Search

Despite knowledge of classical coronary artery disease (CAD) risk factors, the morbidity and mortality associated with this disease remain high. Therefore, new factors that may affect the development of CAD, such as the gut microbiome, are extensively investigated. This study aimed to evaluate gut microbiome composition in CAD patients in relation to the control group. We examined 169 CAD patients and 166 people in the control group, without CAD, matched in terms of age and sex to the study group. Both populations underwent a detailed health assessment. The microbiome analysis was based on the V3–V4 region of the 16S rRNA gene (NGS method). Among 4074 identified taxonomic units in the whole population, 1070 differed between study groups. The most common bacterial types were Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria. Furthermore, a higher Firmicutes/Bacteroidetes ratio in the CAD group compared with the control was demonstrated. Firmicutes/Bacteroidetes ratio, independent of age, sex, CAD status, LDL cholesterol concentration, and statins treatment, was related to altered phosphatidylcholine concentrations obtained in targeted metabolomics. Altered alpha-biodiversity (Kruskal–Wallis test, p = 0.001) and beta-biodiversity (Bray–Curtis metric, p &lt, 0.001) in the CAD group were observed. Moreover, a predicted functional analysis revealed some taxonomic units, metabolic pathways, and proteins that might be characteristic of the CAD patients’ microbiome, such as increased expressions of 6-phospho-β-glucosidase and protein-N(pi)-phosphohistidine-sugar phosphotransferase and decreased expressions of DNA topoisomerase, oxaloacetate decarboxylase, and 6-beta-glucosidase. In summary, CAD is associated with altered gut microbiome composition and function.

Environmental stress is one of the most important factors affecting plant growth and development. Recent studies have shown that epigenetic mechanisms, such as DNA methylation, play a key role in adapting plants to stress conditions. Here, we analyzed the dynamics of changes in the level of DNA methylation in Arabidopsis thaliana (L.) Heynh. (Brassicaceae) under the influence of heat stress. For this purpose, whole-genome sequencing of sodium bisulfite-treated DNA was performed. The analysis was performed at seven time points, taking into account the control conditions, heat stress, and recovery to control conditions after the stress treatment was discontinued. In our study we observed decrease in the level of DNA methylation under the influence of heat stress, especially after returning to control conditions. Analysis of the gene ontology enrichment and regulatory pathways showed that genes characterized by differential DNA methylation are mainly associated with stress response, including heat stress. These are the genes encoding heat shock proteins and genes associated with translation regulation. A decrease in the level of DNA methylation in such specific sites suggests that under the influence of heat stress we observe active demethylation phenomenon rather than passive demethylation, which is not locus specific.

Personalized and precision medicine is gaining recognition due to the limitations by standard diagnosis and treatment; many areas of medicine, from cancer to psychiatry, are moving towards tailored and individualized treatment for patients based on their clinical characteristics and genetic signatures as well as novel imaging techniques. Advances in whole genome sequencing have led to identification of genes involved in a variety of diseases. Moreover, biomarkers indicating severity of disease or susceptibility to treatment are increasingly being characterized. The continued identification of new genes and biomarkers specific to disease subtypes and individual patients is essential and inevitable for translation into personalized medicine, in estimating both, disease risk and response to therapy. Taking into consideration the mostly unsolved necessity of tailored therapy in oncology the innovative project MOBIT (molecular biomarkers for individualized therapy) was designed. The aims of the project are: (i) establishing integrative management of precise tumor diagnosis and therapy including systematic biobanking, novel imaging techniques, and advanced molecular analysis by collecting comprehensive tumor tissues, liquid biopsies (whole blood, serum, plasma), and urine specimens (supernatant; sediment) as well as (ii) developing personalized lung cancer diagnostics based on tumor heterogeneity and integrated genomics, transcriptomics, metabolomics, and radiomics PET/MRI analysis. It will consist of 5 work packages. In this paper the rationale of the Polish MOBIT project as well as its design is presented. (iii) The project is to draw interest in and to invite national and international, private and public, preclinical and clinical initiatives to establish individualized and precise procedures for integrating novel targeted therapies and advanced imaging techniques.

