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MicroRNAs (miRNAs) are ~21 nt 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) ca...
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The Vittiaceae are a small family of aquatic mosses that are defined based on gametophytic traits whose interpretation has led to conflicting taxonomic arrangements. Phylogenetic analyses of two cpDNA regions, trnL-trnF and atpB-rbcL, indicate that Vittia is nested within the Amblystegiaceae s. str., suggesting that the family Vittiaceae should not...
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... Additionally, Species or lineage-specific stress-regulated genes not found in higher plants suggest that these unknown and un-classified transcripts might represent valuable targets for crop plant improvement in a changing climate via targeted gene replacement. 39 . Freshly disrupted protonemata were sub-cultured for 5 days and transferred to fresh medium and cultured over night in Erlenmeyer flasks on shakers at 125 rpm. ...
Changes in the environment, such as those caused by climate change, can exert stress
on plant growth, diversity and ultimately global food security. Thus, focused
efforts to fully understand plant response to stress are urgently needed in order to
develop strategies to cope with the effects of climate change. Because
Physcomitrella patens holds a key evolutionary position bridging the gap
between green algae and higher plants, and because it exhibits a well-developed
stress tolerance, it is an excellent model for such exploration. Here, we have used
Physcomitrella patens to study genome-wide responses to abiotic stress
through transcriptomic analysis by a high-throughput sequencing platform. We report
a comprehensive analysis of transcriptome dynamics, defining profiles of elicited
gene regulation responses to abiotic stress-associated hormone Abscisic Acid (ABA),
cold, drought, and salt treatments. We identified more than 20,000 genes expressed
under each aforementioned stress treatments, of which 9,668 display differential
expression in response to stress. The comparison of Physcomitrella patens
stress regulated genes with unicellular algae, vascular and flowering plants
revealed genomic delineation concomitant with the evolutionary movement to land,
including a general gene family complexity and loss of genes associated with
different functional groups.
... This allows us to modify miRNA sequences and to create artificial miRNAs (amiRNAs) that are able to target any gene of interest and to knockdown its expression at the post-transcriptional level. This method was successfully applied in different seed plants [96][97][98][99] and subsequently adapted for specific gene knockdown in Physcomitrella [100,101]. ...
Small, non-coding RNAs are a distinct class of regulatory RNAs in plants and animals that control a variety of biological processes. In plants, several classes of small RNAs with specific sizes and dedicated functions have evolved through a series of pathways. The major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis. 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 a similar structure, function, and biogenesis as miRNAs, but are derived from long double-stranded RNAs and can often direct DNA methylation at target sequences. Environmental stress factors such as drought, elevated temperature, salinity, and rising carbon dioxide (CO 2) levels affect plant growth and pose a growing threat to sustainable agriculture. This has become a hot issue due to concerns about the effects of climate change on plant resources, biodiversity, and global food security. Besides the roles of small RNAs in growth, development, and maintenance of genome integrity, small RNAs are also important components in plant stress responses. One way in which plants respond to environmental stress is by modifying their gene expression through the use of small RNAs. Thus, understanding how small RNAs regulate gene expression will enable researchers to explore the role of small RNAs in abiotic stress responses for adapting to climate change. Here, we present an overview of small RNA-mediated plant improvement under a changing climate.
... Small RNAs with specific sizes and functions have been identified by high-throughput sequencing in diverse plant species, including Arabidopsis, rice, barley, peanuts, grapevine, olive, Medicago and other plants[13][14][15][16][17][18][19][20][21][22][23][24][25][26]. Comparative studies on small RNAs across species have revealed the existence of highly conserved miRNAs and siRNAs with important regulatory functions in both plants and animals[27,28]. Small RNAs are derived from partially double-stranded RNA (dsRNA) precursors by the action of ribonuclease III-family enzymes designated Dicers and Dicer-like (DCL) proteins[3,29]. The small RNA duplexes generated by Dicer activity have a characteristic 2-nucleotide overhang at the 39 end due to offset cleavage of the complementary strands by Dicers and DCLs. ...
... In this study microRNAs in Red Sea MangrovemiRNA target genes were computationally predicted for both conserved and candidate novel miRNAs, the expression of these miRNAs should cause cleavage of the cognate mRNAs within the region complementary to the miRNA sequences. Target sites in plant mRNAs normally share high sequence complementarity to the respective miRNA[2,27,28]. Normally, in plants, cleavage within a target transcript that is mediated by a 21-nt miRNA occurs between positions 10 and 11 with respect to the miRNA sequence[2,27,28]. To prove this, we performed 59RACE-PCRs to detect specific mRNA cleavage products. ...
... Target sites in plant mRNAs normally share high sequence complementarity to the respective miRNA[2,27,28]. Normally, in plants, cleavage within a target transcript that is mediated by a 21-nt miRNA occurs between positions 10 and 11 with respect to the miRNA sequence[2,27,28]. To prove this, we performed 59RACE-PCRs to detect specific mRNA cleavage products. ...
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, Avicennia marina. 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.
... In addition to GT, gene knockdown can also be obtained via gene silencing techniques such as RNA interference ( 33 ) , or amiRNA ( 34,35 ) . Recently, a system based on RNA interference has been described in order to screen rapidly for temperature sensitive alleles of a given gene ( 36 ) . ...
In this chapter, we review the main organogenesis features and associated regulation processes of the moss Physcomitrella patens (P. patens), the model plant for the Bryophytes. We highlight how the study of this descendant of the earliest plant species that colonized earth, brings useful keys to understand the mechanisms that determine and control both vascular and non vascular plants organogenesis. Despite its simple morphogenesis pattern, P. patens still requires the fine tuning of organogenesis regulators, including hormone signalling, common to the whole plant kingdom, and which study is facilitated by a high number of molecular tools, among which the powerful possibility of gene targeting/replacement. The recent discovery of moss cells reprogramming capacity completes the picture of an excellent model for studying plant organogenesis.
