Comparative Analysis of the Effect of Gamma-, Electron, and Proton Irradiation on Transcriptomic Profile of Hordeum vulgare L. Seedlings: In Search for Molecular Contributors to Abiotic Stress Resilience.

The development of adaptation strategies for crops under ever-changing climate conditions is a critically important food security issue. Studies of barley responses to ionising radiation showed that this evolutionarily ancient stress factor can be successfully used to identify molecular pathways involved in adaptation to a range of abiotic stressors. In order to identify potential molecular contributors to abiotic stress resilience, we examined the transcriptomic profiles of barley seedlings after exposure to γ-rays, electrons, and protons. A total of 553 unique differentially expressed genes with increased expression and 124 with decreased expression were detected. Among all types of radiation, the highest number of differentially expressed genes was observed in electron-irradiated samples (428 upregulated and 56 downregulated genes). Significant upregulation after exposure to the three types of radiation was shown by a set of ROS-responsive genes, genes involved in DNA repair, cell wall metabolism, auxin biosynthesis and signalling, as well as photosynthesis-related genes. Most of these genes are known to be involved in plant ROS-mediated responses to other abiotic stressors, especially with genotoxic components, such as heavy metals and drought. Ultimately, the modulation of molecular pathways of plant responses to ionising radiation may be a prospective tool for stress tolerance programmes.

Plant adaptation to ionizing radiation: Mechanisms and patterns.

Adaptation to environmental stressors is an essential property of plants that allows them, despite an immobile lifestyle, to survive in a changeable environment. The chain of successive events culminating in the final radiobiological reaction begins with the absorption of energy of ionizing radiation in the cell. Starting from stochastic acts of molecular injury formation, radiation damage gradually acquires deterministic features, which are expressed in a limited number of phenomena that complete plant radiation damage. As plants undergo specialization, the differences between plants and animals become more pronounced, leading to distinct responses to radiation. Chronic radiation exposure may activate biological mechanisms resulting in increased radioresistance of the population. The higher the level of radiation exposure and the sensitivity of plants to radiation, the more intensive the selection. Depending on the circumstances, enhanced radioresistance of a population can be achieved in different ways or has not evolved at all. High dose rates of chronic irradiation leаd to selection for the efficiency of repair systems, while low dose rates activate epigenetic mechanisms that lead to the maintenance of oxidative balance, additional synthesis of chaperones, and control of TEs transposition. Due to huge differences in the radiosensitivity of organisms that make up the ecosystem, irradiation can result in disruption of connections between components of ecosystems which may lead to consequences that can differ drastically from those expected at the organismal and population levels. Therefore, the use of ecological knowledge is essential for understanding the responses of populations and ecosystems to radiation exposure.

Comparative analysis of epigenetic variability in two pine species exposed to chronic radiation in the Chernobyl and Fukushima affected zones.

Comparative analysis of epigenetic variability in two pine species affected as a result of the Chernobyl and Fukushima accidents is presented. The absorbed dose rate within the affected Chernobyl sites varies over a wider range (1.5-24.6 µGy/h) than within the Fukushima sites (3.5-6.5 µGy/h). It was shown that chronic irradiation can change the level of whole genome methylation in pine populations, but in different ways. The genomes of Japanese red pines are hypomethylated, and the degree of methylation and hydroxymethylation decreases with an increase in the level of radiation exposure. In contrast, the percentages of genome methylation and hydroxymethylation in Scots pine populations exceed the reference levels. The observed discrepancy in the patterns of genome-wide DNA methylation can be attributed partly to the design of the study (differences in the climate, radiation dose, age and species of the pines) which could affect the results. In the frame of IRAP analysis, a larger number of different bands was observed in the Chernobyl populations compared to the Japanese populations. Both the Japanese and Chernobyl populations are characterized by significant genetic variability. However, the main part of this variability is observed within populations. The dendrograms, based on presence/absence of IRAP fragments and Nei's genetic distances, revealed subdivisions of the Chernobyl and Japanese populations according to the level of radioactive contamination. Analysis of the results presented will improve our understanding of the mechanisms underlying the responses of pine trees to chronic radiation exposure.