Root systems play a pivotal role in coupling with drought stress, which is accompanied with a substantial transcriptome rebuilding in the root tissues. Here, we present the results of global gene expression profiling of roots of two barley genotypes with contrasting abilities to cope with drought that were subjected to a mild level of the stress. We concentrate our analysis on gene expression regulation processes, which allowed the identification of 88 genes from 39 families involved in transcriptional regulation in roots upon mild drought. They include 13 genes encoding transcription factors (TFs) from AP2 family represented by ERFs, DREB, or B3 domain-containing TFs, eight WRKYs, six NACs, five of the HD-domain, MYB or MYB-related, bHLH and bZIP TFs. Also, the representatives of C3H, CPP, GRAS, LOB-domain, TCP, Tiffy, Tubby, and NF-Ys TFs, among others were found to be regulated by the mild drought in barley roots. We found that drought tolerance is accompanied with a lower number of gene expression changes than the amount observed in a susceptible genotype. The better drought acclimation may be related to the activation of transcription factors involved in the maintenance of primary root growth and in the epigenetic control of chromatin and DNA methylation. In addition, our analysis pointed to fives TFs from ERF, LOB, NAC, WRKY and bHLH families that may be important in the mild but not the severe drought response of barley roots.

Environmental stress is one of the most important factors affecting plant growth and development. Recent studies have shown that epigenetic mechanisms, such as DNA methylation, play a key role in adapting plants to stress conditions. Here, we analyzed the dynamics of changes in the level of DNA methylation in Arabidopsis thaliana (L.) Heynh. (Brassicaceae) under the influence of heat stress. For this purpose, whole-genome sequencing of sodium bisulfite-treated DNA was performed. The analysis was performed at seven time points, taking into account the control conditions, heat stress, and recovery to control conditions after the stress treatment was discontinued. In our study we observed decrease in the level of DNA methylation under the influence of heat stress, especially after returning to control conditions. Analysis of the gene ontology enrichment and regulatory pathways showed that genes characterized by differential DNA methylation are mainly associated with stress response, including heat stress. These are the genes encoding heat shock proteins and genes associated with translation regulation. A decrease in the level of DNA methylation in such specific sites suggests that under the influence of heat stress we observe active demethylation phenomenon rather than passive demethylation, which is not locus specific.

Recent studies have led to considerable advances in the identification of genetic variants associated with type 1 and type 2 diabetes. An approach for converting genetic data into a predictive measure of disease susceptibility is to add the risk effects of loci into a polygenic risk score. In order to summarize the recent findings, we conducted a systematic review of studies comparing the accuracy of polygenic risk scores developed during the last two decades. We selected 15 risk scores from three databases (Scopus, Web of Science and PubMed) enrolled in this systematic review. We identified three polygenic risk scores that discriminate between type 1 diabetes patients and healthy people, one that discriminate between type 1 and type 2 diabetes, two that discriminate between type 1 and monogenic diabetes and nine polygenic risk scores that discriminate between type 2 diabetes patients and healthy people. Prediction accuracy of polygenic risk scores was assessed by comparing the area under the curve. The actual benefits, potential obstacles and possible solutions for the implementation of polygenic risk scores in clinical practice were also discussed. Develop strategies to establish the clinical validity of polygenic risk scores by creating a framework for the interpretation of findings and their translation into actual evidence, are the way to demonstrate their utility in medical practice.

