes, including 39 rRNAs, 103 tRNAs, and 5 ncRNAs. The aim of this study was to make a comprehensive genomic exploration that promises to enhance our understanding of the previously understudied and recently documented capabilities of Bacillus paranthracis and to shed light on a potential use of the strain in the industrial production of PC-PLC.

Illuminating the Genomic Landscape of Lactiplantibacillus plantarum PU3-A Novel Probiotic Strain Isolated from Human Breast Milk, Explored through Nanopore Sequencing.

Lactiplantibacillus plantarum stands out as a remarkably diverse species of lactic acid bacteria, occupying a myriad of ecological niches. Particularly noteworthy is its presence in human breast milk, which can serve as a reservoir of probiotic bacteria, contributing significantly to the establishment and constitution of infant gut microbiota. In light of this, our study attempted to conduct an initial investigation encompassing both genomic and phenotypic aspects of the L. plantarum PU3 strain, that holds potential as a probiotic agent. By employing the cutting-edge third-generation Nanopore sequencing technology, L. plantarum PU3 revealed a circular chromosome of 3,180,940 bp and nine plasmids of various lengths. The L. plantarum PU3 genome has a total of 2962 protein-coding and non-coding genes. Our in-depth investigations revealed more than 150 probiotic gene markers that unfold the genetic determinants for acid tolerance, bile resistance, adhesion, and oxidative and osmotic stress. The in vivo analysis showed the strain's proficiency in utilizing various carbohydrates as growth substrates, complementing the in silico analysis of the genes involved in metabolic pathways. Notably, the strain demonstrated a pronounced affinity for D-sorbitol, D-mannitol, and D-Gluconic acid, among other carbohydrate sources. The in vitro experimental verification of acid, osmotic and bile tolerance validated the robustness of the strain in challenging environments. Encouragingly, no virulence factors were detected in the genome of PU3, suggesting its safety profile. In search of beneficial properties, we found potential bacteriocin biosynthesis clusters, suggesting its capability for antimicrobial activity. The characteristics exhibited by L. plantarum PU3 pave the way for promising strain potential, warranting further investigations to unlock its full capacity and contributions to probiotic and therapeutic avenues.

Koelreuteria paniculata Seed Oil-A Rich Natural Source of Unsaturated Fatty Acids and Phytocompounds with DNA Protective Potential.

The present work is focused on the physicochemical characteristics, chemical composition, and some biological activities of Koelreuteria paniculata seed oil. The glyceride oil, obtained with a Soxhlet apparatus by extraction with hexane, was characterized by a relatively high oil content (over 20%), and it is defined as a non-drying oil (iodine value-44 gI2/100 g) with good oxidative stability (over 50 h). There were identified 11 fatty acids, 6 sterols, 3 tocopherols, and 6 phospholipids, as the last group was reported for the first time. The major components among them were-monounsaturated eicosenoic and oleic acids, ß-sitosterol, ß-tocopherol, and phosphatidylcholine. The in vitro tests demonstrated DNA protective activity and a lack of cytotoxicity of the oil, data that has been reported for the first time. The in vitro MTT test of the oil on HT-29 and PC3 cell lines did not indicate antitumor activity. The seed oil studied contains valuable bio-components, which have proven benefits for human health, and that is why it could be used in food, cosmetic, and pharmaceutical products.

16S-rRNA-Based Metagenomic Profiling of the Bacterial Communities in Traditional Bulgarian Sourdoughs.

