Igor Kovalchuk

Molecular Aspects of Plant Adaptation to Life in the Chernobyl Zone

With each passing year since the Chernobyl accident of 1986, more questions arise about the potential for organisms to adapt to radiation exposure. Often this is thought to be attributed to somatic and germline mutation rates in various organisms. We analyzed the adaptability of native Arabidopsis plants collected from areas with different levels of contamination around the Chernobyl nuclear power plant from 1986 to 1992. Notably, progeny of Chernobyl plants resisted higher concentrations of the mutagens Rose Bengal and methyl methane sulfonate. We analyzed the possible molecular mechanisms of their resistance to mutagens and found a more than 10-fold lower frequency of extrachromosomal homologous recombination, significant differences in the expression of radical scavenging (CAT1 and FSD3) and DNA-repair (RAD1 and RAD51-like) genes upon exposure to mutagens (Rose Bengal and x-rays), and a higher level of global genome methylation. This data suggests that adaptation to ionizing radiation is a complex process involving epigenetic regulation of gene expression and genome stabilization that improves plants resistance to environmental mutagens.

Genome-wide variation of the somatic mutation frequency in transgenic plants

In order to analyse the frequency of point mutations in whole plants, several constructs containing single nonsense mutations in the β‐glucuronidase (uidA) gene were used to generate transgenic Arabidopsis thaliana plants. Upon histochemical staining of transgenic plants, sectors indicative of transgene reactivation appeared. Reversion frequencies were in the range of 10−7–10−8 events per base pair, exceeding the previous estimates for other eukaryotes at least 100‐fold. The frequency was dependent on the position of the mutation substrate within the transgene and the position of the transgene within the Arabidopsis genome. An inverse relationship between the level of transgene transcription and mutation frequency was observed in single‐copy lines. DNA‐damaging factors induced the mutation frequency by a factor of up to 56 for UV‐C, a factor of 3 for X‐rays and a factor of 2 for methyl methanesulfonate. This novel plant mutation‐monitoring system allowed us to measure the frequencies of point mutation in whole plants and may be used as an alternative or complement to study the mutagenicity of different environmental factors on the higher eukaryotes genome.

A sensitive transgenic plant system to detect toxic inorganic compounds in the environment

We describe a transgenic plant–based assay to study the genetic effects of heavy metals. Arabidopsis thaliana plants carrying a β-glucuronidase (GUS) marker gene either with a point mutation or as a recombination substrate were used to analyze the frequency of somatic point mutations and homologous recombination in whole plants. Transgenic test plants sown on media contaminated by the salts of the heavy metals Cd2+, Pb2+, Ni2+, Zn2+, Cu2+, and As2O3 exhibited a pronounced uptake-dependent increase in the frequencies of both somatic intrachromosomal recombination and point mutation. The test was applied to monitor the genotoxicity of soils sampled in sites contaminated with several heavy metals. Our results indicate that this is a highly sensitive system for monitoring metal contamination in soils and water.

Wheat mutation rate after Chernobyl

The accident at the Chernobyl nuclear power plant in 1986 has generated concern over the genetic consequences of chronic exposure to radiation. Here we describe a new approach to monitoring germline mutation in plants and find evidence for a remarkably strong induction of germline mutation in wheat upon chronic exposure to ionizing radiation produced by the Chernobyl accident.

The Allium cepa chromosome aberration test reliably measures genotoxicity of soils of inhabited areas in the Ukraine contaminated by the Chernobyl accident