Growth, antioxidant system, and phytohormonal status of barley cultivars contrasting in cadmium tolerance.

Cadmium leads to disturbance of plant growth, and the manifestation of toxicity can vary greatly in different genotypes within one species. In this work we studied the effect of Cd on growth, antioxidant enzyme activity, and phytohormonal status of four barley cultivars (cvs. Simfoniya, Mestnyj, Ca 220702, Malva). According to the earlier study on seedlings, these cultivars were contrast in tolerance to Cd: Simfoniya and Mestnyj are Cd-tolerant and Ca 220702 and Malva are Cd-sensitive. The results presented showed that barley plants accumulated more Cd in straw than in grain. Tolerant cultivars accumulated significantly less Cd in grain than sensitive ones. The leaf area appeared to be a growth parameter susceptible to Cd treatment. The significant differences in leaf area values depended on Cd contamination and were not associated with cultivars' tolerance. Tolerance of cultivars was contingent on the activity of the antioxidant defense system. Indeed, activity of enzymes decreased in sensitive cultivars Ca 220702 and Malva under Cd stress. In contrast, in tolerant cultivars, increased activity of guaiacol peroxidase was revealed. The concentrations of abscisic acid and salicylic acid mostly increased as a result of Cd treatment, while the concentrations of auxins and trans-zeatin either decreased or did not change. The results obtained indicate that antioxidant enzymes and phytohormones play an important role in the response of barley plants to elevated concentrations of cadmium; however, these parameters are not able to explain the differentiation of barley cultivars in terms of tolerance to cadmium at the seedling stage. Therefore, barley intraspecific polymorphism for cadmium resistance is determined by the interplay of antioxidant enzymes, phytohormones, and other factors that require further elucidation.

From tangled banks to toxic bunnies; a reflection on the issues involved in developing an ecosystem approach for environmental radiation protection.

The objective of this paper is to present the results of discussions at a workshop held as part of the International Congress of Radiation Research (Environmental Health stream) in Manchester UK, 2019. The main objective of the workshop was to provide a platform for radioecologists to engage with radiobiologists to address major questions around developing an Ecosystem approach in radioecology and radiation protection of the environment. The aim was to establish a critical framework to guide research that would permit integration of a pan-ecosystem approach into radiation protection guidelines and regulation for the environment. The conclusions were that the interaction between radioecologists and radiobiologists is useful in particular in addressing field versus laboratory issues where there are issues and challenges in designing good field experiments and a need to cross validate field data against laboratory data and vice versa. Other main conclusions were that there is a need to appreciate wider issues in ecology to design good approaches for an ecosystems approach in radioecology and that with the capture of 'Big Data', novel tools such as machine learning can now be applied to help with the complex issues involved in developing an ecosystem approach.

Threshold dose rates for the cytogenetic effects in crested hairgrass populations from the Semipalatinsk nuclear test site, Kazakhstan.

An assessment of cytogenetic effects in crested hairgrass (Koeleria gracilis Pers.) populations was carried out within the Semipalatinsk nuclear test site (Kazakhstan) where combat radioactive substances were tested in 1953-1957. Current levels of radioactive contamination within this site are varied by orders of magnitude, while soil characteristics and heavy metal pollution are similar. The main contribution to the absorbed by plants doses at this site was caused by incorporated 90Sr. The frequency of cytogenetic alterations in crested hairgrass was investigated in a wide range of doses (10-4-13 Gy/growing season) at 100 sampling points. For the first time in the field conditions the shape of the cytogenetic effects - dose rate relationship was evaluated with acceptable accuracy and found to be nonlinear. The frequency of aberrant cells remained practically unchanged up to 49 µGy/h. Exceeding the threshold dose rate lead to a steep increase in the aberrant cells frequency from less than 2% up to 16%. The main contribution to the cytogenetic effects was made by double bridges and fragments. Breakpoints for other types of cytogenetic alterations were also evaluated (7 µGy/h for single fragments and bridges; 74 for double fragments and bridges; 81 for mitotic abnormalities).