Sourdoughs (SDs) are spontaneously formed microbial ecosystems composed of various species of lactic acid bacteria (LAB) and acid-tolerant yeasts in food matrices of cereal flours mixed with water. To date, more than 90 LAB species have been isolated, significantly impacting the organoleptic characteristics, shelf life, and health properties of bakery products. To learn more about the unique bacterial communities involved in creating regional Bulgarian sourdoughs, we examined the metacommunities of five sourdoughs produced by spontaneous fermentation and maintained by backslopping in bakeries from three geographic locations. The 16S rRNA gene amplicon sequencing showed that the former genus Lactobacillus was predominant in the studied sourdoughs (51.0-78.9%). Weissella (0.9-42.8%), Herbaspirillum (1.6-3.8%), Serratia (0.1-11.7%), Pediococcus (0.2-7.5%), Bacteroides (0.1-1.3%), and Sphingomonas (0.1-0.5%) were also found in all 5 samples. Genera Leuconostoc, Enterococcus, Bacillus, and Asaia were sample-specific. It is interesting to note that the genus Weissella was more abundant in wholegrain samples. The greatest diversity at the species level was found in the former genus Lactobacillus, presented in the sourdough samples with 13 species. The UPGMA cluster analysis clearly demonstrated similarity in species' relative abundance between samples from the same location. In addition, we can conclude that the presence of two main clusters-one including samples from mountainous places (the cities of Smolyan and Bansko) and the other including samples from the city of Ruse (the banks of the Danube River)-may indicate the impact of climate and geographic location (e.g., terrain, elevation, land use, and nearby water bodies and their streams) on the abundance of microbiome taxa. As the bacterial population is crucial for bread standardization, we expect the local bakery sector to be interested in the relationship between process variables and their effect on bacterial dynamics described in this research study.

Phenolic Profile, Antioxidant and DNA-Protective Capacity, and Microscopic Characters of Ailanthus altissima Aerial Substances.

Invasive species as sources of natural components are of increasing interest for scientific research. This is the case of Ailanthus altissima, which belongs to the top 100 of the most dangerous invasive plant species in Europe, and which is the subject of the present study. The purpose of the research was to analyze the main phenolic compounds in the flowers, leaves, and stem bark of A. altissima and determine the DNA-protective and antioxidant potential of their ethanolic extracts. HPLC profiling revealed the presence of 6 flavonoids and 10 phenolic acids, of which 15 were found in flowers, 14 in leaves, and 11 in the stem bark. Rutin (5.68 mg/g dw in flowers), hesperidin (2.67 mg/g dw in leaves) and (+)-catechin (2.15 mg/g dw in stem bark) were the best-represented flavonoids. Rosmarinic (10.32 mg/g dw in leaves) and salicylic (6.19 mg/g dw in leaves) acids were predominant among phenolic acids. All plant extracts tested showed in vitro antioxidant activity (determined by DPPH, ABTS, FRAP, and CUPRAC assays) and DNA-protection capacity (assay with supercoiled plasmid DNA-pUC19). The highest antioxidant activity was recorded in the flower parts (in the range from 661 to 893 mmol TE/g dw), followed by the leaves. A DNA protective potential for A. altissima leaf and flower extracts has not been established to date. In addition, the main microscopic diagnostic features of studied plant substances were described, with data for the flower parts being reported for the first time. The present study proves that A. altissima could be a natural source of DNA protection and antioxidants.

Antioxidant and DNA-Protective Potentials, Main Phenolic Compounds, and Microscopic Features of Koelreuteria paniculata Aerial Parts.

Interest in plant extracts as a natural source of antioxidants has grown significantly in recent years. The tree species Koelreuteria paniculata deserves attention due to its wide distribution, good adaptability, and growth to the degree of invasiveness in a number of European countries. The purpose of the present study was to analyze flavonoids and phenolic acids of the ethanol extracts from aerial parts of K. paniculata and to screen their antioxidant and DNA-protective activity. HPLC profiling revealed the presence of five flavonoids, with rutin (4.23 mg/g DW), hesperidin (2.97 mg/g DW), and quercetin (2.66 mg/g DW) as the major ones in the leaves, and (-)-epicatechin (2.69 mg/g DW) in the flower buds. Among the nine phenolic acids identified, rosmarinic, p-coumaric, salicylic, vanillic, and gallic acids were the best represented. All the extracts tested showed in vitro antioxidant activity that was determined by DPPH, ABTS, FRAP, and CUPRAC assays. The highest activity was recorded in the flower parts (in the range from 1133 to 4308 mmol TE/g DW). The DNA-protective capacity of the flower and stem bark extracts from the in vitro nicking assay performed, as well as the main diagnostic microscopic features of the plant substances, are given for the first time. According to the results obtained, the aerial parts of K. paniculata could be valuable sources of natural antioxidants.