Detailed DNA methylation analyses in plant species with large and highly repetitive genomes can be challenging as well as costly. Here, we describe a complete protocol for a high-throughput DNA methylation changes analysis using Methylation-Sensitive Amplification Polymorphism Sequencing (MSAP-Seq; Chwialkowska et al., Front Plant Sci. 8: 2056 (2017)). This method allows detailed information about DNA methylation changes in large and complex genomes to be obtained at a relatively low cost. MSAP-Seq is based on conventional MSAP marker analysis and employs all its basic steps such as restriction cleavage with methylation-sensitive restriction enzyme, ligation of universal adapters, and PCR amplification. However, the traditional gel-based amplicon separation is replaced by direct, global sequencing with next-generation sequencing (NGS) methods. Consequently, MSAP-Seq allows for parallel analysis of hundreds of thousands of different CCGG sites and evaluation of their DNA methylation state. This technique especially targets to genic regions, so it is well suited for large genomes with low gene density, such as barley and other plants with large genomes.

Detailed DNA methylation analyses in plant species with large and highly repetitive genomes can be challenging as well as costly. Here, we describe a complete protocol for a high-throughput DNA methylation changes analysis using Methylation-Sensitive Amplification Polymorphism Sequencing (MSAP-Seq; Chwialkowska et al., Front Plant Sci. 8: 2056 (2017)). This method allows detailed information about DNA methylation changes in large and complex genomes to be obtained at a relatively low cost. MSAP-Seq is based on conventional MSAP marker analysis and employs all its basic steps such as restriction cleavage with methylation-sensitive restriction enzyme, ligation of universal adapters, and PCR amplification. However, the traditional gel-based amplicon separation is replaced by direct, global sequencing with next-generation sequencing (NGS) methods. Consequently, MSAP-Seq allows for parallel analysis of hundreds of thousands of different CCGG sites and evaluation of their DNA methylation state. This technique especially targets to genic regions, so it is well suited for large genomes with low gene density, such as barley and other plants with large genomes.

Highlight Characterization of barley methylome modulation in leaves and roots under water-deficiency stress and following rewatering with respect to organ-specific responses., One of the strategies of plant adaptation to stress is the modulation of gene expression, which may result from the regulation of DNA methylation. This study attempted to characterize and compare the barley methylome of leaves and roots under water-deficiency treatment and in the subsequent rewatering phase. Our results, obtained using methylation-sensitive amplification polymorphism sequencing analysis, indicated that the overall DNA methylation level in the barley genome was high and in general stable under water-deficiency conditions. Nevertheless, numerous differentially methylated sites (DMSs) were induced by stress in the leaves and roots. Equal proportions of novel stress-induced methylation and demethylation events were observed within the genes in the leaves, but new methylations dominated in the roots. Repetitive elements preferentially underwent demethylation in the leaves and novel methylations in the roots. Importantly, rewatering and plant recovery resulted in the reversibility of the majority of stress-induced methylation events, but this process was more efficient in the leaves than in the roots. Different biological processes were enriched within the subsets of the DMSs that were identified in the genic regions of leaves and roots. We assume that the organ specificity of the methylome changes in response to water deficiency might be an important regulatory mechanism that leads to multi-level mechanisms of stress tolerance in barley.

Highlight The root system is the first plant organ that senses a water deficit. Here we have shown evidence that root hairs play a role as sensors of environmental conditions during water stress., An important part of the root system is the root hairs, which play a role in mineral and water uptake. Here, we present an analysis of the transcriptomic response to water deficiency of the wild-type (WT) barley cultivar ‘Karat’ and its root-hairless mutant rhl1.a. A comparison of the transcriptional changes induced by water stress resulted in the identification of genes whose expression was specifically affected in each genotype. At the onset of water stress, more genes were modulated by water shortage in the roots of the WT plants than in the roots of rhl1.a. The roots of the WT plants, but not of rhl1.a, specifically responded with the induction of genes that are related to the abscisic acid biosynthesis, stomatal closure, and cell wall biogenesis, thus indicating the specific activation of processes that are related to water-stress signalling and protection. On the other hand, the processes involved in the further response to abiotic stimuli, including hydrogen peroxide, heat, and high light intensity, were specifically up-regulated in the leaves of rhl1.a. An extended period of severe stress caused more drastic transcriptome changes in the roots and leaves of the rhl1.a mutant than in those of the WT. These results are in agreement with the much stronger damage to photosystem II in the rhl1.a mutant than in its parent cultivar after 10 d of water stress. Taking into account the putative stress sensing and signalling features of the root hair transcriptome, we discuss the role of root hairs as sensors of environmental conditions.