Evolutionary history of angiosperms illustrates extensive and recurrent whole genome duplication (WGD) events. A direct consequence of WGD is establishment of multiple notional sub-genomes within the polyploid cytotypes accompanied with an overall increase in gene copies known as homeologs. Even in diploids, prevalence of multiple, redundantly functioning gene copies is not unusual and is reminiscent of ancient genome duplication events. Functional analysis of such redundant genes poses challenges while using conventional loss- and gain-of-function approaches. Whereas loss-of-function approaches involving withdrawal of gene function entail recombining homozygous mutant alleles at multiple homeologous loci, serial analysis of gain-of-function mutants generated by over-expressing individual gene copies is cumbersome yet important for delineating homeolog-wise contribution to the phenotype. Development of transgene-based gene silencing technologies provided useful alternatives for functional genomics in polyploids. MicroRNAs and small interfering RNAs (siRNAs) were discovered as key regulators of gene expression based on their ability to base-pair with transcripts in a sequence-specific manner. Such a binding down-regulates target genes via transcript cleavage or translation repression, hence the term RNA interference (RNAi). Since mutants mimic loss-of-function phenotypes, siRNA-based gene silencing tools were initially applied in functional genomics and trait modification in plants. However, such prototypes of RNAi technology suffer from widespread off-target silencing. Artificial miRNA-based silencing platform was designed to enhance specificity and minimize off-target silencing for achieving systematic characterization of genes. Through a flexible format, artificial miRNA technology permits specific and efficient silencing of a single or multiple genes by modulating the spectrum of target transcripts. Furthermore, instances of “off-target” silencing are minimized since homogenous population of mature miRNA are more precise relative to heterogenous siRNAs. In refined versions, suitable promoters have been used to regulate expression of artificial miRNAs in select spatio-temporal domains. Constant addition of novel features underpins evolution of artificial miRNA technology and justify its adoption in large-scale gene function studies and trait manipulation. Herein, we provide an overview of the genesis and application of artificial miRNAs to illustrate the impact of the technology over a decade in plant research and crop improvement.
Synthetic Biology is an interdisciplinary approach combining biotechnology, evolutionary biology, molecular biology, systems biology and biophysics. While the exact definition of Synthetic Biology might still be debatable, its focus on design and construction of biological devices that perform useful functions is clear and of great utility to engineering algae. This relies on the re-engineering of biological circuits and optimization of certain metabolic pathways to reprogram algae and introduce new functions in them via the use of genetic modules. Genetic editing tools are primary enabling techniques in Synthetic Biology and this chapter discusses common techniques that show promise for algal gene editing. The genetic editing tools discussed in this chapter include RNA interference (RNAi) and artificial microRNAs, RNA scaffolds, transcription activator-like effector nucleases (TALENs), RNA guided Cas9 endonucleases (CRISPR), and multiplex automated genome engineering (MAGE). DNA and whole genome synthesis is another enabling technology in Synthetic Biology and might present an alternative approach to drastically and readily modify algae. Clear and powerful examples of the potential of whole genome synthesis for algal engineering are presented. Also, the development of relevant computational tools, and genetic part registries has stimulated further advancements in the field and their utility in algal research and engineering is described. For now, the majority of synthetic biology efforts are focused on microbes as many pressing problems, such as sustainability in food and energy production rely on modification of microorganisms. Synthetic modifications of algal strains to enhance desired physiological properties could lead to improvements in their utility.
In contrast to hairpin RNAs, in which heterogeneous small RNAs are processed from double-stranded RNA to have potential off-target effects on endogenous other genes, artificial miRNAs (amiRNAs) have advantages of exquisite specificity and non-transitivity to thus target individual genes and groups of endogenous genes. Earlier studies showed that amiRNA engineering based on osa-miRNA528 precursor could efficiently trigger endogenous gene silencing and modulate agronomic traits in rice. However, both the expression efficiency of heterologous amiRNAs based on osa-miRNA528 precursor and the correlation of copy number with the relative expression level of amiRNAs remain unknown. In the present study, five amiRNAs (S9-1174, S9-1192, S11-864, S11-868 and S11-869) targeting different sites of S9 and S11 negative strands in rice dwarf virus (RDV) genome were constructed using endogenous osa-miRNA528 precursor as backbone. After identification by Northern blot, two amiRNAs (S9-1174 and S9-1192) targeting S9 negative strand in RDV genome were highly expressed, whereas in three tested amiRNAs targeting S11 negative strand in RDV genome, only two amiRNAs (S11-868 and S11-869) were processed efficiently. T0 generation transgenic rice containing amiRNAs (S9-1174, S9-1192, S11-868 and S11-869) exhibited different expression ratios of amiRNAs, accounting for 90.0, 90.0, 66.7 and 77.8 %, respectively. In addition, combination analysis with the relative amiRNA expression levels and its copy number revealed that the relative expression levels of amiRNAs had no relation to the copy number of T-DNA insert in transgenic rice.
Virus-induced gene silencing using artificial microRNAs (MIR VIGS) is a newly developed technique for plant reverse genetic studies. Traditional virus-induced gene silencing (VIGS) assays introduce a large gene fragment, which is expressed and then converted into small RNAs by the endogenous siRNA-based gene silencing machinery of the plant host. By contrast, MIR VIGS uses well-designed miRNAs to induce RNA-mediated silencing of the target gene. Using a single artificial miRNA can provide greater specificity by reducing off-target effects. Here, we describe a detailed protocol for MIR VIGS in Nicotiana benthamiana using a modified Cabbage leaf curl virus (CaLCuV)-based vector.
