Role of RNA Interference (RNAi) in the Moss Physcomitrella patens

RNA interference (RNAi) is a mechanism that regulates genes by either transcriptional (TGS) or posttranscriptional gene silencing (PTGS), required for genome maintenance and proper development of an organism. Small non-coding RNAs are the key players in RNAi and have been intensively studied in eukaryotes. In plants, several classes of small RNAs with specific sizes and dedicated functions have evolved. The major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis. miRNAs are synthesized from a short hairpin structure while siRNAs are derived from long double-stranded RNAs (dsRNA). Both miRNA and siRNAs control the expression of cognate target RNAs by binding to reverse complementary sequences mediating cleavage or translational inhibition of the target RNA. They also act on the DNA and cause epigenetic changes such as DNA methylation and histone modifications. In the last years, the analysis of plant RNAi pathways was extended to the bryophyte Physcomitrella patens, a non-flowering, non-vascular ancient land plant that diverged from the lineage of seed plants approximately 450 million years ago. Based on a number of characteristic features and its phylogenetic key position in land plant evolution P. patens emerged as a plant model species to address basic as well as applied topics in plant biology. Here we summarize the current knowledge on the role of RNAi in P. patens that shows functional overlap with RNAi pathways from seed plants, and also unique features specific to this species

An integrative Raman microscopy-based workflow for rapid in situ analysis of microalgal lipid bodies

Additional file 1. Schematic diagram of Raman imaging technique

Using nuclear run-on transcription assays in RNAi studies

RNA interference (RNAi) is a mechanism regulating gene transcript levels by either transcriptional gene silencing or by posttranscriptional gene silencing, which act in the genome maintenance and the regulation of gene expression which is typically inferred from measuring transcript abundance. Nuclear “run-on” (or “run-off”) transcription assays have been used to obtain quantitative information about the relative rates of transcription of different genes in nuclei isolated from a particular tissue or organ. Basically, these assays exploit the activity of RNA polymerases to synthesize radiolabeled transcripts that then can be hybridized to filter-bound, cold, excess single-stranded DNA probes representing genes of interest. The protocol presented here streamlines, adapts and optimizes nuclear run-on transcription assays for use in RNAi studies

Use of Northern blotting for specific detection of small RNA molecules in transgenic plants

Small RNAs (20-24 nucleotides long and non protein coding) have been increasingly investigated. They were responsible for phenomena described as RNA interference (RNAi), co-suppression, gene silencing or quelling. Major classes of small RNAs include MicroRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biosynthesis. MiRNAs control the expression of cognate target genes by binding to reverse complementary sequences resulting in cleavage or translational inhibition of the target RNA. SiRNAs have similar structure, function and biogenesis as miRNAs, siRNAs derive from long double stranded RNA of transgenes, endogenous repeat sequences or transposons. Understanding these fundamental processes requires the sensitive and specific detection of small RNA species. In this report, we present a simple northern blot protocol for small RNAs in transgenic plants

Biological function of RNA interference (RNAi) pathways in the moss Physcomitrella patens (Hedw.) Bruch & Schimp.

