Detection and analysis of UV-induced mutations in the chromosomal DNA of Arabidopsis.

Under natural conditions, plants are exposed to solar ultraviolet (UV) radiation, which damages chromosomal DNA. Although plant responses to UV-induced DNA damage have recently been elucidated in detail, revealing a set of DNA repair mechanisms and translesion synthesis (TLS), limited information is currently available on UV-induced mutations in plants. We previously reported the development of a supF-based system for the detection of a broad spectrum of mutations in the chromosomal DNA of Arabidopsis. In the present study, we used this system to investigate UV-induced mutations in plants. The irradiation of supF-transgenic plants with UV-C (500 and 1000 J/m2) significantly increased mutation frequencies (26- and 45-fold, respectively). G:C to A:T transitions (43-67% of base substitutions) dominated in the mutation spectrum and were distributed throughout single, tandem, and multiple base substitutions. Most of these mutations became undetectable with the subsequent illumination of UV-irradiated plants with white light for photoreactivation (PR). These results indicated that not only G:C to A:T single base substitutions, but also tandem and multiple base substitutions were caused by two major UV-induced photoproducts, cyclobutane-type pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4 PPs). In contrast, a high proportion of A:T to T:A transversions (56% of base substitutions) was a characteristic feature of the mutation spectrum obtained from photoreactivated plants. These results define the presence of the characteristic feature of UV-induced mutations, and provide insights into DNA repair mechanisms in plants.

Rapid Birth or Death of Centromeres on Fragmented Chromosomes in Maize.

Comparative genomics has revealed common occurrences in karyotype evolution such as chromosomal end-to-end fusions and insertions of one chromosome into another near the centromere, as well as many cases of de novo centromeres that generate positional polymorphisms. However, how rearrangements such as dicentrics and acentrics persist without being destroyed or lost remains unclear. Here, we sought experimental evidence for the frequency and timeframe for inactivation and de novo formation of centromeres in maize (Zea mays). The pollen from plants with supernumerary B chromosomes was gamma-irradiated and then applied to normal maize silks of a line without B chromosomes. In ∼8,000 first-generation seedlings, we found many B-A translocations, centromere expansions, and ring chromosomes. We also found many dicentric chromosomes, but a fraction of these show only a single primary constriction, which suggests inactivation of one centromere. Chromosomal fragments were found without canonical centromere sequences, revealing de novo centromere formation over unique sequences; these were validated by immunolocalization with Thr133-phosphorylated histone H2A, a marker of active centromeres, and chromatin immunoprecipitation-sequencing with the CENH3 antibody. These results illustrate the regular occurrence of centromere birth and death after chromosomal rearrangement during a narrow window of one to potentially only a few cell cycles for the rearranged chromosomes to be recognized in this experimental regime.

Epigenetic factors of individual radiosensitivity and adaptive capacity.

Purpose: Studying the relationship between epigenetic variability with different individual radiosensitivity and adaptive capacity.Material and method: Using a simple and convenient experimental model - maize seedlings with different germination terms and epigenetic patterns - the hypothesis was tested that homogeneous genetically but epigenetic different organisms have different radiosensitivity and radioadaptive capacity. Differences in the DNA methylation profiles of individual subpopulations of seedlings were used as a marker of epigenetic differences and the yield of chromosomal aberration was used as an indicator of DNA vulnerability and its changes under different UV-C irradiation modes. In two series of experiments involving а UV-C acute single and exposure according to the scheme 'adaptive - challenging', the investigation of possible biological importance of epigenetic polymorphism has been performed. The study used a cytogenetic analysis of the yield of chromosomal aberrations and restriction analysis followed by ITS-ISSR- PCR.Results: Significant differences have been established in chromosome aberration yield and DNA methylation profile in control and under UV-C exposure for seedlings of subpopulations differing in time of germination. The differences in the DNA methylation profiles and the yield of chromosomal aberrations in the control subpopulations of seedlings of different germination term indicate the influence of the DNA methylation profile on DNA damage by regular metabolic factors, such as thermal vibrations or reactive oxygen species (ROS). This phenomenon can be explained with different chromatin conformation determining structural or 'passive' resistance, which provides different DNA availability to damage. Methylation switching into de novo under different mode radiation exposure could become a marker of gene expression changes due to induced repair and protecting.Conclusions: The obtained data indicate the importance of epigenetic factors in determining the radio-resistance and adaptive capacity of organisms. It points out that the epigenetic mechanisms that determine the choice of the metabolic pattern also contribute to the individual radiosensitivity and adaptive capacity of the organisms. This contribution is determined by two ways. First, the DNA methylation profile affects the initial damage processes and secondly, the type of methylation switching into de novo is associated with the further development of protection and repair processes.

