MicroRNAs potentially targeting DDR-related genes are differentially expressed upon exposure to γ-rays during seed germination in wheat.

DNA damage response (DDR), a complex network of cellular pathways that cooperate to sense and repair DNA lesions, is regulated by several mechanisms, including microRNAs. As small, single-stranded RNA molecules, miRNAs post-transcriptionally regulate their target genes by mRNA cleavage or translation inhibition. Knowledge regarding miRNAs influence on DDR-associated genes is still scanty in plants. In this work, an in silico analysis was performed to identify putative miRNAs that could target DDR sensors, signal transducers and effector genes in wheat. Selected putative miRNA-gene pairs were tested in an experimental system where seeds from two wheat mutant lines were irradiated with 50 Gy and 300 Gy gamma(γ)-rays. To evaluate the effect of the treatments on wheat germination, phenotypic and molecular (DNA damage, ROS accumulation, gene/miRNA expression profile) analyses have been carried out. The results showed that in dry seeds ROS accumulated immediately after irradiation and decayed soon after while the negative impact on seedling growth was supported by enhanced accumulation of DNA damage. When a qRT-PCR analysis was performed, the selected miRNAs and DDR-related genes were differentially modulated by the γ-rays treatments in a dose-, time- and genotype-dependent manner. A significant negative correlation was observed between the expression of tae-miR5086 and the RAD50 gene, involved in double-strand break sensing and homologous recombination repair, one of the main processes that repairs DNA breaks induced by γ-rays. The results hereby reported can be relevant for wheat breeding programs and screening of the radiation response and tolerance of novel wheat varieties.

Exogenous Cytokinin 4PU-30 Modulates the Response of Wheat and Einkorn Seedlings to Ultraviolet B Radiation.

Abiotic stress is responsible for a significant reduction in crop plant productivity worldwide. Ultraviolet (UV) radiation is a natural component of sunlight and a permanent environmental stimulus. This study investigated the distinct responses of young wheat and einkorn plants to excessive UV-B radiation (180 min at λmax 312 nm) following foliar pretreatment with 1 µM synthetic cytokinin 4PU-30. Results demonstrated that UV radiation significantly amplified hydrogen peroxide levels in both wheat and einkorn, with einkorn exhibiting a more pronounced increase compared to wheat. This elevation indicated the induction of oxidative stress by UV radiation in the two genotypes. Intensified antioxidant enzyme activities and the increased accumulation of typical stress markers and non-enzyme protectants were evidenced. Transcriptional activity of genes encoding the key antioxidant enzymes POX, GST, CAT, and SOD was also investigated to shed some light on their genetic regulation in both wheat and einkorn seedlings. Our results suggested a role for POX1 and POX7 genes in the UV-B tolerance of the two wheat species as well as a cytokinin-stimulated UV-B stress response in einkorn involving the upregulation of the tau subfamily gene GSTU6. Based on all our findings, it could be concluded that 4PU-30 had the potential of alleviating oxidative stress by attenuating the symptoms of superfluous UV-B illumination in the two examined plant species.

Morphological, Pathological and Genetic Diversity of the Colletotrichum Species, Pathogenic on Solanaceous Vegetable Crops in Bulgaria.

Colletotrichum species are among the most devastating plant pathogens in a wide range of hosts. Their accurate identification requires a polyphasic approach, including geographical, ecological, morphological, and genetic data. Solanaceous crops are of significant economic importance for Bulgarian agriculture. Colletotrichum-associated diseases pose a serious threat to the yield and quality of production but are still largely unexplored. The aim of this study was to identify and characterize 26 pathogenic Colletotrichum isolates that threaten solanaceous crops based on morphological, pathogenic, and molecular data. DNA barcodes enabled the discrimination of three main taxonomic groups: C. acutatum, C. gloeosporioides, and C. coccodes. Three different species of acutatum complex (C. nymphaeae, C. godetiae, and C. salicis) and C. cigarro of the gloeosporioides complex were associated with fruit anthracnose in peppers and tomatoes. The C. coccodes group was divided in two clades: C. nigrum, isolated predominantly from fruits, and C. coccodes, isolated mainly from roots. Only C. salicis and C. cigarro produced sexual morphs. The species C. godetiae, C. salicis, and C. cigarro have not previously been reported in Bulgaria. Our results enrich the knowledge of the biodiversity and specific features of Colletotrichum species, which are pathogenic to solanaceous hosts, and may serve as a scientific platform for efficient disease control and resistance breeding.

Cotinus coggygria Scop. induces cell cycle arrest, apoptosis, genotoxic effects, thermodynamic and epigenetic events in MCF7 breast cancer cells.