Transcriptome Analysis Reveals Dynamic Cultivar-Dependent Patterns of Gene Expression in Potato Spindle Tuber Viroid-Infected Pepper.

Potato spindle tuber viroid (PSTVd) infects various plants. PSTVd pathogenesis is associated with interference with the cellular metabolism and defense signaling pathways via direct interaction with host factors or via the transcriptional or post-transcriptional modulation of gene expression. To better understand host defense mechanisms to PSTVd infection, we analyzed the gene expression in two pepper cultivars, Capsicum annuum Kurtovska kapia (KK) and Djulunska shipka (DS), which exhibit mild symptoms of PSTVd infection. Deep sequencing-based transcriptome analysis revealed differential gene expression upon infection, with some genes displaying contrasting expression patterns in KK and DS plants. More genes were downregulated in DS plants upon infection than in KK plants, which could underlie the more severe symptoms seen in DS plants. Gene ontology enrichment analysis revealed that most of the downregulated differentially expressed genes in both cultivars were enriched in the gene ontology term photosynthesis. The genes upregulated in DS plants fell in the biological process of gene ontology term defense response. We validated the expression of six overlapping differentially expressed genes that are involved in photosynthesis, plant hormone signaling, and defense pathways by quantitative polymerase chain reaction. The observed differences in the responses of the two cultivars to PSTVd infection expand the understanding of the fine-tuning of plant gene expression that is needed to overcome the infection.

Analysis of the complete mitochondrial genome sequence of the resurrection plant Haberlea rhodopensis.

Haberlea rhodopensis is a paleolithic tertiary relict species that belongs to the unique group of resurrection plants sharing remarkable tolerance to desiccation. When exposed to severe drought stress, this species shows an ability to maintain structural integrity of its deactivated photosynthetic apparatus, which easily reactivates upon rehydration. In addition to its homoiochlorophyllous nature, the resurrection capability of H. rhodopensis is of particular importance to the global climate change mitigation. In this study, we sequenced, assembled, and analyzed the mitochondrial (mt) genome of H. rhodopensis for the first time. The master circle has a typical circular structure of 484 138 bp in length with a 44.1% GC content in total. The mt genome of H. rhodopensis contains 59 genes in total, including 35 protein-coding, 21 tRNAs, and 3 rRNAs genes. 7 tandem repeats and 85 simple sequence repeats (SSRs) are distributed throughout the mt genome. The alignment of 20 plant mt genomes confirms the phylogenetic position of H. rhodopensis in the Lamiales order. Our comprehensive analysis of the complete mt genome of H. rhodopensis is a significant addition to the limited database of organelle genomes of resurrection species. Comparative and phylogenetic analysis provides valuable information for a better understanding of mitochondrial molecular evolution in plants.

Can Biomarkers Respond Upon Freshwater Pollution?-A Moss-Bag Approach.