Microtubule organization defects in Arabidopsis thaliana.

Microtubules (MT) are critical cytoskeletal filaments that have several functions in cell morphogenesis, cell division, vesicle transport and cytoplasmic separation in the spatiotemporal regulation of eukaryotic cells. Formation of MT requires the co-interaction of MT nucleation and α-ß-tubulins, as well as MT-associated proteins (MAP). Many key MAP contributing to MT nucleation and elongation are essential for MT nucleation and regulation of MT dynamics, and are conserved in the plant kingdom. Therefore, the deletion or decrease of γ-tubulin ring complex (γTuRC) components and related MAP, such as the augmin complex, NEDD1, MZT1, EB1, MAP65, etc., in Arabidopsis thaliana results in MT organizational defects in the spindle and phragmoplast MT, as well as in chromosome defects. In addition, similar defects in MT organization and chromosome structure have been observed in plants under abiotic stress conditions, such as under high UV-B radiation. The MT can sense the signal from UV-B radiation, resulting in abnormal MT arrangement. Further studies are required to determine whether the abnormal chromosomes induced by UV-B radiation can be attributed to the involvement of abnormal MT arrays in chromosome migration after DNA damage.

Detecting Brachypodium distachyon Chromosomes Bd4 and Bd5 in MH- and X-Ray-Induced Micronuclei Using mcFISH.

Micronuclei are biomarkers of genotoxic effects and chromosomal instability. They are formed when chromosome fragments or whole chromosomes fail to disjoin into daughter nuclei. We present qualitative and quantitative analyses of the involvement of specific chromosome regions of chromosomes Bd4 and Bd5 in the formation of micronuclei of Brachypodium distachyon root tip cells following maleic hydrazide (MH) treatment and X-radiation. This is visualised by cytomolecular approaches using bacterial artificial chromosome (BAC)-based multicolour fluorescence in situ hybridisation (mcFISH) in combination with 5S and 25S rDNA probes. The results showed that the long arm of submetacentric chromosome Bd4 forms micronuclei at twice the frequency of its short arm, suggesting that the former is more prone to double-strand breaks (DSBs). In contrast, no difference was observed in the frequency of micronuclei derived from the long and short arms of submetacentric chromosome Bd5. Interestingly, the proximal region of the short arm of Bd5 is more prone to DSBs than its distal part. This demonstrates that 5S rDNA and 35S rDNA loci are not "hot spots" for DNA breaks after the application of these mutagens.

Cytological Effect of Gamma Radiation on Selected Mutants of Wheat Triticum aestivum L. in M3 Generation.

BACKGROUND AND OBJECTIVE: Wheat (Triticum aestivum L.) offers some unique opportunities for the induction and exploitation of agronomic value. The use of gamma radiation has been proven to be an effective method to induce genetic variation in crops. We aimed to determine genetically stable mutants of wheat which could be utilized for breeding purposes. MATERIALS AND METHODS: We did a cytological investigation of induced mutant's behavior and chiasma frequency. Selected mutant types induced in dry and soaked seeds were treated with different doses of gamma rays. Each treated sample and control were subjected to cytological examination of the fixed pollen mother cells in various meiotic stages. RESULTS: The percentage of the total abnormal cells significantly increased in one mutant and significantly decreased in the other mutant. The percentage of total abnormal cells did not diminish from the first to the second meiotic division. The types of meiotic anomalies found included laggards (56.51%), univalent (9.43%), stickiness (45.45%) and bridges (19.32%). There were genotypic differences in the frequency of occurrence of multivalent (trivalent and quadrivalents). A marked reduction in the number of rod and ring bivalent/cell in some genotypes were noticed. The frequency of chiasmata per pollen mother cell was reduced subsequently. Depression index of mutants was negative compared with controls or treatments except for a few genotypes. CONCLUSION: Selected mutants of wheat tend to be cytologically stable and can therefore, be utilized for breeding purposes.

