Andrzej Pacak

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

Summary Selected barley miRNAs and their targets are regulated upon heat stress. Splicing of introns carrying miRNAs was induced by heat and correlated with the accumulation of mature miRNA.

Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in Brachypodium distachyon and oat.

BackgroundGene silencing vectors based on Barley stripe mosaic virus (BSMV) are used extensively in cereals to study gene function, but nearly all studies have been limited to genes expressed in leaves of barley and wheat. However since many important aspects of plant biology are based on root-expressed genes we wanted to explore the potential of BSMV for silencing genes in root tissues. Furthermore, the newly completed genome sequence of the emerging cereal model species Brachypodium distachyon as well as the increasing amount of EST sequence information available for oat (Avena species) have created a need for tools to study gene function in these species.ResultsHere we demonstrate the successful BSMV-mediated virus induced gene silencing (VIGS) of three different genes in barley roots, i.e. the barley homologues of the IPS1, PHR1, and PHO2 genes known to participate in Pi uptake and reallocation in Arabidopsis. Attempts to silence two other genes, the Pi transporter gene HvPht1;1 and the endo-β-1,4-glucanase gene HvCel1, in barley roots were unsuccessful, probably due to instability of the plant gene inserts in the viral vector. In B. distachyon leaves, significant silencing of the PHYTOENE DESATURASE (BdPDS) gene was obtained as shown by photobleaching as well as quantitative RT-PCR analysis. On the other hand, only very limited silencing of the oat AsPDS gene was observed in both hexaploid (A. sativa) and diploid (A. strigosa) oat. Finally, two modifications of the BSMV vector are presented, allowing ligation-free cloning of DNA fragments into the BSMV-γ component.ConclusionsOur results show that BSMV can be used as a vector for gene silencing in barley roots and in B. distachyon leaves and possibly roots, opening up possibilities for using VIGS to study cereal root biology and to exploit the wealth of genome information in the new cereal model plant B. distachyon. On the other hand, the silencing induced by BSMV in oat seemed too weak to be of practical use. The new BSMV vectors modified for ligation-free cloning will allow rapid insertion of plant gene fragments for future experiments.

Arabidopsis microRNA expression regulation in a wide range of abiotic stress responses

Arabidopsis microRNA expression regulation was studied in a wide array of abiotic stresses such as drought, heat, salinity, copper excess/deficiency, cadmium excess, and sulfur deficiency. A home-built RT-qPCR mirEX platform for the amplification of 289 Arabidopsis microRNA transcripts was used to study their response to abiotic stresses. Small RNA sequencing, Northern hybridization, and TaqMan® microRNA assays were performed to study the abundance of mature microRNAs. A broad response on the level of primary miRNAs (pri-miRNAs) was observed. However, stress response at the level of mature microRNAs was rather confined. The data presented show that in most instances, the level of a particular mature miRNA could not be predicted based on the level of its pri-miRNA. This points to an essential role of posttranscriptional regulation of microRNA expression. New Arabidopsis microRNAs responsive to abiotic stresses were discovered. Four microRNAs: miR319a/b, miR319b.2, and miR400 have been found to be responsive to several abiotic stresses and thus can be regarded as general stress-responsive microRNA species.

Organellar inheritance in liverworts: An example of Pellia borealis

Liverwort Pellia borealis is an allopolyploid species that originated after the hybridization and chromosome doubling of two cryptic species; Pellia epiphylla species N and Pellia epiphylla species S. A sequence comparison of chloroplast tRNAUCCGly, tRNAUUULys gene introns, the mitochondrial tRNAGCUSer gene intron, and the first intron of the coxIII gene in the case of three liverwort species studied revealed that the chloroplast and mitochondrial sequences are identical in P. borealis and P. epiphylla species N but different from homologous P. epiphylla species S sequences. Both mitochondria and chloroplasts of P. borealis were thus inherited from one parent—P. epiphylla species N. Studies on 14 different populations of P. borealis gave the same result. These are the first data on organellar transmission in liverworts, the earliest land plants. Moreover, we show that the intron sequences of some organellar genes, until now not used in any systematic studies, could be very good markers in studying taxonomic relationships in closely related species and reconstructing historical events.

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.

Developmentally regulated expression and complex processing of barley pri-microRNAs

BackgroundMicroRNAs (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.ResultsTo 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.ConclusionsSeven 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.

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

BackgroundMicroRNAs 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.DescriptionThe 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.ConclusionsThe 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 Role of the P1BS Element Containing Promoter-Driven Genes in Pi Transport and Homeostasis in Plants

Inorganic phosphate (Pi) is an easily accessible form of phosphorus for plants. Plant Pi uptake is usually limited however by slow Pi diffusion through the soil which strongly adsorps phosphate species. Plants have developed mechanisms to increase Pi availability. There are also abiotic (phosphate level) and biotic (e.g., mycorrhizal) factors regulating the expression of Pi-responsive genes. Transcription factors binding to the promoters of Pi-responsive genes activate different pathways of Pi transport, distribution, and homeostasis maintenance. Pi metabolism involves not only functional proteins but also microRNAs and other non-coding RNAs.

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.

Organellar inheritance in the allopolyploid moss Rhizomnium pseudopunctatum

Earlier isozyme studies have proved that the moss Rhizomnium pseudopunctatum is an allopolyploid species whose progenitors are the haploid species R. magnifolium and R. gracile. A sequence comparison of chloroplast tRNA L e u (UAA) and tRNA G l y (UCC) gene introns, as well as mitochondrial fragments of nad5 and nad4 gene introns and exons in all three species reveals that the nucleotide sequences studied are almost identical in R. magnifolium and R. pseudopunctatum but differ in R. gracile. Both chloroplasts and mitochondria of R. pseudopunctatum were therefore probably inherited from one parent: R. magnifolium. To our knowledge, this is the first report of uniparental transmission of organelles in mosses.