The accident on the Chernobyl Nuclear Power Plant reactor IV in April 1986 led to the release of an enormous amount of radioactive material into the biosphere and to the formation of a complex pattern of nuclear contamination over a large area. As a consequence more than 5 million km2 of the soil in the Ukraine became contaminated with more than 1 Ci/km2 [1,2]. An assessment of the genetic consequences of the nuclear pollution is one of the most important problems. We applied the Allium cepa test to estimate the impact on plant chromosomes of nuclear pollution in the inhabited zones of the Ukraine. We tested soil from the obligatory resettlement zone (zone 2), where the mean density of pollution is 15-40 Ci/km2; zones of enhanced radiological control-zone 3, 5-15 Ci/km2 and zone 4, 1-5 Ci/km2. We found a dose-dependent increase in the fraction of aberrant mitoses from control values of 1.6 +/- 0.9% up to 23.8 +/- 5.0%, and a corresponding monotonous decrease of the mitotic index from 49.4 +/- 4.8% to a limiting value of 22.5 +/- 4.0% at pollution levels exceeding 35 Ci/km2 (activity of the soil samples exceeding 6000 Bq/kg, respectively). We observed a strong, significant correlation of 137Cs activity of soil samples with the percentage of chromosomal abnormalities, r = 0.97 (P < 0.05), and with the mitotic index, r = -0.93 (P < 0.05), in the roots of A. cepa, respectively. The results showed high toxicity and genotoxicity of radioactively polluted soils and confirmed the efficiency of the A. cepa test as a quick and inexpensive biological test for ecological and genetic risk assessment in the Chernobyl zones.

Transgenic plants are sensitive bioindicators of nuclear pollution caused by the Chernobyl accident

To evaluate the genetic consequences of radioactive contamination originating from the nuclear reactor accident of Chernobyl on indigenous populations of plants and animals, it is essential to determine the rates of accumulating genetic changes in chronically irradiated populations. An increase in germline mutation rates in humans living close to the Chernobyl Nuclear Power Plant site, and a two- to tenfold increase in germline mutations in barn swallows breeding in Chernobyl have been reported. Little is known, however, about the effects of chronic irradiation on plant genomes. Ionizing radiation causes double-strand breaks in DNA, which are repaired via illegitimate or homologous recombination. We make use of Arabidopsis thaliana plants carrying a β-glucuronidase marker gene as a recombination substrate to monitor genetic alterations in plant populations, which are caused by nuclear pollution of the environment around Chernobyl. A significant (p<0.05) increase in somatic intrachromosomal recombination frequencies was observed at nuclear pollution levels from 0.1–900 Ci/km2, consistent with an increase in chromosomal aberrations. This bioindicator may serve as a convenient and ethically acceptable alternative to animal systems.

Plants experiencing chronic internal exposure to ionizing radiation exhibit higher frequency of homologous recombination than acutely irradiated plants

Ionizing radiation (IR) is a known mutagen responsible for causing DNA strand breaks in all living organisms. Strand breaks thus created can be repaired by different mechanisms, including homologous recombination (HR), one of the key mechanisms maintaining genome stability [A. Britt, DNA damage and repair in plants, Annu. Rev. Plant. Phys. Plant Mol. Biol., 45 (1996) 75-100; H. Puchta, B. Hohn, From centiMorgans to basepairs: homologous recombination in plants, Trends Plant Sci., 1 (1996) 340-348.]. Acute or chronic exposure to IR may have different influences on the genome integrity. Although in a radioactively contaminated environment plants are mostly exposed to chronic pollution, evaluation of both kinds of influences is important. Estimation of the frequency of HR in the exposed plants may serve as an indication of genome stability. We used previously generated Arabidopsis thaliana and Nicotiana tabacum plants, transgenic for non-active versions of the beta-glucoronidase gene (uidA) [P. Swoboda, S. Gal, B. Hohn, H. Puchta, Intrachromosomal homologous recombination in whole plants, EMBO J., 13 (1994) 484-489; H. Puchta, P. Swoboda, B. Hohn, Induction of homologous DNA recombination in whole plants, Plant, 7 (1995) 203-210.] serving as a recombination substrate, to study the influence of acute and chronic exposure to IR on the level of HR as example of genome stability in plants. Exposure of seeds and seedlings to 0.1 to 10.0 Gy 60Co resulted in increased HR frequency, although the effect was more pronounced in seedlings. For the study of the influence of chronic exposure to IR, plants were grown on two chemically different types of soils, each artificially contaminated with equal amounts of 137Cs. We observed a strong and significant correlation between the frequency of HR in plants, the radioactivity of the soil samples and the doses of radiation absorbed by plants (in all cases r0.9, n=6, P<0.05). In addition, we noted that plants grown in soils with different chemical composition, but equal radioactivity, exhibited different levels of HR, dependent upon the absorbed dose of radiation. Remarkably, we observed a much higher frequency of HR in plants exposed to chronic irradiation when compared to acutely irradiated plants. Although acute application of 0.1-0.5 Gy did not lead to an increase of frequency of HR, the chronic exposure of the plants to several orders of magnitude lower dose of 200 muGy led to a 5-6-fold induction of the frequency of HR as compared to the control.