Impact of Mutagens on DNA Replication in Barley Chromosomes.

Replication errors that are caused by mutagens are critical for living cells. The aim of the study was to analyze the distribution of a DNA replication pattern on chromosomes of the H. vulgare 'Start' variety using pulse 5-ethynyl-2'-deoxyuridine (EdU) labeling, as well as its relationship to the DNA damage that is induced by mutagenic treatment with maleic hydrazide (MH) and γ ray. To the best of our knowledge, this is the first example of a study of the effects of mutagens on the DNA replication pattern in chromosomes, as well as the first to use EdU labeling for these purposes. The duration of the cell cycle of the Hordeum vulgare 'Start' variety was estimated for the first time, as well as the influence of MH and γ ray on it. The distribution of the signals of DNA replication along the chromosomes revealed relationships between DNA replication, the chromatin structure, and DNA damage. MH has a stronger impact on replication than γ ray. Application of EdU seems to be promising for precise analyses of cell cycle disturbances in the future, especially in plant species with small genomes.

Inducement and identification of chromosome introgression and translocation of Gossypium australe on Gossypium hirsutum.

BACKGROUND: We previously reported the development of a set of Gossypium hirsutum-G. australe alien chromosome addition lines. Naturally, however, G. hirsutum-G. australe chromosome exchanges were very limited, impeding the stable transference of useful genes from G. australe (G2G2 genome) into the most cultivated cotton, G. hirsutum (AADD). RESULTS: In the present report, the pollen from a pentaploid (2n = AADDG2) of G. hirsutum-G. australe was irradiated with seven different doses ranging from 10 to 40 Grays and used to pollinate emasculated flowers of G. hirsutum over three consecutive years. Irradiation greatly increased the genetic recombination rates of the G. hirsutum and G. australe chromosomes and a total of 107 chromosome introgression individuals in 192 GISH-negative (with no GISH signal on chromosome) survived individuals, 11 chromosome translocation individuals (containing 12 chromosome translocation events) and 67 chromosome addition individuals were obtained in 70 GISH-positive (with GISH signal(s) on chromosome(s)) survived individuals, which are invaluable for mining desirable genes from G. australe. Multicolor genomic in situ hybridization results showed that there were three types of translocation, whole arm translocation, large alien segment translocation and small alien segment translocation, and that all translocations occurred between the G2-genome and the A-subgenome chromosomes in G. hirsutum. We also found that higher doses induced much higher rates of chromosome variation but also greatly lowered the seed viability and seedling survivability. CONCLUSIONS: Irradiation has been successfully employed to induce chromosome introgressions and chromosome translocations and promote chromosome exchanges between cultivated and wild species. In addition, by balancing the rates of chromosome introgression and translocation to those of seed set, seed germination, and seedling rates in the M1 generation, we conclude that the dosage of 20 Grays is the most suitable. The established methodology may guide the utilization of the tertiary gene pool of Gossypium species such as G. australe in cotton breeding in the future.

Dissecting Plant Chromosomes by the Use of Ionizing Radiation.

Radiation treatment of genomes is used to generate chromosome breaks for numerous applications. This protocol describes the preparation of seeds and the determination of the optimal level of irradiation dosage for the creation of a radiation hybrid (RH) population. These RH lines can be used to generate high-resolution physical maps for the assembly of sequenced genomes as well as the fine mapping of genes. This procedure can also be used for mutation breeding and forward/reverse genetics.

Flow cytometry may allow microscope-independent detection of holocentric chromosomes in plants.