Molecular markers play an important role in identifying the species variation, characterizing the genic diversity, and also linking the identified markers to trait of interest. Genome- and transcriptome-derived molecular markers have been widely used to understand the geographical diversity and have also played a major role in the development of high-density linkage maps. In the present protocol, we present a detailed protocol on bioinformatics approaches towards the whole-genome and transcriptome-assisted simple sequence repeats (SSRs) marker mining in Brachypodium distachyon and identification of orthologus SSRs and their validation in Brachypodium ecotypes. We also present a protocol for the validation of the identified markers.

Molecular markers play an important role in identifying the species variation, characterizing the genic diversity, and also linking the identified markers to trait of interest. Genome- and transcriptome-derived molecular markers have been widely used to understand the geographical diversity and have also played a major role in the development of high-density linkage maps. In the present protocol, we present a detailed protocol on bioinformatics approaches towards the whole-genome and transcriptome-assisted simple sequence repeats (SSRs) marker mining in Brachypodium distachyon and identification of orthologus SSRs and their validation in Brachypodium ecotypes. We also present a protocol for the validation of the identified markers.

Non-small-cell lung cancer (NSCLC) represents a heterogeneous group of malignancies consisting essentially of adenocarcinoma (ADC) and squamous cell carcinoma (SCC). Although the diagnosis and treatment of ADC and SCC have been greatly improved in recent decades, there is still an urgent need to identify accurate transcriptome profile associated with the histological subtypes of NSCLC. The present study aims to identify the key dysregulated pathways and genes involved in the development of lung ADC and SCC and to relate them with the clinical traits. The transcriptional changes between tumour and normal lung tissues were investigated by RNA-seq. Gene ontology (GO), canonical pathways analysis with the prediction of upstream regulators, and weighted gene co-expression network analysis (WGCNA) to identify co-expressed modules and hub genes were used to explore the biological functions of the identified dysregulated genes. It was indicated that specific gene signatures differed significantly between ADC and SCC related to the distinct pathways. Of identified modules, four and two modules were the most related to clinical features in ADC and SCC, respectively. CTLA4, MZB1, NIP7, and BUB1B in ADC, as well as GNG11 and CCNB2 in SCC, are novel top hub genes in modules associated with tumour size, SUVmax, and recurrence-free survival. Our research provides a more effective understanding of the importance of biological pathways and the relationships between major genes in NSCLC in the perspective of searching for new molecular targets.

Background The aim of study was to collect and analyze data on relapsing-remitting multiple sclerosis (RRMS) patients receiving disease modifying therapies (DMTs) in Poland. Material and methods The observational, multi-center with prospective data collection study included all RRMS patients receiving DMTs reimbursed by the National Health Fund (NFZ) in Poland, monitored by the Therapeutic Program Monitoring System (SMPT). The demographic profile, disability status and treatment modalities were analyzed. Results Data from 12,341 patients were collected, including 70.1% women and 29.9% men, therein 11,653 patients in the first-line and 1026 in second-line therapeutic DMTs programme. Proportion female to male was 2.36 in the first-line and 1.9 in the second-line. The mean age was 36.4 years (±SD 10.6) in the first-line and 35.1 years (±SD 9.7) in the second line. The mean time of observation in the first line was 3.7 years (±SD 2.4) and in the second line was 2.8 years (±SD 1.3). The mean time from the first symptoms to MS diagnosis was 24.54 months (median 7.23), and from MS diagnosis to treatment: 39.35 months (median 10.12). There was a positive correlation between time to start treatment and higher the initial EDSS score (R 0.29, p Conclusions In Poland, only about one third of all MS patients is treated with DMTs. There is a need to improve the quality of MS patients healthcare and their access to DMTs.

Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare. However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop plants with large and complex genomes.