Current plant-derived anticancer therapeutics aim to reach higher effectiveness, to potentiate chemosensitivity and minimize the toxic side effects compared to conventional chemotherapy. Cotinus coggygria Scop. is a herb with high pharmacological potential, widely applied in traditional phytotherapy. Our previous study revealed that leaf aqueous ethanolic extract from C. coggygria exerts in vitro anticancer activity on human breast, ovarian and cervical cancer cell lines. The objective of the present research was to investigate possible molecular mechanisms and targets of the antitumor activity of the extract in breast cancer MCF7 cells through analysis of cell cycle and apoptosis, clonogenic ability assessment, evaluation of the extract genotoxic capacity, characterization of cells thermodynamic properties, and analysis on the expression of genes involved in cellular epigenetic processes. The obtained results indicated that in MCF7 cells C. coggygria extract causes S phase cell cycle arrest and triggers apoptosis, reduces colony formation, induces DNA damage, affects cellular thermodynamic parameters, and tends to inhibit the relative expression of DNMT1, DNMT3a, MBD3, and p300. Further studies on the targeted molecules and the extract anti-breast cancer potential on animal experimental model system, need to be performed in the future.

Efficient removal of cyclobutane pyrimidine dimers in barley: differential contribution of light-dependent and dark DNA repair pathways.

Barley stress response to ultraviolet radiation (UV) has been intensively studied at both the physiological and morphological level. However, the ability of barley genome to repair UV-induced lesions at the DNA level is far less characterized. In this study, we have investigated the relative contribution of light-dependent and dark DNA repair pathways for the efficient elimination of cyclobutane pyrimidine dimers (CPDs) from the genomic DNA of barley leaf seedlings. The transcriptional activity of barley CPD photolyase gene in respect to the light-growth conditions and UV-C irradiation of the plants has also been analyzed. Our results show that CPDs induced in the primary barley leaf at frequencies potentially damaging DNA at the single-gene level are removed efficiently and exclusively by photorepair pathway, whereas dark repair is hardly detectable, even at higher CPD frequency. A decrease of initially induced CPDs under dark is observed but only after prolonged incubation, suggesting the activation of light-independent DNA damage repair and/or tolerance mechanisms. The green barley seedlings possess greater capacity for CPD photorepair than the etiolated ones, with efficiency of CPD removal dependent on the intensity and quality of recovering light. The higher repair rate of CPDs measured in the green leaves correlates with the higher transcriptional activity of barley CPD photolyase gene. Visible light and UV-C radiation affect differentially the expression of CPD photolyase gene particularly in the etiolated leaves. We propose that the CPD repair potential of barley young seedlings may influence their response to UV-stress.

DNA damage and repair in plants - from models to crops.

The genomic integrity of every organism is constantly challenged by endogenous and exogenous DNA-damaging factors. Mutagenic agents cause reduced stability of plant genome and have a deleterious effect on development, and in the case of crop species lead to yield reduction. It is crucial for all organisms, including plants, to develop efficient mechanisms for maintenance of the genome integrity. DNA repair processes have been characterized in bacterial, fungal, and mammalian model systems. The description of these processes in plants, in contrast, was initiated relatively recently and has been focused largely on the model plant Arabidopsis thaliana. Consequently, our knowledge about DNA repair in plant genomes - particularly in the genomes of crop plants - is by far more limited. However, the relatively small size of the Arabidopsis genome, its rapid life cycle and availability of various transformation methods make this species an attractive model for the study of eukaryotic DNA repair mechanisms and mutagenesis. Moreover, abnormalities in DNA repair which proved to be lethal for animal models are tolerated in plant genomes, although sensitivity to DNA damaging agents is retained. Due to the high conservation of DNA repair processes and factors mediating them among eukaryotes, genes and proteins that have been identified in model species may serve to identify homologous sequences in other species, including crop plants, in which these mechanisms are poorly understood. Crop breeding programs have provided remarkable advances in food quality and yield over the last century. Although the human population is predicted to "peak" by 2050, further advances in yield will be required to feed this population. Breeding requires genetic diversity. The biological impact of any mutagenic agent used for the creation of genetic diversity depends on the chemical nature of the induced lesions and on the efficiency and accuracy of their repair. More recent targeted mutagenesis procedures also depend on host repair processes, with different pathways yielding different products. Enhanced understanding of DNA repair processes in plants will inform and accelerate the engineering of crop genomes via both traditional and targeted approaches.

Karyotype reconstruction modulates the sensitivity of barley genome to radiation-induced DNA and chromosomal damage.