Two chromosomal structures, known as monocentric and holocentric chromosomes, have evolved in eukaryotes. Acentric fragments of monocentric chromosomes are unequally distributed to daughter cells and/or lost, while holocentric fragments are inherited normally. In monocentric species, unequal distribution should generate chimeras of cells with different nuclear DNA content. We investigated whether such differences in monocentric species are detectable by flow cytometry (FCM) as (i) a decreased nuclear DNA content and (ii) an increased coefficient of variance (CV) of the G1 peak after gamma radiation-induced fragmentation. We compared 13 monocentric and 9 holocentric plant species. Unexpectedly, monocentrics and holocentrics did not differ with respect to parameters (i) and (ii) in their response to gamma irradiation. However, we found that the proportion of G2 nuclei was highly elevated in monocentrics after irradiation, while holocentrics were negligibly affected. Therefore, we hypothesize that DNA-damaging agents induce cell cycle arrest leading to endopolyploidy only in monocentric and not (or to much lesser extent) in holocentric plants. While current microscope-dependent methods for holocentrism detection are unreliable for small and numerous chromosomes, which are common in holocentrics, FCM can use somatic nuclei. Thus, FCM may be a rapid and reliable method of high-throughput screening for holocentric candidates across plant phylogeny.

INDUCED CYTOMICTIC VARIATIONS AND SYNCYTE FORMATION DURING MICROSPOROGENESIS IN PHASEOLUS VULGARIS L.

The intercellular translocation of chromatin material along with other cytoplasmic contents among the proximate meiocytes lying in close contact with each other commonly referred as cytomixis was reported during microsporogenesis in Phaseolus vulgaris L., a member of the family Fabaceae. The phenomenon of cytomixis was observed at three administered doses of gamma rays viz. 100, 200, 300 Gy respectively in the diploid plants of Phaseolus vulgaris L. The gamma rays irradiated plants showed the characteristic feature of inter-meiocyte chromatin/chromosomes transmigration through various means.such as channel formation, beak formation or by direct adhesion between the PMC's (Pollen mother cells). The present study also reports the first instance of syncyte formation induced via cytomictic transmigration in Phaseolus vulgaris L. Though the frequency of syncyteformation was rather low yet these could play a significant role in plant evolution. It is speculated that syncyte enhances the ploidy level of plants by forming 2n gametes and may lead to the production ofpolyploid plants. The phenomenon of cytomixis shows a gradual inclination along with the increasing treatment doses of gamma rays. The preponderance of cytomixis was more frequent during meiosis I as compared to meiosis II. An interesting feature noticed during the present study was the channel formation among the microspores and fusion among the tetrads due to cell wall dissolution. The impact of this phenomenon is also visible on the development of post-meiotic products. The formation of heterosized pollen grains; a deviation from the normal pollen grains has also been reported. The production of gametes with unbalanced chromosomes is of utmost importance and should be given more attention in future studies as they possess the capability of inducing variations at the genomic level and can be further utilized in the improvement of germplasm.

Identification of Novel Chromosomal Aberrations Induced by (60)Co-γ-Irradiation in Wheat-Dasypyrum villosum Lines.

Mutations induced by radiation are widely used for developing new varieties of plants. To better understand the frequency and pattern of irradiation-induced chromosomal rearrangements, we irradiated the dry seeds of Chinese Spring (CS)-Dasypyrum villosum nullisomic-tetrasomic (6A/6D) addition (6V) line (2n = 44), WD14, with (60)Co-γ-rays at dosages of 100, 200, and 300 Gy. The M0 and M1 generations were analyzed using Feulgen staining and non-denaturing fluorescence in situ hybridization (ND-FISH) by using oligonucleotide probes. Abnormal mitotic behavior and chromosomes with structural changes were observed in the M0 plants. In all, 39 M1 plants had structurally changed chromosomes, with the B genome showing the highest frequency of aberrations and tendency to recombine with chromosomes of the D genome. In addition, 19 M1 plants showed a variation in chromosome number. The frequency of chromosome loss was considerably higher for 6D than for the alien chromosome 6V, indicating that 6D is less stable after irradiation. Our findings suggested that the newly obtained γ-induced genetic materials might be beneficial for future wheat breeding programs and functional gene analyses.

Modulating crossover positioning by introducing large structural changes in chromosomes.