The cell nucleus harbors a large number of proteins involved in transcription, RNA processing, chromatin remodeling, nuclear signaling, and ribosome assembly. The nuclear genome of the model alga Chlamydomonas reinhardtii P. A. Dang. was recently sequenced, and many genes encoding nuclear proteins, including transcription factors and transcription regulators, have been identified through computational discovery tools. However, elucidating the specific biological roles of nuclear proteins will require support from biochemical and proteomics data. Cellular preparations with enriched nuclei are important to assist in such analyses. Here, we describe a simple protocol for the isolation of nuclei from Chlamydomonas, based on a commercially available kit. The modifications done in the original protocol mainly include alterations of the differential centrifugation parameters and detergent-based cell lysis. The nuclei-enriched fractions obtained with the optimized protocol show low contamination with mitochondrial and plastid proteins. The protocol can be concluded within only 3 h, and the proteins extracted can be used for gel-based and non-gel-based proteomic approaches.

The specialized root epidermis cells of higher plants produce long, tubular outgrowths called root hairs. Root hairs play an important role in nutrient and water uptake, and they serve as a valuable model in studies of plant cell morphogenesis. More than 1,300 articles that describe the biological processes of these unique cells have been published to date. As new fields of root hair research are emerging, the number of new papers published each year and the volumes of new relevant data are continuously increasing. Therefore, there is a general need to facilitate studies on root hair biology by collecting, presenting, and sharing the available information in a systematic, curated manner. Consequently, in this paper, we present a comprehensive database of root hair genomics, iRootHair, which is accessible as a Web-based service. The current version of the database includes information about 153 root hair-related genes that have been identified to date in dicots and monocots along with their putative orthologs in higher plants with sequenced genomes. In order to facilitate the use of the iRootHair database, it is subdivided into interrelated, searchable sections that describe genes, processes of root hair formation, root hair mutants, and available references. The database integrates bioinformatics tools with a focus on sequence identification and annotation. iRootHair is a unique resource for root hair research that integrates the large volume of data related to root hair genomics in a single, curated, and expandable database that is freely available at www.iroothair.org.

The cell nucleus harbors a large number of proteins involved in transcription, RNA processing, chromatin remodeling, nuclear signaling, and ribosome assembly. The nuclear genome of the model alga Chlamydomonas reinhardtii P. A. Dang. was recently sequenced, and many genes encoding nuclear proteins, including transcription factors and transcription regulators, have been identified through computational discovery tools. However, elucidating the specific biological roles of nuclear proteins will require support from biochemical and proteomics data. Cellular preparations with enriched nuclei are important to assist in such analyses. Here, we describe a simple protocol for the isolation of nuclei from Chlamydomonas, based on a commercially available kit. The modifications done in the original protocol mainly include alterations of the differential centrifugation parameters and detergent-based cell lysis. The nuclei-enriched fractions obtained with the optimized protocol show low contamination with mitochondrial and plastid proteins. The protocol can be concluded within only 3 h, and the proteins extracted can be used for gel-based and non-gel-based proteomic approaches.

We report here that ORS1, a previously uncharacterized member of the NAC transcription factor family, controls leaf senescence in Arabidopsis thaliana. Overexpression of ORS1 accelerates senescence in transgenic plants, whereas its inhibition delays it. Genes acting downstream of ORS1 were identified by global expression analysis using transgenic plants producing dexamethasone-inducible ORS1-GR fusion protein. Of the 42 up-regulated genes, 30 (similar to 70%) were previously shown to be up-regulated during age-dependent senescence. We also observed that 32 (similar to 76%) of the ORS1-dependent genes were induced by long-term (4 d), but not short-term (6 h) salinity stress (150 mM NaCl). Furthermore, expression of 16 and 24 genes, respectively, was induced after 1 and 5 h of treatment with hydrogen peroxide (H(2)O(2)), a reactive oxygen species known to accumulate during salinity stress. ORS1 itself was found to be rapidly and strongly induced by H(2)O(2) treatment in both leaves and roots. Using in vitro binding site selection, we determined the preferred binding motif of ORS1 and found it to be present in half of the ORS1-dependent genes. ORS1 is a paralog of ORE1/ANAC092/AtNAC2, a previously reported regulator of leaf senescence. Phylogenetic footprinting revealed evolutionary conservation of the ORS1 and ORE1 promoter sequences in different Brassicaceae species, indicating strong positive selection acting on both genes. We conclude that ORS1, similarly to ORE1, triggers expression of senescence-associated genes through a regulatory network that may involve cross-talk with salt- and H(2)O(2)-dependent signaling pathways.

