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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 theirmRNA 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 fertilizerbased 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 β-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. Key Message: The 5′-UTR of the barley PHOSPHATE 2 gene contains two P1BS motifs that can bind the transcription factor (TF) PHR1 (PHOSPHATE STARVATION RESPONSE 1) and the newly identified TF APL (ALTERED PHLOEM DEVELOPMENT) [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

CBP20 (Cap-Binding Protein 20) encodes a small subunit of the cap-binding complex (CBC), which is involved in the conserved cell processes related to RNA metabolism in plants and, simultaneously, engaged in the signaling network of drought response, which is dependent on ABA. Here, we report the enhanced tolerance to drought stress of barley mutant in the HvCBP20 gene manifested at the morphological, physiological, and transcriptomic levels. Physiological analyses revealed differences between the hvcbp20.ab mutant and its WT in response to a water deficiency. The mutant exhibited a higher relative water content (RWC), a lower stomatal conductance and changed epidermal pattern compared to the WT after drought stress. Transcriptome analysis using the Agilent Barley Microarray integrated with observed phenotypic traits allowed to conclude that the hvcbp20.ab mutant exhibited better fitness to stress conditions by its much more efficient and earlier activation of stress-preventing mechanisms. The network hubs involved in the adjustment of hvcbp20.ab mutant to the drought conditions were proposed. These results enabled to make a significant progress in understanding the role of CBP20 in the drought stress response. [ABSTRACT FROM AUTHOR]

Phosphorus (P) in plants is taken from soil as an inorganic phosphate (Pi) and is one of the most important macroelements in growth and development. Plants actively react to Pi starvation by the induced expression of Pi transporters, MIR399, MIR827, and miR399 molecular sponge – IPS1 genes and by the decreased expression of the ubiquitin-conjugating enzyme E2 (PHOSPHATE2 – PHO2) and Pi sensing and transport SPX-MFS genes. The PHO2 protein is involved in the degradation of Pi transporters PHT1;1 (from soil to roots) and PHO1 (from roots to shoots). The decreased expression of PHO2 leads to Pi accumulation in shoots. In contrast, the pho1 mutant shows a decreased level of Pi concentration in shoots. Finally, Pi starvation leads to decreased Pi concentration in all plant tissues. Little is known about plant Pi homeostasis in other abiotic stress conditions. We found that, during the first hour of heat stress, Pi accumulated in barley shoots but not in the roots, and transcriptomic data analysis as well as RT-qPCR led us to propose an explanation for this phenomenon. Pi transport inhibition from soil to roots is balanced by lower Pi efflux from roots to shoots directed by the PHO1 transporter. In shoots, the PHO2 mRNA level is decreased, leading to an increased Pi level. We concluded that Pi homeostasis in barley during heat stress is maintained by dynamic changes in Pi-related genes expression. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.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. [ABSTRACT FROM AUTHOR]

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 30 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Gene 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. Results: Here 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-g component. Conclusions: Our 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. [ABSTRACT FROM AUTHOR]

Previous studies using isozymes have established that the moss Plagiomnium curvatulum is an allopolyploid species that originated from hybridization and chromosome doubling of Plagiomnium elatum and Plagiom- nium ellipticum. It has so far remained unknown which species is the donor of chioroplasts and mitochondria to P. curvatulum. Studies of organellar inheritance in bryophytes suggest that uniparental transmission was involved. Comparison of nucleotide sequence data of three chloroplast and three mitochondrial DNA markers from P. curvatulum and its parental species reveal that the chloroplast and mitochondrial sequences of P. cur- vatulum and P. ellipticum are identical and differ from homologous sequences of P. datum. Seven samples from five different populations of P. curvatulum all gave the same result. Both mitochondria and chloroplasts of P. curvatulum were inherited from P. ellipticum. The nucleotide substitution rate calculated for homologous organellar nucleotide sequences is 4.57 times higher in chloroplasts than in mitochondria. Moreover, substi- tution rates differ considerably between chloroplast and mitochondrial homologous intron sequences when allopolyploid species and their parental haploids from different genera of bryophytes are compared. [ABSTRACT FROM AUTHOR]

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 tRNALeu(UAA) and tRNAGly(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. [ABSTRACT FROM AUTHOR]

The study was undertaken as an attempt at explaining interrelations between the presently found much diversified isoforms of ACC synthase, a key enzyme of the ethylene synthesis pathway in higher plants. The results of our study analysed on a background of current knowledge implied some not yet discussed, physiological and evolutionary aspects of the plant ACS isozymes. The computer methods applied are based on analysis of phenetic data. The subjects of the study were 159 synthases from higher plants and the only one known from the fungus Penicillium citrinum. The phenograms of 95 full-length sequence ACC synthases from higher plants and the synthase from Penicillium citrinum were made for cDNA and polypeptides by the two different techniques: UPGMA and Kitsch’s method and were almost identical. They indicate the presence of three significantly different types of ACS plant isozymes denoted as type A, type B and A3 group. A number of arguments are given showing that the synthases denoted as A1 and A2 group, hitherto treated by many authors as separate evolutionary lineages, are of the same type A. The presence of a new poorly recognised and probably evolutionary separate group of synthases, denoted as A3, is for the first time evidenced. The type B isozymes are shown to comprise two distinct groups B1 and B2, and B1 group can be divided into subgroups. A comparison of the proportion of genes encoding different type synthases in taxa distinguished by molecular systematicians has shown that similar sets and numbers of genes of type A and A3 group synthases are conserved in the genomes of eudicots and noneudicots. In the genomes of noneudicots the genes of B1 (B1a and B1b) group synthaseswere not found. The genes of B1(B1a and B1b) group in full diversity were established to occur in Rosidae and as B1a subgroup in Asteridae, the subclasses representing eudicots. It is evidenced that the C-terminal region of the enzyme, hitherto treated as highly variable, and the last aa residue are conserved within the B type and A1 synthases. The acidic character of the polypeptides and the lack of conservation of the last aa residue in the synthases of A2 group are explained by the loss of the 3′-terminal fragment by some A type genes. The C-terminal region of B type and A1 group synthases was found to contain a fragment similar to the so-called MAPK-docking domain, which suggests that these isozymes are controlled by the processes of phosphorylation. The aa residues forming the catalytically important three-dimensional structure called the hydrophobic pocket for the adenine ring of SAM can differ slightly in the particular type or groups of ACS. Moreover, A3 group differs from the other synthase groups by the aa residues required for correct orientation of PLP in the active site. Probably, particular ACS groups can differ in the kinetic features and the half-life. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The random amplified polymorphic DNA (RAPD) technique was used to study the genomic relationships of three liverworts from the genus Pellia: P. epiphylla, P. borealis and P. neesiana. Altogether 150 characters (150 DNA fragments obtained using PCR) were scored. These characters were used to create a matrix of pairwise distances between all the pairs of taxa. Both distance (UPGMA, Fitch–Margoliash and Neighbor–Joining) and binary character state (Wagner and Camin Sokal parsimony and compatibility) methods were applied for trees' construction. Our results strongly support distinction of the recently discovered sibling species of P. epiphylla – species N and P. epiphylla – species S, which have an allopatric distribution in Poland (N – North, S – South Poland). Moreover, our data also supports the hypothesis of a hybrid origin (alloploid) of the polyploid P. borealis from P.epiphylla−N×P.epiphylla−S. P. neesiana was excluded as a donor of either of the genomes of P. borealis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]