Germination and Growth Characteristics of nud Knockout and win1 Knockout Barley Lines under Salt Stress.

Hordeum vulgare genes NUD (HvNUD) and WIN1 (HvWIN1) play a regulatory role in cuticle organization. Because the cuticle is a key evolutionary acquisition of plants for protection against environmental factors, a knockout (KO) of each gene may alter their ability to adapt to unfavorable conditions. A potential pleiotropic effect of HvNUD or HvWIN1 gene mutations can be assessed under salt stress. Initial developmental stages are the most sensitive in living organisms; therefore, we evaluated salt tolerance of nud KO and win1 KO barley lines at the seedling stage. Air-dried barley grains of the KO lines and of a wild-type (WT) line were germinated in NaCl solutions (50, 100, or 150 mM). Over 30 physiological and morphological parameters of seedlings were assessed. Potential pleiotropic effects of the HvNUD gene KO under salt stress included the stimulation of root growth (which was lower under control conditions) and root necrosis. The pleiotropic effects of the HvWIN1 gene KO under the stressful conditions manifested themselves as maintenance of longer root length as compared to the other lines; stable variation of most of morphological parameters; lack of correlation between root lengths before and after exposure to NaCl solutions, as well as between shoot lengths; and the appearance of twins. Salt tolerance of the analyzed barley lines could be ranked as follows: nud KO > win1 KO ≈ WT, where nud KO lines were the most salt-tolerant. A comparison of effects of salinity and ionizing radiation on nud KO and win1 KO barley lines indicated differences in tolerance of the lines to these stressors.

Investigation of biological-rhythm patterns: physiological and biochemical effects in herbaceous plants exposed to low-level chronic radiation - part 1: nonirradiated seeds.

HYPOTHESIS: Because reactive oxygen species are involved in the regulation of biological rhythms, we hypothesized that intra-annual variability of seed progeny quality at low doses of ionizing radiation (LDIRs) would differ from that of background plants. MATERIALS AND METHODS: We conducted 12 consecutive experiments using the roll culture method by germinating seeds (monthly for 3 weeks) of six herbaceous plant species (Bromus inermis, Geum aleppicum, Plantago major, Rumex confertus, Silene latifolia, and Taraxacum officinale) growing under conditions of chronic radiation in the East Ural Radioactive Trace (EURT). We assessed physiological (seed viability and abnormality frequency) and biochemical (low-molecular-weight antioxidants, LMWAs) parameters of seedlings. RESULTS: Total absorbed dose rates of maternal plants (TADRplants) and seed embryos (TADRseeds) in the EURT exceeded background levels by 1-3 and 1-2 orders of magnitude, respectively. Nonlinear dependencies on TADR were mainly characteristic of physiological and biochemical parameters. For most populations of the studied species (B. inermis, G. aleppicum, R. confertus, and S. latifolia), seedling survival and root length decreased in the autumn-winter period, while the frequency of abnormal seedlings increased. The content of LMWAs could be ranked as R. confertus > B. inermis > G. aleppicum > S. latifolia, in good agreement with the presence of anthocyanin pigmentation in the plants. The lowest synthesis of antioxidants in seedlings was observed in winter. A high LMWA content promoted growth and reduced the frequency of abnormal seedlings. CONCLUSIONS: These results underscore a multistage nature of the impact of LDIRs on intra-annual biological rhythms in plants. High heterogeneity in reference group 'wild grasses' and diversity of their radiobiological effects should help to develop methods of radiation protection for natural ecosystems and facilitate approaches used by the International Commission on Radiological Protection.HighlightsAbsorbed dose rates for six plant species in the East Ural Radioactive Trace (EURT) area range from 0.11 to 73.89 µGy h-s (plants) and 0.11 to 6.88 µGy h-s (seed embryos).Intra-annual rhythms of physiological and biochemical parameters in the EURT zone differ from those in background seedlings.Plants in the EURT area exhibit a wide range of trait variability, asynchrony of the manifestation of the effects, nonlinear dose-response relations, and hormesis.A high content of low-molecular-weight antioxidants (LMWAs) is associated with low frequency of developmental abnormalities and high viability of seed progeny.

Multiannual Assessment of Quality of Plantago major L. Seed Progeny from Kyshtym Radiation Accident Area: Weather-Dependent Effects.

