Comet-FISH for the evaluation of plant DNA damage after mutagenic treatments

The aim of this study was to perform a comparative investigation of the actions of three mutagens that are widely used in plantmutagenesis 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 ofDNA damage and repairwithin specificDNA 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

Cytomolecular analysis of ribosomal DNA evolution in a natural allotetraploid brachypodium hybridum and its putative ancestors - dissecting complex repetitive structure of intergenic spacers

Nucleolar dominance is an epigenetic phenomenon associated with nuclear 35S rRNA genes and consists in selective suppression of gene loci inherited from one of the progenitors in the allopolyploid. Our understanding of the exact mechanisms that determine this process is still fragmentary, especially in case of the grass species. This study aimed to shed some light on the molecular basis of this genome-specific inactivation of 35S rDNA loci in an allotetraploid Brachypodium hybridum (2n = 30), which arose from the interspecific hybridization between two diploid ancestors that were very similar to modern B. distachyon (2n = 10) and B. stacei (2n = 20). Using fluorescence in situ hybridization with 25S rDNA and chromosome-specific BAC clones as probes we revealed that the nucleolar dominance is present not only in meristematic root-tip cells but also in differentiated cell fraction of B. hybridum. Additionally, the intergenic spacers (IGSs) from both of the putative ancestors and the allotetraploid were sequenced and analyzed. The presumptive transcription initiation sites, spacer promoters and repeated elements were identified within the IGSs. Two different length variants, 2.3 and 3.5 kb, of IGSs were identified in B. distachyon and B. stacei, respectively, however only the IGS that had originated from B. distachyon-like ancestor was present in the allotetraploid. The amplification pattern of B. hybridum IGSs suggests that some genetic changes occurred in inactive B. stacei-like rDNA loci during the evolution of the allotetraploid. We hypothesize that their preferential silencing is an effect of structural changes in the sequence rather than just the result of the sole inactivation at the epigenetic leve

Isolation and characterization of microsatellite loci in Pulsatilla patens (L.) Mill. (Ranunculaceae) a rare and endangered plant species in Europe

Pulsatilla patens is a rare and endangered plant species in many areas of Europe and protected under the Bern Convention and it is listed in Annex II and Annex IV to the Habitats Directive. In this study we developed 12 novel microsatellite loci using via 454 sequencing. We determined 11,220 contigs with a length of 156–11,384 bp. Within this dataset, we identified 319 SSR motifs in 301 contigs. All markers were genotyped on 56 individuals from three populations located in Poland. The number of alleles and expected heterozygosity were 2–12 (mean 3.7) and 0.142–0.820 (0.541 on average) respectively. The markers described in this study will be useful for evaluating genetic diversity of P. patens populations, could be applied to investigate the biological aspects and to develop effective conservation programs for the European populations of this species. Pulsatilla patens (L.) Mill. (Ranunculaceae) is a long-lived perennial herb. It is a lowland species with a circumpolar range, found in all three continents in the northern hemisphere (Hulten and Fries 1986). Pulsatilla patens is widespread in Central and Eastern Europe, with its western range extending to Sweden and Germany (Akeroyd 1993). In North America, the species occurs mostly in the Central and Western United States, in Central and Northwestern Canada and in Eastern Alaska. Pulsatilla patens shows a preference for dry, sun-exposed sites. In Europe, it can be found in thermophilous grasslands and coniferous forest. Within its range in Europe, P. patens is considered to be critically endangered in many areas. The species is protected under the Bern Convention, and it is listed in Annex II and Annex IV to the Habitats Directive (Council of Europe 1979; European Communities 2004) due to a small number of localities, low abundance and the gradual disappearance of populations. The species decline is related to changes in land use, especially in forestry practices where efficient wildfire prevention and termination of cattle grazing in forests have led to the formation of a continuous moss layer or strongly grass dominated vegetation, which severely hinders the regeneration of P. patens (Kalamees et al. 2005). Long-term protection and management plans aimed at preserving P. patens populations should involve habitat and environmental monitoring as well as the quantification of genetic diversity within and among populations. Microsatellite markers, also referred to as simple sequence repeat (SSR) markers, are widely used in ecological studies and can also be employed to investigate the genetic diversity of populations. In this study, we developed nuclear microsatellite markers for P. patens with the involvement of GS Junior next generation sequencing (Roche 454 Life Sciences, Branford, CT, USA)

Water-deficiency conditions differently modulate the methylome of roots and leaves in barley (Hordeum vulgare L.)