Moss-bags were applied to study the effect of contamination in three standing water bodies in Bulgaria (Kardzhali, Studen Kladenets and Zhrebchevo Reservoirs), the first two with old industrial contamination and the last polluted with short-chain chlorinated paraffins (SCCPs). Fontinalis antipyretica Hedw. collected from background (unpolluted) site was placed in cages for a period of 30 days. The present study examined whether inorganic and organic pollution detected with moss-bags resulted in corresponding differences in molecular, chemical and micromorphological markers. Suppressed large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL) expression was assessed in moss-bags from two of the reservoirs, contaminated with heavy metals. There was a decrease of the total phenolic content (TPC) in the moss-bags, which provides a basis for further studies of the chemical content of aquatic mosses. Fontinalis antipyretica also showed a response through leaf micromorphological characteristics. In the all three reservoirs, an increase of the twig leaf cell number was recorded (p ≤ 0.01 for Kardzhali and p ≤ 0.001 for Studen Kladenets and Zhrebchevo reservoirs), as well as of the stem leaf cell number in Zhrebchevo Reservoir (p ≤ 0.001). On the contrary, the width of the cells decreased in the studied anthropogenically impacted reservoirs. All three studied groups of biomarkers (molecular, chemical and micromorphological) appeared to be sensitive to freshwater pollution. The results achieved indicated that rbcL gene expression, TPC, cell number and size are promising biomonitoring tools.

Transposon-associated polymorphisms of stress-responsive gene promoters in selected accessions of Arabidopsis thaliana.

Genetic diversity caused by transposable element movement can play an important role in plant adaptation to local environments. Regarding genes, transposon-induced alleles were mostly related to gene bodies and a few of them to promoter regions. In this study, promoter regions of 9 stress-related genes were searched for transposable element insertions in 12 natural accessions of Arabidopsis thaliana. The promoter screening was performed via PCR amplification with primers designed to flank transposable element insertions in the promoter regions of the reference accession Col-0. Transposable element-associated insertion/deletion (indel) polymorphisms were identified in 7 of the 12 promoter loci across studied accessions that can be developed further as molecular markers. The transposable element absence in the promoter regions of orthologous genes in A. lyrata indicated that the insertion of these transposable elements in A. thaliana lineage had occurred after its divergence from A. lyrata. Sequence analysis of the promoter regions of CML41 (Calmodulin-like protein 41) and CHAP (chaperone protein dnaJ-related) confirmed the indel polymorphic sites in four accessions - Col-0, Wassilewskija, Shahdara, and Pirin. The observed indel polymorphism of the CHAP promoter region was associated with specific gene expression profiles in the different accessions grown at a normal and elevated temperature in a plant growth chamber. The collected data can be a starting point for gene expression profiling studies under conditions resembling the natural habitats of accessions.

Metagenomic profiling of the microbial freshwater communities in two Bulgarian reservoirs.

Microorganisms inhabiting freshwater environments are an integral part of the aquatic ecosystems. Very few data are available regarding the profiles of the microbial communities in the reservoirs in Bulgaria, despite their key role in the biogeochemical processes. In the present study, we provide the first comprehensive metagenomic analysis on the planktonic bacterial diversity of two large and economically important Bulgarian reservoirs (Batak and Tsankov Kamak) using next-generation sequencing of 16S ribosomal RNA gene (16S rRNA). Analysis of the metagenomic amplicon datasets, including quality filtering, clustering of Operational Taxonomic Units and taxonomy assignment revealed that 78.45% of the microbial communities between the two reservoirs were overlapping. The diversity (H) and Pielou's evenness (J) indices declined along the longitudinal axis of both reservoirs. The estimated values for the Shannon diversity index are typically observed in oligotrophic lakes. The microbial communities of both reservoirs were dominated by Proteobacteria, followed by Actinobacteria and Bacteroidetes all comprised over 95% of the relative abundance, regardless of the reservoir's large hydrogeological differences. The bacterioplankton was characterized by high phylogenetic heterogeneity in the taxonomic structure, being distributed among 211 genera. The genera Limnohabitans and Rhodoferax held the absolute predominance, implying their significance in the aquatic food webs. The obtained data can contribute to the better systematic understanding of the microbial diversity of freshwater environments.

Chloroplast Genome Analysis of Resurrection Tertiary Relict Haberlea rhodopensis Highlights Genes Important for Desiccation Stress Response.