The effects of low-dose radiation that are observed in plant populations in radioactively contaminated areas are variable. One of the reasons is the influence of fluctuating weather conditions and the interaction of radiation with weather factors. This article summarizes results of 12-year research on the viability and radioresistance of greater plantain (Plantago major L.) seed progeny growing in the East Ural Radioactive Trace (EURT) zone and in control (nonradioactive) areas, with consideration of weather conditions' variability. The EURT was formed by the Kyshtym accident, which occurred in 1957 at the Mayak Production Association. Absorbed dose rates of P. major parental plants in the pollution gradient were 14.5-165.9 µGy h-1, which correspond to a low-dose range. Seed progeny quality was evaluated as seed weight, the survival rate, and root length of 21-day seedlings. Interannual variability in the studied parameters was high, and their ranges overlapped between EURT groups of seeds and control groups in most cases. The number of significant correlations between the parameters of seed quality and weather conditions was higher in EURT groups than in control populations. In the control groups of seeds, 88.9% of correlations were negative, whereas in the EURT groups, 78.5% were positive.

Seedling Biometry of nud Knockout and win1 Knockout Barley Lines under Ionizing Radiation.

The genes NUD and WIN1 play a regulatory role in cuticle organization in barley. A knockout (KO) of each gene may alter plant mechanisms of adaptation to adverse environmental conditions. A putative pleiotropic effect of NUD or WIN1 gene mutations in barley can be assessed in a series of experiments in the presence or absence of a provoking factor. Ionizing radiation is widely used in research as a provoking factor for quantifying adaptive potential of living organisms. Our aim was to evaluate initial stages of growth and development of barley lines with a KO of NUD or WIN1 under radiation stress. Air-dried barley grains with different KOs and wild-type control (WT) were exposed to γ-radiation at 50, 100, or 200 Gy at a dose rate of 0.74 R/min. Approximately 30 physiological parameters were evaluated, combined into groups: (1) viability, (2) radiosensitivity, and (3) mutability of barley seed progeny. Seed germination, seedling survival, and shoot length were similar among all barley lines. Naked nud KO lines showed lower weights of seeds, roots, and seedlings and shorter root length as compared to win1 KO lines. The shoot-to-root length ratio of nud KO lines' seedlings exceeded that of win1 KO and WT lines. In terms of the number of seedlings with leaves, all the KO lines were more sensitive to pre-sowing γ-irradiation. Meanwhile, the radioresistance of nud KO lines (50% growth reduction dose [RD50] = 318-356 Gy) and WT plants (RD50 = 414 Gy) judging by seedling weight was higher than that of win1 KO lines (RD50 = 201-300 Gy). Resistance of nud KO lines to radiation was also demonstrated by means of root length (RD50 = 202-254 Gy) and the shoot-to-root length ratio. WT seedlings had the fewest morphological anomalies. In nud KO lines, mainly alterations of root shape were found, whereas in win1 KO lines, changes in the color and shape of leaves were noted. Thus, seedlings of nud KO lines are characterized mainly by changes in the root system (root length, root number, and root anomalies). For win1 KO lines, other parameters are sensitive (shoot length and alterations of leaf shape and color). These data may indicate a pleiotropic effect of genes NUD and WIN1 in barley.

Evaluation of the genetic structure of Bromus inermis populations from chemically and radioactively polluted areas using microsatellite markers from closely related species.

Hypothesis: The anthropogenic effects can be manifested in a decrease in genetic diversity and the population differentiation, and increase in the frequencies of rare and/or private alleles.Materials and methods: We have selected a collection of primers for B. inermis, consisting of 21 microsatellite loci from B. sterilis, B. tectorum and Triticum aestivum.Results: Only 38% of SSR primers showed good transferability and were used for B. inermis population studies from technogenic pollution areas. We revealed 42 alleles at eight loci, and the number of alleles per locus varied from one to 13 per populations. The percentage of polymorphic loci in B. inermis populations was 48.44%. A total of 22 rare, 14 private and 9 both rare and private alleles were reported. There were no correlations between geographic and genetic distances. Only 6.8% of the genetic variability was distributed among B. inermis populations.Conclusion: There was no decrease in genetic diversity found in B. inermis populations growing under anthropogenic stress. No significant differences in the number of rare and private alleles in the background and impact populations of B. inermis were found. The smooth brome is characterized by low differentiation of the populations. Possible reasons for this phenomenon are discussed.

Phytochemical Analysis of Phenolics, Sterols, and Terpenes in Colored Wheat Grains by Liquid Chromatography with Tandem Mass Spectrometry.