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

Stimulatory Effect of Xenobiotics on Oxidative Electron Transport of Chemolithotrophic Nitrifying Bacteria Used as Biosensing Element

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)

Automatic analysis of 2D polyacrylamide gels in the diagnosis of DNA polymorphisms

Introduction: The analysis of polyacrylamide gels is currently carried out manually or automatically. In the automatic method, there are limitations related to the acceptable degree of distortion of lane and band continuity. The available software cannot deal satisfactorily with this type of situations. Therefore, the paper presents an original image analysis method devoid of the aforementioned drawbacks.Material: This paper examines polyacrylamide gel images from Li-Cor DNA Sequencer 4300S resulting from the use of the electrophoretic separation of DNA fragments. The acquired images have a resolution dependent on the length of the analysed DNA fragments and typically it is MG×NG=3806×1027 pixels. The images are saved in TIFF format with a grayscale resolution of 16 bits/pixel. The presented image analysis method was performed on gel images resulting from the analysis of DNA methylome profiling in plants exposed to drought stress, carried out with the MSAP (Methylation Sensitive Amplification Polymorphism) technique.Results: The results of DNA polymorphism analysis were obtained in less than one second for the Intel Core™ 2 Quad CPU [email protected], 8GB RAM. In comparison with other known methods, specificity was 0.95, sensitivity = 0.94 and AUC (Area Under Curve) = 0.98.Conclusions: It is possible to carry out this method of DNA polymorphism analysis on distorted images of polyacrylamide gels. The method is fully automatic and does not require any operator intervention. Compared with other methods, it produces the best results and the resulting image is easy to interpret. The presented method of measurement is used in the practical analysis of polyacrylamide gels in the Department of Genetics at the University of Silesia in Katowice, Poland

Isolation and characterization of Simple Sequence Repeats (SSR) Markers from the moss genus Orthotrichum using a small throughput pyrosequencing machine

Here, we report the results of next-generation sequencing on the GS Junior system to identify a large number of microsatellites from the epiphytic moss Orthotrichum speciosum. Using a combination of a total (non-enrichment) genomic library and small-scale 454 pyrosequencing, we determined 5382 contigs whose length ranged from 103 to 5445 bp. In this dataset we identified 92 SSR (simple sequence repeats) motifs in 89 contigs. Forty-six of these had flanking regions suitable for primer design. We tested PCR amplification, reproducibility, and the level of polymorphism of 46 primer pairs for Orthotrichum speciosum using 40 individuals from two populations. As a result, the designed primers revealed 35 polymorphic loci with more than two alleles detected. This method is cost- and time-effective in comparison with traditional approaches involving cloning and sequencing

No time to waste : transcriptome study reveals that drought tolerance in barley may be attributed to stressed-like expression patterns that exist before the occurrence of stress

Plant survival in adverse environmental conditions requires a substantial change in the metabolism, which is reflected by the extensive transcriptome rebuilding upon the occurrence of the stress. Therefore, transcriptomic studies offer an insight into the mechanisms of plant stress responses. Here, we present the results of global gene expression profiling of roots and leaves of two barley genotypes with contrasting ability to cope with drought stress. Our analysis suggests that drought tolerance results from a certain level of transcription of stress-influenced genes that is present even before the onset of drought. Genes that predispose the plant to better drought survival play a role in the regulatory network of gene expression, including several transcription factors, translation regulators and structural components of ribosomes. An important group of genes is involved in signaling mechanisms, with significant contribution of hormone signaling pathways and an interplay between ABA, auxin, ethylene and brassinosteroid homeostasis. Signal transduction in a drought tolerant genotype may be more efficient through the expression of genes required for environmental sensing that are active already during normal water availability and are related to actin filaments and LIMdomain proteins, which may function as osmotic biosensors. Better survival of drought may also be attributed to more effective processes of energy generation and more efficient chloroplasts biogenesis. Interestingly, our data suggest that several genes involved in a photosynthesis process are required for the establishment of effective drought response not only in leaves, but also in roots of barley. Thus, we propose a hypothesis that root plastids may turn into the anti-oxidative centers protecting root macromolecules from oxidative damage during drought stress. Specific genes and their potential role in building upa drought-tolerant barley phenotype is extensively discussedwith special emphasis on processes that take place in barley roots. When possible, the interconnections between particular factors are emphasized to drawa broader picture of the molecular mechanisms of drought tolerance in barle

DNA Demethylation in Response to Heat Stress in Arabidopsis thaliana

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

Transcriptome analysis reveals the role of the root hairs as environmental sensors to maintain plant functions under water-deficiency conditions

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