Developmental programs in animals and plants are controlled by RNA interference (RNAi) via small non-coding RNAs regulating mRNA stability and translation, and chromatin modifications. MiRNAs are a specific class of small RNAs, generated by Dicer proteins, that down-regulate the expression of target genes by base-pairing to their cognate mRNAs in the RNA-induced silencing complex (RISC). In addition, miRNAs initiate the release of ta-siRNAs from precursor molecules TASRNAs. In contrast to the seed plant Arabidopsis thaliana the genome of the moss Physcomitrella patens encodes two DICER-LIKE1 proteins, PpDCL1a and PpDCL1b. In this study we found that PpDCL1a is the functional equivalent of the Arabidopsis thaliana DICER-LIKE1 protein required for the biogenesis of miRNAs and ta-siRNAs. Unlike AtDCL1 and PpDCL1a, PpDCL1b is not required for miRNA biogenesis, but is essential for miRNA-directed targetRNA cleavage and subsequent generation of transitive siRNAs and ta-siRNAs. In PpDCL1b mutant lines miRNA accumulation was unaffected but cleavage of targetRNAs was abolished. Instead, miRNA:targetRNA duplexes, hypermethylation of genomic loci encoding targetRNAs, and reduced levels of targetRNAs occurred. In Physcomitrella patens wild type we observed epigenetic silencing of a miRNA target gene in response to the phytohormone abscisic acid indicating that miRNAs control their targets not only at the post-transcriptional but also at the transcriptional level. Based on these data from Physcomitrella patens a model is proposed for the gene-specific control of transcription factors upon dysfunctions of the RNAi machinery. We anticipate similar control mechanisms to exist in other eukaryotes as well. Artificial miRNAs (amiRNAs) have been mainly used to downregulate single or multiple protein coding genes.amiRNAs can be generated by exchanging the miRNA/miRNA* sequence within miRNA precursor genes, while maintaining the pattern of matches and mismatches in the foldback. Thus, for functional gene analysis, amiRNAs can be designed to target any gene of interest. The moss Physcomitrella patens exhibits the unique feature of a highly efficient homologous recombination mechanism, which allows for the generation of targeted gene knockout lines. However, the completion of the Physcomitrella genome sequence necessitates the development of alternative techniques to speed up such reverse genetics analyses, and to allow for more flexible inactivation of genes or groups of genes. To prove the adaptability of amiRNA expression in Physcomitrella two amiRNAs were designed, the first one targeting the gene PpFtsZ2-1, which is indispensable for chloroplast division, and the second one targeting the gene PpGNT1 encoding an N-acetylglucosaminyltransferase. The PpFtsZ2-1-amiRNA and PpGNT1-amiRNA were expressed from the Arabidopsis thaliana miR319a precursor fused to a constitutive promoter. Transgenic Physcomitrella lines harboring the overexpression constructs showed precise processing of the PpFtsZ2-1-amiRNA and PpGNT1-amiRNA, and an efficient knock-down of the PpFtsZ2-1 and PpGNT1 genes. Furthermore, chloroplast division was impeded in PpFtsZ2-1-amiRNA lines which phenocopied PpFtsZ2-1 knockout mutants. We also provide evidence for the amplification of the initial amiRNA signal by secondary transitive siRNAs, although these siRNAs do not seem to have a major effect on the sequence-related PpFtsZ2-2 mRNA, confirming specificity of the amiRNA approach. We provide evidence that amiRNAs can be used as an alternative tool for studying gene functions in Physcomitrella patens

Effect of nitrogen form, plant spacing and water regime on lettuce plants (Lactuca sativa L.)

A field study was conducted in Jordan Valley and Al-Jubeiha, Jordan, during 2000/2001 to study the optimum planting density, form of nitrogen and irrigation regime for lettuce cv. Amar. Seeds were sown one month before transplanting. A total of 100 kg pure N/ha was applied in the form of Ca(NO3)2, (NH4)2SO4, and CO(NH2)2. The N fertilizer was applied at 3 different times during plant development (25 kg N/ha applied at 3 weeks after transplanting, 25 kg N/ha applied at 5 weeks after transplanting, and 50 kg N/ha applied at 7 weeks after transplanting), and in-row spacing of 15, 20 and 25 cm were used. Two irrigation regimes were evaluated in the trials, i.e. regime 1 (~40 litre m-2 week-1, which was higher by two-fold than the amount of irrigation supplied under regime 2) and regime 2. At harvesting, the vertical and horizontal diameters of the head, number of leaves, leaf area, fresh and dry weights of head, and total fresh yield were determined. The effect of N form on production and vegetative parameters of lettuce head (vertical and horizontal diameter, number of leaves, leaf area, and fresh and dry weights) followed the order (NH4)2SO4 > Ca(NO3)2 > CO(NH2)2. Plant spacing also had a highly significant (p<0.01) effect on vegetative and yield components, but a spacing of 20 cm apart generally showed the highest values, followed by 25 cm then 15 cm. Regarding the effect of irrigation regime on vegetative and yield components, the amount of irrigation water had induced significant differences among the values of these parameters. The plants irrigated under regime 1 exhibited greater growth response than those irrigated under regime 2. Lettuce showed the best response under (NH4)2SO4 + 20 cm spacing + 1 irrigation under regime 1