The colored grain of wheat (Triticum aestivum L.) contains a large number of polyphenolic compounds that are biologically active ingredients. The purpose of this work was a comparative metabolomic study of extracts from anthocyaninless (control), blue, and deep purple (referred to here as black) grains of seven genetically related wheat lines developed for the grain anthocyanin pigmentation trait. To identify target analytes in ethanol extracts, high-performance liquid chromatography was used in combination with Bruker Daltonics ion trap mass spectrometry. The results showed the presence of 125 biologically active compounds of a phenolic (85) and nonphenolic (40) nature in the grains of T. aestivum (seven lines). Among them, a number of phenolic compounds affiliated with anthocyanins, coumarins, dihydrochalcones, flavan-3-ols, flavanone, flavones, flavonols, hydroxybenzoic acids, hydroxycinnamic acids, isoflavone, lignans, other phenolic acids, stilbenes, and nonphenolic compounds affiliated with alkaloids, carboxylic acids, carotenoids, diterpenoids, essential amino acids, triterpenoids, sterols, nonessential amino acids, phytohormones, purines, and thromboxane receptor antagonists were found in T. aestivum grains for the first time. A comparative analysis of the diversity of the compounds revealed that the lines do not differ from each other in the proportion of phenolic (53.3% to 70.3% of the total number of identified compounds) and nonphenolic compounds (46.7% to 29.7%), but diversity of the compounds was significantly lower in grains of the control line. Even though the lines are genetically closely related and possess similar chemical profiles, some line-specific individual compounds were identified that constitute unique chemical fingerprints and allow to distinguish each line from the six others. Finally, the influence of the genotype on the chemical profiles of the wheat grains is discussed.

Genetic polymorphism of Plantago major populations from the radioactive and chemical polluted areas.

The variability of nine microsatellite loci was studied for Plantago major L. populations from radioactive (East-Ural Radioactive Trace, EURT) and chemical (Karabash Copper Smelter, KCS) contaminated areas (Urals, Russia). The absorbed dose rates in the EURT area were 178-1455 times higher than background, and the indices of the total toxic load in the KCS area were 13-42 times higher than background values. In total, 65 alleles were identified in P. major populations, while the number of alleles per locus in the EURT and KCS samples was lower than in the background samples. The expected heterozygosity in all loci significantly exceeded the observed, indicating a high level of inbreeding. The largest number of rare alleles (11-21) was found in background samples, of which 3-7 alleles were private. In the technogenically disturbed zones, 8-11 rare alleles (1-2 private) were noted. The Bayesian analysis (K = 3) showed that no unique groups were found in any of the areas; descendants of all founders (pioneers) were represented in each population, but in different proportions. However, only 4.1% of the variability was distributed between local P. major populations (FST = 0.041) and 95.9% was concentrated within the samples. A pairwise comparison revealed genetic differentiation between all EURT samples. In the KCS area, there was no significant differentiation in pairs of samples that were at a distance of 3-4 km from each other. For samples from the KCS and background sites, the Mantel test showed a statistically significant relationship between geographical and genetic distances, therefore, the intensity of migration flows between these areas is high. For samples from the EURT and background areas, no such dependence was found. In both impact zones, P. major populations showed reduced genetic diversity. This article discusses the causes of this phenomenon.

Biochemical and genetic polymorphism of Bromopsis inermis populations under chronic radiation exposure.

MAIN CONCLUSION: For the subsequent assessment of the genetic mechanisms responsible for the resistance of plants to chronic irradiation, the analysis of RAPD-cDNA with the subsequent isolation, cloning, and sequencing of expressed polymorphic sequences is a promising technique. A study was conducted on Bromopsis inermis populations that have been growing for a long time in the EURT area. Using RAPD primers, we studied the genetic spectra of plants. In analysing the UPGMA algorithm, we identified two well-distinguishable clusters with a high level of bootstrap support (> 85%): background samples hit the first, and impact samples hit the second. Our data indicate a decrease in diversity in the most polluted population, as well as the appearance of new alleles in chronically irradiated samples of the B. inermis. Smooth brome seedlings were characterised by the content of anthocyanins, comparable with other types of cereals. In the gradient of chronic irradiation, the relative content of anthocyanins was not significantly changed. For the first time, the partial nucleotide sequences of the key genes of anthocyanin biosynthesis (Chi and F3h) in the brome were determined, these sequences were found to be 191 and 356 bp in length, respectively, and were cloned and sequenced. Three copies of the Chi gene were identified in the B. inermis genome. One copy (BiChi-1) clustered with the sequences of the Aegilops tauschii gene (D genome), and the other two copies (BiChi-2 and BiChi-3) formed a separate cluster in the Pooideae subfamily adjacent to Hordeum vulgare. In the copy of BiChi-1, a complete deletion of intron 1 was detected. For the F3h gene, one copy of the B. inermis gene was obtained, which forms a separate branch in the subfamily Pooideae.

Radiosensitivity and mutability of wheat seed progeny cultivated under adverse environments.