BACKGROUND: Crossing over assures the correct segregation of the homologous chromosomes to both poles of the dividing meiocyte. This exchange of DNA creates new allelic combinations thus increasing the genetic variation present in offspring. Crossovers are not uniformly distributed along chromosomes; rather there are preferred locations where they may take place. The positioning of crossovers is known to be influenced by both exogenous and endogenous factors as well as structural features inherent to the chromosome itself. We have introduced large structural changes into Arabidopsis chromosomes and report their effects on crossover positioning. RESULTS: The introduction of large deletions and putative inversions silenced recombination over the length of the structural change. In the majority of cases analyzed, the total recombination frequency over the chromosomes was unchanged. The loss of crossovers at the sites of structural change was compensated for by increases in recombination frequencies elsewhere on the chromosomes, mostly in single intervals of one to three megabases in size. Interestingly, two independent cases of induced structural changes in the same chromosomal interval were found on both chromosomes 1 and 2. In both cases, compensatory increases in recombination frequencies were of similar strength and took place in the same chromosome region. In contrast, deletions in chromosome arms carrying the nucleolar organizing region did not change recombination frequencies in the remainder of those chromosomes. CONCLUSIONS: When taken together, these observations show that changes in the physical structure of the chromosome can have large effects on the positioning of COs within that chromosome. Moreover, different reactions to induced structural changes are observed between and within chromosomes. However, the similarity in reaction observed when looking at chromosomes carrying similar changes suggests a direct causal relation between induced change and observed reaction.

[Cytogenetic studies on submerged plants from the Yenisei river area in the zone of radioactive contamination].

Cytogenetic studies on three species of submerged plants from different parts of the Yenisei river area subjected to radioactive impact of the Krasnoyarsk Mining-and-Chemical Plant and the Electrochemical Factory have been conducted. A high level of irregularities in anatelophase and metaphase of mitoses has been revealed in test samples compared to the control: agglutination and fragmentation of chromosomes, lagging chromosomes, bridges, fragments, misdivisions, and others. The natuie of the disorders indicates that they are related in part to the direct damage to the chromosome structure and in part to damage to the spindle.

The rice RAD51C gene is required for the meiosis of both female and male gametocytes and the DNA repair of somatic cells.

The RecA/RAD51 family of rice (Oryza sativa) consists of at least 13 members. However, the functions of most of these members are unknown. Here the functional characterization of one member of this family, RAD51C, is reported. Knockout (KO) of RAD51C resulted in both female and male sterility in rice. Transferring RAD51C to the RAD51C-KO line restored fertility. Cytological analyses showed that the sterility of RAD51C-KO plants was associated with abnormal early meiotic processes in both megasporocytes and pollen mother cells (PMCs). PMCs had an absence of normal pachytene chromosomes and had abnormal chromosome fragments. The RAD51C-KO line showed no obvious difference from wild-type plants in mitosis in the anther wall cells, which was consistent with the observation that the RAD51C-KO line did not have obviously abnormal morphology during vegetative development. However, the RAD51C-KO line was sensitive to different DNA-damaging agents. These results suggest that RAD51C is essential for reproductive development by regulating meiosis as well as for DNA damage repair in somatic cells.

Construction of whole genome radiation hybrid panels and map of chromosome 5A of wheat using asymmetric somatic hybridization.

To explore the feasibility of constructing a whole genome radiation hybrid (WGRH) map in plant species with large genomes, asymmetric somatic hybridization between wheat (Triticum aestivum L.) and Bupleurum scorzonerifolium Willd. was performed. The protoplasts of wheat were irradiated with ultraviolet light (UV) and gamma-ray and rescued by protoplast fusion using B. scorzonerifolium as the recipient. Assessment of SSR markers showed that the radiation hybrids have the average marker retention frequency of 15.5%. Two RH panels (RHPWI and RHPWII) that contained 92 and 184 radiation hybrids, respectively, were developed and used for mapping of 68 SSR markers in chromosome 5A of wheat. A total of 1557 and 2034 breaks were detected in each panel. The RH map of chromosome 5A based on RHPWII was constructed. The distance of the comprehensive map was 2103 cR and the approximate resolution was estimated to be ∼501.6 kb/break. The RH panels evaluated in this study enabled us to order the ESTs in a single deletion bin or in the multiple bins cross the chromosome. These results demonstrated that RH mapping via protoplast fusion is feasible at the whole genome level for mapping purposes in wheat and the potential value of this mapping approach for the plant species with large genomes.

Genome-wide analysis of mutations in mutant lineages selected following fast-neutron irradiation mutagenesis of Arabidopsis thaliana.