Haberlea rhodopensis is a paleolithic tertiary relict species, best known as a resurrection plant with remarkable tolerance to desiccation. When exposed to severe drought stress, H. rhodopensis shows an ability to maintain the structural integrity of its photosynthetic apparatus, which re-activates easily upon rehydration. We present here the results from the assembly and annotation of the chloroplast (cp) genome of H. rhodopensis, which was further subjected to comparative analysis with the cp genomes of closely related species. H. rhodopensis showed a cp genome size of 153,099 bp, harboring a pair of inverted repeats (IR) of 25,415 bp separated by small and large copy regions (SSC and LSC) of 17,826 and 84,443 bp. The genome structure, gene order, GC content and codon usage are similar to those of the typical angiosperm cp genomes. The genome hosts 137 genes representing 70.66% of the plastome, which includes 86 protein-coding genes, 36 tRNAs, and 4 rRNAs. A comparative plastome analysis with other closely related Lamiales members revealed conserved gene order in the IR and LSC/SSC regions. A phylogenetic analysis based on protein-coding genes from 33 species defines this species as belonging to the Gesneriaceae family. From an evolutionary point of view, a site-specific selection analysis detected positively selected sites in 17 genes, most of which are involved in photosynthesis (e.g., rbcL, ndhF, accD, atpE, etc.). The observed codon substitutions may be interpreted as being a consequence of molecular adaptation to drought stress, which ensures an evolutionary advantage to H. rhodopensis.

High-temperature effect on genes engaged in DNA methylation and affected by DNA methylation in Arabidopsis.

Along with its essential role in the maintenance of genome integrity, DNA methylation takes part in regulation of genes which are important for plant development and stress response. In plants, DNA methylation process can be directed by small RNAs in process known as RNA-directed DNA methylation (RdDM) involving two plant-specific RNA polymerases - PolIV and PolV. The aim of the present study was to investigate the effect of heat stress on the expression of genes encoding key players in DNA methylation - DNA methyltransferase (MET1, CMT3, and DRM2), the largest subunits of PoIIV and PolV (NRPD1 and NRPE1 respectively) and the DNA demethylase ROS1. We also examined the high-temperature effect on two protein-coding genes - At3g50770 and At5g43260 whose promoters contain transposon insertions and are affected by DNA-methylation, as well as on the AtSN1, a SINE-like retrotransposon. To assess the involvement of PolIV and PolV in heat stress response, the promoter methylation status and transcript levels of these genes were compared between wild type and double mutant lacking NRPD1 and NRPE1. The results demonstrate coordinated up-regulation of the DRM2, NRPD1 and NRPE1 in response to high temperature and suggest that PolIV and/or PolV might be required for the induction of DRM2 expression under heat stress. The ROS1 expression was confirmed to be suppressed in the mutant lacking active PolIV and PolV that might be a consequence of abolished DNA methylation. The increased expression of At3g50770 in response to elevated temperature correlated with reduced promoter DNA methylation, while the stress response of At5g43260 did not show inverse correlation between promoter methylation and gene expression. Our results also imply that PolIV and/or PolV could regulate gene expression under stress conditions not only through RdDM but also by acting in other regulatory processes.

Insight into small RNA abundance and expression in high- and low-temperature stress response using deep sequencing in Arabidopsis.

Small RNA profiling and assessing its dependence on changing environmental factors have expanded our understanding of the transcriptional and post-transcriptional regulation of plant stress responses. Insufficient data have been documented earlier to depict the profiling of small RNA classes in temperature-associated stress which has a wide implication for climate change biology. In the present study, we report a comparative assessment of the genome-wide profiling of small RNAs in Arabidopsis thaliana using two conditional responses, induced by high- and low-temperature. Genome-wide profiling of small RNAs revealed an abundance of 21 nt small RNAs at low temperature, while high temperature showed an abundance of 21 nt and 24 nt small RNAs. The two temperature treatments altered the expression of a specific subset of mature miRNAs and displayed differential expression of a number of miRNA isoforms (isomiRs). Comparative analysis demonstrated that a large number of protein-coding genes can give rise to differentially expressed small RNAs following temperature shifts. Low temperature caused accumulation of small RNAs, corresponding to the sense strand of a number of cold-responsive genes. In contrast, high temperature stimulated the production of small RNAs of both polarities from genes encoding functionally diverse proteins.