Double-Strand Break Repair in Plants Is Developmentally Regulated

In this study, we analyzed double-strand break (DSB) repair in Arabidopsis (Arabidopsis thaliana) at various developmental stages. To analyze DSB repair, we used a homologous recombination (HR) and point mutation reversion assays based on nonfunctional β-glucuronidase reporter genes. Activation of the reporter gene through HR or point mutation reversion resulted in the appearance of blue sectors after histochemical staining. Scoring of these sectors at 3-d intervals from 2 to 31 d post germination (dpg) revealed that, although there was a 100-fold increase in the number of genomes per plant, the recombination frequency only increased 30-fold. This translates to a recombination rate at 31 dpg (2.77 × 10−8) being only 30% of the recombination rate at 2 dpg (9.14 × 10−8). Conversely, the mutation frequency increased nearly 180-fold, resulting in a 1.8-fold increase in mutation rate from 2 to 31 dpg. Additional analysis of DSBs over the early developmental stages revealed a substantial increase in the number of strand breaks per unit of DNA. Furthermore, RNA analysis of Ku70 and Rad51, two key enzymes in two different DSB repair pathways, and further protein analysis of Ku70 revealed an increase in Ku70 levels and a decrease of Rad51 levels in the developing plants. These data suggest that DSB repair mechanisms are developmentally regulated in Arabidopsis, whereby the proportion of breaks repaired via HR substantially decreases as the plants mature.

Repair of Damaged DNA by Arabidopsis Cell Extract

All living organisms have to protect the integrity of their genomes from a wide range of genotoxic stresses to which they are inevitably exposed. However, understanding of DNA repair in plants lags far behind such knowledge in bacteria, yeast, and mammals, partially as a result of the absence of efficient in vitro systems. Here, we report the experimental setup for an Arabidopsis in vitro repair synthesis assay. The repair of plasmid DNA treated with three different DNA-damaging agents, UV light, cisplatin, and methylene blue, after incubation with whole-cell extract was monitored. To validate the reliability of our assay, we analyzed the repair proficiency of plants depleted in AtRAD1 activity. The reduced repair of UV light– and cisplatin-damaged DNA confirmed the deficiency of these plants in nucleotide excision repair. Decreased repair of methylene blue–induced oxidative lesions, which are believed to be processed by the base excision repair machinery in mammalian cells, may indicate a possible involvement of AtRAD1 in the repair of oxidative damage. Differences in sensitivity to DNA polymerase inhibitors (aphidicolin and dideoxy TTP) between plant and human cell extracts were observed with this assay.

Extremely complex pattern of microsatellite mutation in the germline of wheat exposed to the post-Chernobyl radioactive contamination.

The molecular structure of rare variants at 13 microsatellite loci found in a population of wheat plants grown for one generation in the heavily contaminated 30 km exclusion zone around the Chernobyl Nuclear Power Plant and in a control population was compared. Evidence for rare alterations (variants) was obtained for all 13 loci, including gain and loss of repeats, as well as the complete loss of microsatellite bands. The ratio between gains and losses among variants in the control group was similar to that in the exposed group. Sequencing of variants at six microsatellite loci found in the exposed population revealed extremely complex pattern of germline mutations, including complete deletions of loci, a bias towards mutations with gains and losses of multiple repeat units, and relatively frequent insertions of DNA of unknown origin. The occurrence of large deletions at two loci may be attributed to direct and inverted repeats sequences located just upstream and downstream of the array. The results of our study also suggest that the majority of mutations within the studied wheat microsatellite loci are represented by gains and losses of multiple repeat units, implying that a simple model of replication slippage cannot account for mutation events at these loci. Our data also support the conclusion that the spectra of spontaneous and radiation-induced mutation in wheat may be similar.