A Pipeline for Non-model Organisms for de novo Transcriptome Assembly, Annotation, and Gene Ontology Analysis Using Open Tools: Case Study with Scots Pine.

RNA sequencing (RNA-seq) has opened up the possibility of studying virtually any organism at the whole transcriptome level. Nevertheless, the absence of a sequenced and accurately annotated reference genome may be an obstacle for applying this technique to non-model organisms, especially for those with a complex genome. While de novo transcriptome assembly can circumvent this problem, it is often computationally demanding. Furthermore, the transcriptome annotation and Gene Ontology enrichment analysis without an automatized system is often a laborious task. Here we describe step-by-step the pipeline that was used to perform the transcriptome assembly, annotation, and Gene Ontology analysis of Scots pine (Pinus sylvestris), a gymnosperm species with complex genome. Using only free software available for the scientific community and running on a standard personal computer, the pipeline intends to facilitate transcriptomic studies for non-model species, yet being flexible to be used with any organism.

Multifaceted effects of chronic radiation exposure in Japanese red pines from Fukushima prefecture.

Despite many studies carried out to date, the long-term effects of chronic exposure on plants and animals inhabiting the territories affected by the Fukushima Dai-Ichi NPP accident remain the subject of scientific discussions. Our investigations were performed on Japanese red pine, the native tree species that is widely spread in the radioactive contaminated areas. Earlier observations revealed the radiation-induced cancellation of the apical dominance in young trees of this species. To understand the mechanism of such transformation, we evaluated the morphometric parameters of needles, the frequency of cytogenetic abnormalities, and the concentrations of the major classes of phytohormones in several natural populations of young red pine trees growing under different exposure conditions in Fukushima prefecture. No significant relationships between the morphometric parameters of needles and dose rates at the experimental sites were revealed. The frequencies of aberrant cells in the needle's intercalary meristem and the frequencies of cancellation of the apical dominance in the young pine populations in the radioactive contaminated areas were significantly higher than in the reference population. However, only cytogenetic abnormalities increased with the dose rate. We have not found the relation between the frequency of cytogenetic abnormalities in needles and cancellation of the apical dominance in the individual trees. In this paper, for the first time, it is shown that chronic radiation exposure changes the concentration ratio of the major classes of phytohormones in the needles of Japanese red pine. Given the complete lack of information about the most important regulatory system of plants in chronically irradiated populations, this study fills a substantial gap in our knowledge. Finally, our findings indicated that the most probable causes of the cancellation of apical dominance observed in chronically exposed Japanese red pines are radiation damage to the apical meristems of the trees and changes in their phytohormonal balance.

Scots pine as a promising indicator organism for biomonitoring of the polluted environment: A case study on chronically irradiated populations.

In this paper the main results of long-term (2003-2016) observations on Scots pine populations inhabiting sites affected by the Chernobyl accident are presented. Populations growing for many years under chronic radiation exposure are characterized by the enhanced mutation rates, increased genetic diversity, changes in the gene expression and in the level of genome-wide methylation, alterations in the temporal dynamics of cytogenetic abnormalities and genetic structure of populations. However, significant changes at the genetic level had no effects on enzymatic activity, morphological abnormalities, and reproductive ability of pine trees. The results presented increase our understanding of the long-term effects of chronic radiation exposure on plant populations in the wild nature and provide important information for the management and monitoring of radioactively contaminated territories.

The response profile to chronic radiation exposure based on the transcriptome analysis of Scots pine from Chernobyl affected zone.

Radioactive contamination of the natural areas is one of the most long-lasting anthropogenic impacts on the environment. Scots pine (Pinus sylvestris L.) is a promising organism for radiation-related research because of its high radiosensitivity, but the genome size of Pinacea species has imposed obstacles for high-throughput studies so far. In this work, we conducted the analysis of the de novo assembled transcriptome of Scots pine populations growing in the Chernobyl-affected zone, which is still today contaminated with radionuclides because of the accident at the nuclear power plant in 1986. The transcriptome profiles indicate a clear pattern of adaptive stress response, which seems to be dose-dependent. The transcriptional response indicates a continuous modulation of the cellular redox system, enhanced expression of chaperones and histones, along with the control of ions balance. Interestingly, the activity of transposable element families is inversely correlated to the exposure levels to radiation. These adaptive responses, which are triggered by radiation doses 30 times lower than the one accepted as a safe for biota species by international regulations, suggest that the environmental management in radiation protection should be reviewed.