Molecular cloning and characterization of cDNAs of the superoxide dismutase gene family in the resurrection plant Haberlea rhodopensis.

Resurrection plants can tolerate almost complete water loss in their vegetative parts. The superoxide dismutases (SODs) are essential enzymes of defense against the oxidative damage caused by water stress. Here, we cloned and characterized cDNAs of the SOD gene family in the resurrection plant Haberlea rhodopensis. Seven full-length cDNAs, and their partial genomic clones, were obtained by combination of degenerate PCR, RT-PCR and RACE. The derived amino acid sequences exhibited a very high degree of similarity to cytosolic Cu,Zn-SODs (HrCSD2, HrCSD3), chloroplastic Cu,Zn-SODs (HrCSD5), other Cu,Zn-SODs (HrCSD4), Mn-SODs (HrMSD) and Fe-SODs (HrFSD). One cDNA turned out to be a pseudogene (HrCSD1). All identified SOD genes were found expressed at transcriptional level--the HrCSD2, HrCSD5, HrMSD and HrFSD were constitutively expressed in all organs, while the HrCSD3 and HrCSD4 were organ-specific. The transcripts of the housekeeping SOD genes were detected at significant levels even in air-dry leaves. The multigene SOD family of H. rhodopensis is the first studied SOD family amongst resurrection plant species. Our finding of well expressed SOD transcripts in fully dehydrated leaves correlates with retention of SOD activity, and with the ability of H. rhodopensis to revive upon rehydration. Because of the endemic relict nature of that species, our findings may help to further elucidate the evolutionary relationships among different SOD isoforms from distinct plant species.

Implementation of a de novo genome-wide computational approach for updating Brachypodium miRNAs.

Plant microRNAs (miRNAs) are single-stranded 20-22 nt small RNAs (sRNA) that are produced from their own genes. We have developed a de novo genome-wide approach for the computational identification of novel plant miRNAs based on the integration of the complete genome sequence with sRNA libraries. It comprises three modules - the clustering module identifies genomic regions that have two closely-located unidirectional sRNA clusters, the mirplan module explores the secondary structure of the genomic regions, and the duplex module predicts miRNA/miRNA* duplexes. We applied our approach to the Brachypodium genome and publicly available sRNA libraries and predicted 102 miRNAs. Our results extend the list of known miRNAs with 58 novel miRNAs and define the genomic loci of all predicted miRNAs. Because this approach considers specific features of plant miRNAs, it can be employed for the analysis of the genome and sRNA libraries generated for plant species to achieve systematic miRNA discovery.

Identification of RNA-dependent DNA-methylation regulated promoters in Arabidopsis.

RNA-dependent DNA methylation (RdDM) is an important regulatory event involved in repressive epigenetic modifications that can trigger transcriptional gene silencing (TGS). The criteria we used to pick out promoter sequences targeted by RdDM in Arabidopsis thaliana were the main RdDM hallmark properties: 24nt siRNAs as inducers of DNA methylation and transposable elements (TE) as one of the major targets of RdDM. Those genes whose promoters comprised overlapping sites for 24nt siRNA hits, TE and DNA methylation (siRNA/TE/Methylation overlapping regions), were defined as candidates that might be silenced by RdDM. On this basis two gene sets were created which include abiotic and biotic stress-responsive genes whose promoters may be silenced by RdDM. The DNA methylation status of the At3g50770 (CML41) promoter - one of the selected candidates, was experimentally assayed, and it showed dependence on the RdDM-associated Polymerase IV and Polymerase V. A publicly available 24nt siRNA-centered database called starPRO was developed that allows users easily to discover whether a particular promoter sequence is related to RdDM-associated features such as 24nt siRNA-target sites, TE, tandem repeats and DNA methylation.