The potential of cytologically reconstructed barley line D-2946 to cope with the major lesions that hamper genome integrity, namely DNA single- and double-strand breaks was investigated. Strand breaks induced by γ-rays and Li ions were assessed by neutral and alkaline comet assay. Repair capacity after bleomycin treatment was evaluated by agarose gel electrophoresis under neutral and alkaline conditions. Frequencies of radiation-induced chromosome aberrations were also determined. Results indicate that radiation-mediated constitutive rearrangement of the chromosome complement has led to a substantial modulation of the sensitivity of barley genome towards DNA strand breaks, produced by ionising radiation, Li ion implantation and bleomycin in an agent-specific manner, as well as of the clastogenic response to γ-rays. Based on these findings, reconstructed barley karyotype D-2946 can be considered a candidate radio-sensitive line with reduced ability to maintain genome integrity with respect to both DNA and chromosomal damage.

Processing of DNA double strand breaks by alternative non-homologous end-joining in hyperacetylated chromatin.

BACKGROUND: Mammalian cells employ at least two subpathways of non-homologous end-joining for the repair of ionizing radiation induced DNA double strand breaks: The canonical DNA-PK-dependent form of non-homologous end-joining (D-NHEJ) and an alternative, slowly operating, error-prone backup pathway (B-NHEJ). In contrast to D-NHEJ, which operates with similar efficiency throughout the cell cycle, B-NHEJ operates more efficiently in G2-phase. Notably, B-NHEJ also shows strong and as of yet unexplained dependency on growth activity and is markedly compromised in serum-deprived cells, or in cells that enter the plateau-phase of growth. The molecular mechanisms underpinning this response remain unknown. Since chromatin structure or changes in chromatin structure are prime candidate-B-NHEJ-modulators, we study here the role of chromatin hyperacetylation, either by HDAC2 knockdown or treatment with the HDAC inhibitor TSA, on the repair by B-NHEJ of IR-induced DSBs. RESULTS: siRNA-mediated knockdown of HDAC2 fails to provoke histone hyperacetylation in Lig4-/- MEFs and has no detectable effect on B-NHEJ function. Treatment with TSA that inhibits multiple HDACs causes efficient, reversible chromatin hyperacetylation in Lig4-/- MEFs, as well as in human HCT116 Lig4-/- cells and the human glioma cell line M059K. The IR yield of DSBs in TSA-treated cells remains similar to that of untreated cells despite the expected chromatin relaxation. In addition, chromatin hyperacetylation leaves unchanged repair of DSBs by B-NHEJ in irradiated exponentially growing, or plateau-phase cells. Notably, under the experimental conditions employed here, chromatin hyperacetylation fails to detectably modulate B-NHEJ in M059K cells as well. CONCLUSIONS: In summary, the results show that chromatin acetylation or deacetylation does not affect the kinetics of alternative NHEJ in all types of cells examined both in exponentially growing and serum deprived cultures. We conclude that parameters beyond chromatin acetylation determine B-NHEJ efficiency in the plateau-phase of growth.

Efficient repair of bleomycin-induced double-strand breaks in barley ribosomal genes.

Ability of barley ribosomal genes to cope with damage produced in vivo by the radiomimetic agent bleomycin was investigated. Repair kinetics of bleomycin-induced double-strand breaks in ribosomal and total genomic DNA was compared. Induction and repair of double-strand breaks in defined regions of the ribosomal genes was also analyzed. Preferential sensitivity of barley linker DNA towards bleomycin treatment in vivo was established. Relatively higher yield of initially induced double-strand breaks in genomic DNA in comparison to ribosomal DNA was also found. Fragments containing intergenic spacers of barley rRNA genes displayed higher sensitivity to bleomycin than the coding sequences. No heterogeneity in the repair of DSB between transcribed and non-transcribed regions of ribosomal genes was detected. Data indicate that DSB repair in barley rDNA, although more efficient than in genomic DNA, does not correlate with the activity of nucleolus organizer regions.

Induction and recovery of double-strand breaks in barley ribosomal DNA.

Barley nucleolus organizing regions (NORs) were previously found to behave as prominent aberration hot-spots after treatment with some restriction endonucleases. The ability of MspI for directed induction of double-strand breaks in barley ribosomal DNA was further analyzed. Ionizing radiation-produced strand breakage within the ribosomal gene clusters was also a subject of investigation. Reconstructed barley karyotypes T1586 and T35 with normal and increased expression of rRNA genes were utilized to evaluate the relationship between transcriptional activity and damage induction. Scanning densitometry of the hybridization profiles revealed that MspI is generating double-strand breaks in barley rDNA with efficiency being independent from the NOR activity. Damage induction observed after treatment with gamma-rays was also not influenced by the transcriptional status of the ribosomal genes. A tendency towards restoration of rDNA integrity after irradiation of both germinating and dry seeds was observed which is indicative for the efficient recovery of double-strand breaks in barley ribosomal DNA.