The tubercular badger and the uncertain curve:- The need for a multiple stressor approach in environmental radiation protection.

This article presents the results of a workshop held in Stirling, Scotland in June 2018, called to examine critically the effects of low-dose ionising radiation on the ecosphere. The meeting brought together participants from the fields of low- and high-dose radiobiology and those working in radioecology to discuss the effects that low doses of radiation have on non-human biota. In particular, the shape of the low-dose response relationship and the extent to which the effects of low-dose and chronic exposure may be predicted from high dose rate exposures were discussed. It was concluded that high dose effects were not predictive of low dose effects. It followed that the tools presently available were deemed insufficient to reliably predict risk of low dose exposures in ecosystems. The workshop participants agreed on three major recommendations for a path forward. First, as treating radiation as a single or unique stressor was considered insufficient, the development of a multidisciplinary approach is suggested to address key concerns about multiple stressors in the ecosphere. Second, agreed definitions are needed to deal with the multiplicity of factors determining outcome to low dose exposures as a term can have different meanings in different disciplines. Third, appropriate tools need to be developed to deal with the different time, space and organisation level scales. These recommendations permit a more accurate picture of prospective risks.

'Omic' technologies as a helpful tool in radioecological research.

This article presents a brief review of the modern 'omic' technologies, namely genomics, epigenomics, transcriptomics, proteomics, and metabolomics, as well as the examples of their possible use in radioecology. For each technology, a short description of advances, limitations, and instrumental applications is given. In addition, the review contains examples of successful use of 'omic' technologies in the assessment of biological effects of pollutants in the field conditions.

Chronic radiation exposure modifies temporal dynamics of cytogenetic but not reproductive indicators in Scots pine populations.

Over a period of 13 years (2003-2015), reproductive and cytogenetic effects are investigated in Scots pine populations growing in the Bryansk region of Russia radioactively contaminated as a result of the Chernobyl accident. In reference populations, the frequencies of cytogenetic abnormalities are shown to change with time in a cyclic manner. In chronically exposed populations, the cyclic patterns in temporal dynamics of cytogenetic abnormalities appear to be disturbed. In addition, a tendency to decrease in the frequencies of cytogenetic abnormalities with time as well as an increase in their variability with dose rate is revealed. In contrast, no significant impact of chronic radiation exposure on the time dynamics of reproductive indexes is detected. Finally, long-term observations on chronically exposed Scots pine populations revealed qualitative differences in the temporal dynamics of reproductive and cytogenetic indicators.

Radiation exposure of barley seeds can modify the early stages of plants' development.

The reactions of barley seeds (Nur and Grace varieties) in terms of the root and sprout lengths, germination and root mass were studied after γ-irradiation with doses in the range of 2-50 Gy. The dose range in which plants' growth stimulation occurs (16-20 Gy) was identified. It was shown that increased size of seedlings after irradiation with stimulating doses was due to the enhancing pace of development rather than an earlier germination. The activity of the majority of the enzymes studied increased in the range of doses that cause stimulation of seedlings development. The influences of the dose rate, the quality of seeds, their moisture and time interval between irradiation and initiation of germination on the manifestation of the effects of radiation were investigated. The experimental data on the effect of γ-irradiation on seedlings development were significantly better explained by mathematical models that take into account the hormetic effect.

Influence of long-term chronic exposure and weather conditions on Scots pine populations.

Over a period of 8 years (2007-2014), we were evaluating seed quality and morphological abnormalities in Scots pine trees affected as a result of the Chernobyl accident. The calculated dose rates for the trees at the study sites varied from background values at the reference sites to 40 mGy/year at the most contaminated site. We investigated whether radioactive contamination and/or weather factors could decrease the reproductive capacity or increase the frequency of morphological abnormalities of needles in pine trees. Scots pine seeds are characterized by high interannual variability of viability, which is largely determined by weather conditions. No consistent differences in reproductive capacity were detected between the impacted and reference populations. Brachyblasts with three needles were found only in the affected populations; however, their frequency was very low and only at the very border of significance at the p < 0.10 level.