Expression of artificial microRNAs in Physcomitrella patens

MicroRNAs (miRNAs) are ~21nt long small RNAs transcribed from endogenous MIR genes which form precursor RNAs with a characteristic hairpin structure. MiRNAs control the expression of cognate target genes by binding to reverse complementary sequences resulting in cleavage or translational inhibition of the target RNA. Artificial miRNAs (amiRNAs) can be generated by exchanging the miRNA/miRNA* sequence of endogenous MIR precursor genes, while maintaining the general pattern of matches and mismatches in the foldback. Thus, for functional gene analysis amiRNAs can be designed to target any gene of interest

Physcomitrella patens small RNA pathways

Small, non-coding RNAs (sRNAs) are a distinct class of regulatory RNAs in plants and animals controlling a variety of biological processes. Given the great impact of sRNAs in biology, recent studies in model seed plant species, particu-larly in A. thaliana, focused on the identification, biogenesis and functional analysis of sRNAs. In seed plants, several classes of sRNAs with specific sizes and dedicated functions have evolved through a series of pathways, namely microR-NAs (miRNAs), repeat-associated small interfering RNAs (ra-siRNAs), natural antisense transcript-derived small interfering RNAs (nat-siRNAs), and trans-acting small interfering RNAs (ta-siRNAs). In the last years, the analysis of plant sRNA pathways was extended to the bryophyte P. patens, a non-flowering, non-vascular ancient land plant, that diverged from the lineage of seed plants approxi-mately 450 million years ago. Based on a number of characteristic features and its phylogenetic key position in land plant evolution P. patens emerged as a plant model species to address basic as well as applied topics in plant biology. The analysis of P. patens sRNA pathways was recently advanced by the deep sequencing of sRNA libraries, the release of the P. patens genome that allowed the mapping of sRNA producing loci, and first molecular analyses of P. patens mutants with targeted disruption of genes encoding essential components of endogenous sRNA pathways. Even though the major sRNA pathways are evolutionarily conserved in P. patens there are particular differences in the functional components of sRNA pathways and the biological function of sRNAs. These include a specific amplification of initial miRNA and ta-siRNA signals by the generation of transitive siRNAs, deviating functions and specificities of DICER-LIKE proteins and an epigenetic gene silencing pathway that is triggered by miRNAs. Further, the conservation of miRNA biogenesis in P. patens was used to establish specific gene silencing by the expression of artificial miRNAs suited for functional gene analysis by reverse genetics approaches. These findings underline that P. patens serves as a valuable model system to study the evolution, diversity, and function of plant sRNAs. Here we summarise the current knowledge on different sRNA biogenesis pathways, their biological relevance and the expres-sion of artificial miRNAs in P. patens

Identification and Analysis of Red Sea Mangrove (<i>Avicennia marina</i>) microRNAs by High-Throughput Sequencing and Their Association with Stress Responses

<div><p>Although RNA silencing has been studied primarily in model plants, advances in high-throughput sequencing technologies have enabled profiling of the small RNA components of many more plant species, providing insights into the ubiquity and conservatism of some miRNA-based regulatory mechanisms. Small RNAs of 20 to 24 nucleotides (nt) are important regulators of gene transcript levels by either transcriptional or by posttranscriptional gene silencing, contributing to genome maintenance and controlling a variety of developmental and physiological processes. Here, we used deep sequencing and molecular methods to create an inventory of the small RNAs in the mangrove species, <i>Avicennia marina</i>. We identified 26 novel mangrove miRNAs and 193 conserved miRNAs belonging to 36 families. We determined that 2 of the novel miRNAs were produced from known miRNA precursors and 4 were likely to be species-specific by the criterion that we found no homologs in other plant species. We used qRT-PCR to analyze the expression of miRNAs and their target genes in different tissue sets and some demonstrated tissue-specific expression. Furthermore, we predicted potential targets of these putative miRNAs based on a sequence homology and experimentally validated through endonucleolytic cleavage assays. Our results suggested that expression profiles of miRNAs and their predicted targets could be useful in exploring the significance of the conservation patterns of plants, particularly in response to abiotic stress. Because of their well-developed abilities in this regard, mangroves and other extremophiles are excellent models for such exploration.</p> </div