Root hairs are long tubular outgrowths of specialized root epidermal cells that play an important role in plant nutrition and water uptake. They are also an important model in studies of higher plant cell differentiation. In contrast to the model dicot Arabidopsis thaliana, currently very little is known about the genetic and molecular basis of root hair formation in monocots, including major cereals. To elucidate candidate genes controlling this developmental process in barley, we took advantage of the recently established Affymetrix GeneChip Barley1 Genome Array to carry out global transcriptome analyses of hairless and root hair primordia-forming roots of two barely mutant lines. Expression profiling of the root-hairless mutant rhl1.a and its wild type parent variety ‘Karat’ revealed 10 genes potentially involved in the early step of root hair formation in barley. Differential expression of all identified genes was confirmed by quantitative reverse transcription-polymerase chain reaction. The genes identified encode proteins associated with the cell wall and membranes, including one gene for xyloglucan endotransglycosylase, three for peroxidase enzymes and five for arabinogalactan protein, extensin, leucine-rich-repeat protein, phosphatidylinositol phosphatidylcholine transfer protein and a RhoGTPase GDP dissociation inhibitor, respectively. The molecular function of one gene is unknown at present. The expression levels of these genes were strongly reduced in roots of the root-hairless mutant rhl1.a compared to the parent variety, while expression of all 10 genes was similar in another mutant, i.e. rhp1.b, that has lost its ability to develop full root hairs but still forms hairs blocked at the primordium stage, and its wild type relative. This clearly indicates that the new genes identified are involved in the initiation of root hair morphogenesis in barley.

In this study, a novel repetitive sequence pTaq10 was isolated from the Taq I digest of the genomic DNA of the pseudocereal Chenopodium quinoa. Sequence analysis indicated that this 286-bp monomer is not homologous to any known retroelement sequence. FISH and Southern blot analysis showed that this sequence is characterized by an interspersed genomic organization. After FISH, hybridization signals were observed as small dots spread throughout all of the chromosomes. pTaq hybridization signals were excluded from 45S rRNA gene loci, but they partly overlapped with 5S rDNA loci. pTaq10 is not a species-specific sequence, as it was also detected in C. berlandieri.

This study compared the meiotic recombination frequency between wheat doubled haploid (DH) populations obtained through two different methods, maize pollination (MP♀) and anther culture (AC♂). The comparison was based on a genetic linkage analysis, performed with DNA markers. Thirty-five polymorphic markers (15 SSR, 15 AFLP, 5 RAPD) were screened in MP♀ and AC♂ doubled haploids populations, derived from the same hybrid genotype (F1 of ‘Eta’ × ‘Darkhan 15’). Nine linkage groups, comprising 35 loci (the MP♀ lines) and 31 loci (the AC♂ lines), were constructed. The linkage groups in both DH populations showed identical orders of markers, except for one group mapping to chromosome 6B. The MP♀ and AC♂ linkage maps differed significantly in recombination frequencies for corresponding intervals. In total, the AC♂ linkage map (495.5 cM) was 40.5% longer than the MP♀ map (352.8 cM), indicating a significantly higher meiotic recombination rate in pollen mother cells. The enhancement in recombination was visible in five of nine linkage groups, and in 7 intervals between individual loci out of 19 compared. Moreover, for 6 other intervals a lack of linkage was observed in the AC♂ population, as compared to the MP♀ map.