Radiation exposure in the remote period after the Chernobyl accident caused oxidative stress and genetic effects in Scots pine populations.

Even 30 years after the Chernobyl accident, biological effects of irradiation are observed in the chronically exposed Scots pine populations. Chronic radiation exposure at dose rates above 50 mGy∙yr-1 caused oxidative stress and led to the increase of antioxidants concentrations in these populations. Genetic variability was examined for 6 enzymes and 14 enzymatic loci of 6 Scots pine populations. Dose rates over 10 mGy∙yr-1 caused the increased frequency of mutations and changes in genetic structure of Scots pine populations. However, the same dose rates had no effect on enzymatic activities. The results indicate that even relatively low dose rates of radiation can be considered as an ecological factor which should be taken into account for ecological management and radiation protection of biota species.

Ecological effects of exposure to enhanced levels of ionizing radiation.

Irradiation of plants and animals can result in disruption of ecological relationships between the components of ecosystems. Such effects may act as triggers of perturbation and lead to consequences that may differ essentially from expected ones based on effects observed at the organismal level. Considerable differences in ecology and niches occupied by different species lead to substantial differences in doses of ionizing radiation absorbed by species, even when they all are present in the same environment at the same time. This is especially evident for contamination with α-emitting radionuclides. Radioactive contamination can be considered an ecological factor that is able to modify the resistance in natural populations. However, there are radioecological situations when elevated radioresistance does not evolve or persist. The complexity and non-linearity of the structure and functioning of ecosystems can lead to unexpected consequences of stress effects, which would appear harmless if they were assessed within the narrower context of organism-based traditional radioecology. Therefore, the use of ecological knowledge is essential for understanding responses of populations and ecosystems to radiation exposure. Integration of basic ecological principles in the design and implementation of radioecological research is essential for predicting radiation effects under rapidly changing environmental conditions.

Addressing ecological effects of radiation on populations and ecosystems to improve protection of the environment against radiation: Agreed statements from a Consensus Symposium.

This paper reports the output of a consensus symposium organized by the International Union of Radioecology in November 2015. The symposium gathered an academically diverse group of 30 scientists to consider the still debated ecological impact of radiation on populations and ecosystems. Stimulated by the Chernobyl and Fukushima disasters' accidental contamination of the environment, there is increasing interest in developing environmental radiation protection frameworks. Scientific research conducted in a variety of laboratory and field settings has improved our knowledge of the effects of ionizing radiation on the environment. However, the results from such studies sometimes appear contradictory and there is disagreement about the implications for risk assessment. The Symposium discussions therefore focused on issues that might lead to different interpretations of the results, such as laboratory versus field approaches, organism versus population and ecosystemic inference strategies, dose estimation approaches and their significance under chronic exposure conditions. The participating scientists, from across the spectrum of disciplines and research areas, extending also beyond the traditional radioecology community, successfully developed a constructive spirit directed at understanding discrepancies. From the discussions, the group has derived seven consensus statements related to environmental protection against radiation, which are supplemented with some recommendations. Each of these statements is contextualized and discussed in view of contributing to the orientation and integration of future research, the results of which should yield better consensus on the ecological impact of radiation and consolidate suitable approaches for efficient radiological protection of the environment.

Genetic diversity in Scots pine populations along a radiation exposure gradient.

Polymorphisms of antioxidant enzymes were studied in the endosperm and embryos of seeds from Scots pine populations inhabiting sites in the Bryansk region of Russia radioactively contaminated as a result of the Chernobyl accident. Chronic radiation exposure at dose rates from 0.8 µGy/h led to a significant increase in the rate of enzymatic loci mutations. The main parameters of genetic variability of the affected Scots pine populations had considerably higher values than those from the reference site. Changes in the genetic makeup of Scots pine populations were observed at dose rates greater than 10.4 µGy/h. However, the higher mutation rate had no effect on the activities of antioxidant enzymes.