Ionizing radiation has long been known to induce heritable mutagenic change in DNA sequence. However, the genome-wide effect of radiation is not well understood. Here we report the molecular properties and frequency of mutations in phenotypically selected mutant lines isolated following exposure of the genetic model flowering plant Arabidopsis thaliana to fast neutrons (FNs). Previous studies suggested that FNs predominantly induce deletions longer than a kilobase in A. thaliana. However, we found a higher frequency of single base substitution than deletion mutations. While the overall frequency and molecular spectrum of fast-neutron (FN)-induced single base substitutions differed substantially from those of "background" mutations arising spontaneously in laboratory-grown plants, G:C>A:T transitions were favored in both. We found that FN-induced G:C>A:T transitions were concentrated at pyrimidine dinucleotide sites, suggesting that FNs promote the formation of mutational covalent linkages between adjacent pyrimidine residues. In addition, we found that FNs induced more single base than large deletions, and that these single base deletions were possibly caused by replication slippage. Our observations provide an initial picture of the genome-wide molecular profile of mutations induced in A. thaliana by FN irradiation and are particularly informative of the nature and extent of genome-wide mutation in lines selected on the basis of mutant phenotypes from FN-mutagenized A. thaliana populations.

[Genetic effects of root extracts of Glycyrrhiza glabra L. on different test-systems].

The antimutagenic and geroprotective activities of root extracts of Glycyrrhiza glabra have been demonstrated both on plant test systems--Allium fistulosum L., Allium cepa L., Vicia faba L. and on animals--Vistar rats. The possibilities of the mobilization of Glycyrrhiza glabra root extracts as antimutagenic agents are discussed.

Hybrid inflorescences derived from gamma-fusion of Arabidopsis thaliana with Bupleurum scorzonerifolium.

In our early experiments, a variety of Bupleurum scorzonerifolium-like somatic hybrid plants were obtained from protoplast fusion between Arabidopsis thaliana and UV-treated/untreated B. scorzonerifolium. To compare the effects of UV and γ-ray irradiation on the B. scorzonerifolium partner and obtain Arabidopsis-like hybrids, we designed a novel combination of somatic hybridization between A. thaliana and B. scorzonerifolium. Before protoplast isolation and fusion, the suspension cells of B. scorzonerifolium were irradiated by gamma ray ((60)Co, 50 Gy with 1.3 Gy min(-1)). Both parental protoplasts lost regeneration capacity, but over 100 somatic hybrids restored the capacity and developed to Arabidopsis-like inflorescences and flowers with some characteristics of B. scorzonerifolium. Some hybrid flowers showed yellow sepal, petal, or carpel, whose color was similar to the petal of B. scorzonerifolium; the others had silique of Arabidopsis with angularity of B. scorzonerifolium, and their parts possessed five stamens, the same as B. scorzonerifolium. Cytological analysis showed that three hybrids had Arabidopsis-like karyotypes. Random Amplified Polymorphic DNA (RAPD) and Simple Sequence Repeats (SSR) profiles revealed that both parental fragments were amplified from these hybrids. These results indicated chromatin introgression from B. scorzonerifolium to A. thaliana, which may be related to the complementation of hybrid inflorescence and flower generation.

[The chiral mutagens: cytogenetic effects on higher plants].

The paper covers investigation of cytogenetic activity of chiral mutagens and their specific effects on the plant cells chromosomes of soft winter wheat (Triticum aestivum L.). Comparative analysis of cytogenetic activity of chiral NEU: S(+)1-N-nitroso- 1-N-methyl-3-N-sec-buthylureas (S(+)NMsBU) and R(-)1-N-nitroso- 1N-methyl-3-Nsec-buthylureas (R(-)NMsBU) on winter wheat was performed. As it was shown by the frequency of chromosomal aberrations the S(+) stereoisomer was twice more active than R(-). In addition to typical anaphase aberrations (fragments, bridges, lagging chromosomes) the numerous mitosis pathologies were revealed - K-mitoses, hyperspiralization and despiralization of chromosomes, unequal allocation of chromosomes between the daughter nuclei, mass fragmentation, nondisjunction and chromosome adhesion, three-pole mitoses, etc. Neither of the mentioned pathologies was observed under the action of NEU and gamma-rays.