Root hairs are specialized epidermal cells that play a role in the uptake of water and nutrients from the rhizosphere and serve as a site of interaction with soil microorganisms. The process of root hair formation is well characterized in Arabidopsis (Arabidopsis thaliana); however, there is a very little information about the genetic and molecular basis of root hair development in monocots. Here, we report on isolation and cloning of the β-expansin (EXPB) gene HvEXPB1, tightly related to root hair initiation in barley (Hordeum vulgare). Using root transcriptome differentiation in the wild-type/root-hairless mutant system, a cDNA fragment present in roots of wild-type plants only was identified. After cloning of full-length cDNA and genomic sequences flanking the identified fragment, the subsequent bioinformatics analyses revealed homology of the protein coded by the identified gene to the EXPB family. Reverse transcription-PCR showed that expression of HvEXPB1 cosegregated with the root hair phenotype in F2 progeny of the cross between the hairless mutant rhl1.a and the wild-type Karat parent variety. Expression of the HvEXPB1 gene was root specific; it was expressed in roots of wild-type forms, but not in coleoptiles, leaves, tillers, and spikes. The identified gene was active in roots of two other analyzed root hair mutants: rhp1.a developing root hair primordia only and rhs1.a with very short root hairs. Contrary to this, a complete lack of HvEXPB1 expression was observed in roots of the spontaneous root-hairless mutant bald root barley. All these observations suggest a role of the HvEXPB1 gene in the process of root hair formation in barley.

The aim of this study was to perform a comparative investigation of the actions of three mutagens that are widely used in plant mutagenesis using the comet-FISH technique. The comet-FISH technique was used for the analysis of DNA damage and the kinetics of repair within specific DNA sequences. FISH with rDNA and telomeric/centromeric DNA probes was applied to comets that were obtained from an alkaline/neutral comet assay. Migration within specific DNA sequences was analysed after treatment with two chemical mutagens-maleic hydrazide (MH) and N-nitroso-N-methylurea (MNU), and γ-rays. Barley was used as a model plant in this study. The possible utility of specific DNA sequences in a comparative assessment of the distribution of DNA damage within a plant genome was evaluated. This study proved that the comet-FISH technique is suitable for a detailed quantification of DNA damage and repair within specific DNA sequences in plant mutagenesis. The analysis of FISH signals demonstrated that the involvement of specific DNA sequences in DNA damage was different and was dependent on the mutagen used. We showed that 5S rDNA and telomeric DNA sequences are more sensitive to mutagenic treatment, which was expressed by a stronger fragmentation and migration in comparison to the other probes used in the study. We found that 5S rDNA and telomeric DNA probes are more suitable for testing the genotoxicity of environmental factors. A comparison of the involvement of specific chromosome domains in direct DNA breakage/repair and in chromosome aberration formation after mutagen treatment indicates the compatibility of the results.

Electron transport chain (ETCh) of ammonium (AOB) and nitrite oxidizing bacteria (NOB) participates in oxidation of ammonium to nitrate (nitrification). Operation of ETCh may be perturbed by a range of water-soluble xenobiotics. Therefore, consortia of nitrifying bacteria may be used as a biosensor to detect water contamination. A surprising feature of this system is an increase of oxygen consumption, detected in the presence of certain inhibitors of ETCh. Thus, to shed light on the mechanism of this effect (and other differences between inhibitors) we monitored separately respiration of the bacteria of the first (AOB - Nitrosomonas) and second (NOB -Nitrobacter) stages of nitrification. Furthermore, we measured plasma membrane potential and the level of reduction of NAD(P)H. We propose a novel model of ETCh in NOB to explain the role of reverse electron transport in the stimulation of oxygen consumption (previously attributed to hormesis).

Root hairs are an important model in studies of cell differentiation and development in higher plants. The function of NADPH oxidase-related reactive oxygen species (ROS) in root hair development has been reported extensively in studies on Arabidopsis. In this study, we investigated the mechanism of the initiation of root hair formation, mediated by the peroxidase-dependent production of the highly reactive hydroxyl radical in barley (Hordeum vulgare L.). The distribution of ROS, including the hydroxyl radical (OH) and superoxide (O(2)(-)) was assessed using hydroxyphenyl fluorescein and nitroblue tetrazolium chloride, respectively, in the roots of wild-type plants and two root-hair mutants: root-hairless (rhl1.a) and with root hair growth blocked at the primordium stage (rhp1.b). Peroxidase-dependent OH accumulation was linked to root hair initiation and growth in plants where root hair formation was at least initiated, whereas OH was not detectable in the epidermis of the root-hairless mutant rhl1.a. O(2)(-) distribution in the roots of rhl1.a and rhp1.b mutants was not impaired and did not influence the root hair phenotype. Peroxidase inhibitor treatments of wild-type roots dramatically reduced the ability of growing roots to form root hairs and thus phenocopied the root-hairless phenotype. Expression of two candidate peroxidase genes, HvPRX45 and HvPRX2, was analyzed and their possible role in root hair-specific production of hydroxyl radicals was discussed. We propose a model of a two-step, coordinated ROS formation process in root hair cells that involves root hair-specific peroxidase(s) and root hair-specific NADPH oxidase necessary for a proper root hair formation in barley.

We used comet-fluorescence in situ hybridization (FISH) in the model plant species Crepis capillaris following exposure of seedlings to maleic hydrazide (MH). FISH with 5S and 25S rDNA probes was applied to comets obtained under alkaline conditions to establish whether these DNA regions were preferentially involved in comet tail formation. MH treatment induced significant fragmentation of nuclear DNA and of rDNA loci. A 24-h post-treatment recovery period allowed a partial reversibility of MH-induced damage on nuclear and rDNA regions. Analyses of FISH signals demonstrated that rDNA sequences were always involved in tail formation and that 5S rDNA was more frequently present in the tail than 25S rDNA, regardless of treatment. The involvement of 25S rDNA in nucleolus formation and differences in chromatin structure between the two loci may explain the different susceptibility of the 25S and 5S rDNA regions to migrate into the tail. This work is the first report on the application of FISH to comet preparations from plants to analyze the distribution and repair of DNA damage within specific genomic regions after mutagenic treatment. Moreover, our work suggests that comet-FISH in plants may be a useful tool for environmental monitoring assessment. Environ. Mol. Mutagen., 2012. © 2012 Wiley Periodicals, Inc.

We report here that ORS1, a previously uncharacterized member of the NAC transcription factor family, controls leaf senescence in Arabidopsis thaliana. Overexpression of ORS1 accelerates senescence in transgenic plants, whereas its inhibition delays it. Genes acting downstream of ORS1 were identified by global expression analysis using transgenic plants producing dexamethasone-inducible ORS1–GR fusion protein. Of the 42 up-regulated genes, 30 (∼70%) were previously shown to be up-regulated during age-dependent senescence. We also observed that 32 (∼76%) of the ORS1-dependent genes were induced by long-term (4 d), but not short-term (6 h) salinity stress (150 mM NaCl). Furthermore, expression of 16 and 24 genes, respectively, was induced after 1 and 5 h of treatment with hydrogen peroxide (H2O2), a reactive oxygen species known to accumulate during salinity stress. ORS1 itself was found to be rapidly and strongly induced by H2O2 treatment in both leaves and roots. Using in vitro binding site selection, we determined the preferred binding motif of ORS1 and found it to be present in half of the ORS1-dependent genes. ORS1 is a paralog of ORE1/ANAC092/AtNAC2, a previously reported regulator of leaf senescence. Phylogenetic footprinting revealed evolutionary conservation of the ORS1 and ORE1 promoter sequences in different Brassicaceae species, indicating strong positive selection acting on both genes. We conclude that ORS1, similarly to ORE1, triggers expression of senescence-associated genes through a regulatory network that may involve cross-talk with salt- and H2O2-dependent signaling pathways.