This research analysed the growth process dynamics of soft wheat (Triticum aestivum L.) seeds cultivated in contrasting microclimatic conditions. We used acute gamma irradiation (5-50 Gy) as a provocative factor to detect hidden differences in the adaptive potential of seeds cultivated under adverse conditions (wet and cool field season) in comparison to seeds obtained under controlled conditions (hydroponic greenhouse). Seeds harvested from wheat plants cultivated in challenging field conditions demonstrated lower weight; moreover, their offspring also had a lower weight and seedling survival rate, as well as a delay in the formation of the fourth - sixth roots. The discrepancy in growth characteristics increased from the beginning to the end of the experiments and was particularly pronounced in offspring cultivated under adverse conditions throughout the entire experiment. The offspring of control seeds were more radioresistant than their field seed counterparts. At the same time, the "field" seeds were characterised by stimulation of growth and development of seedlings in their responses to irradiation. Few seedlings grown from "greenhouse" seeds exhibited evidence of root necrosis and twisted roots. Among the field plants, unusual developmental anomalies for 'greenhouse' seeds were encountered, including the disruption of gravitropism, thickening of roots, changes in the form of coleoptiles and leaves, and necrotic coleoptiles. Gamma irradiation stimulated an increase in the number of seedlings with various developmental disorders. In the case of seed progeny grown under adverse conditions, developmental anomalies were more frequent following irradiation relative to optimal conditions.

Radionuclide uptake and dose assessment of 14 herbaceous species from the east-Ural radioactive trace area using the ERICA Tool.

The evaluation of radiation exposure in 14 species of herbaceous plants from the East-Ural Radioactive Trace (EURT) zone was performed, using the ERICA Tool, v. 1.2. Recent (up to 2015) levels of radionuclide activity concentration were measured in soil and vegetative plant mass. 239,240Pu content was used for the first time to estimate external dose rates for herbaceous plant species along the pollution gradient. In addition, a new approach to assessing the geometry of objects was adopted, including not only aboveground but also underground plant organs. This improved approach to the evaluation of radiation exposure confirms previous findings that herbaceous plant populations currently exist under low-level chronic exposure in the EURT area. This reassessment based on new data suggests a 48-977-fold increase in the total dose rate per plant organism at the most polluted site compared to background areas. The highest capacity for the transfer of 90Sr and 137Cs was observed in Taraxacum officinale and Plantago major. In these species, the total dose rate per plant exceeded 150 µGy h-1 due to 90Sr + 137Cs + 239,240Pu radionuclide anthropogenic pollution in the EURT zone. All estimated total dose rates per plant were below the dose rate screening value of 400 µGy h-1.

Temporal variability of the quality of Taraxacum officinale seed progeny from the East-Ural radioactive trace: is there an interaction between low level radiation and weather conditions?

HYPOTHESIS: The multiple stressors, in different combinations, may impact differently upon seed quality, and low-level doses of radiation may enhance synergistic or antagonistic effects. RESULTS: During 1991-2014 we investigated the quality of the dandelion (Taraxacum officinale s.l.) seed progeny growing under low-level radiation exposure at the East-Ural Radioactive Trace (EURT) area (result of the Kyshtym accident, Russia), and in plants from areas exposed to background radiation. The viability of the dandelion seed progeny was assessed according to chronic radiation exposure, accounting for the variability of weather conditions among years. Environmental factors (temperature, precipitation, and their ratio in different months) can modify the radiobiological effects. We found a wide range of possible responses to multiple stressors: inhibition, stimulation, and indifferent effects in different seasons. CONCLUSION: The intraspecific variability of the quality of dandelion seed progeny was greatly increased under conditions of low doses of chronic irradiation. Temperature was the most significant factor for seed progeny formation in the EURT zone, whereas the sums of precipitation and ratios of precipitation to temperature dominantly affected organisms from the background population.

Genetic variation in natural Melandrium album populations exposed to chronic ionizing radiation.

The effect of radiation pollution on genetic variation in natural populations of Melandrium album was investigated at the head part of the East-Ural Radioactive Trace (EURT) and background areas. The highest genetic differentiation estimated using F ST was revealed between compared pairs of the background and impact samples in populations of M. album. The highest rate of polymorphism was observed at the closest to nuclear accident, Impact-1 site. The unique alleles (Mdh-3104, Pgi-2106, Lap 105, Mdh-296, and Dia 94) were discovered at the EURT. Individuals from chronically low-level irradiated sites were genetically closer than to plants from background sites using Nadhdh locus. The increase of the frequency of unique homozygous and heterozygous genotypes was identified in populations of M. album growing under chronic radiation exposure conditions. The largest contribution to the group of unique heterozygous genotypes at the EURT was made by three loci - Lap, Pgi-2, and Nadhdh; the main role in interpopulation differentiation of samples was made by the alleles Sod-2115, Skdh 100, and Nadhdh 100. Our results provide evidence for the correlation between the increase of genetic variation other than the «genetic erosion¼ and chronic radiation exposure factor in natural plant populations.

The influence of Plagiorchis mutationis larval infection on the cellular immune response of the snail host Lymnaea stagnalis.

The effects of trematode Plagiorchis mutationis parasitism on the cellular immune responses of the snail host Lymnaea stagnalis were investigated. The number of spreading blood cells (hemocytes) from infected snails was significantly less (69.5%) than in uninfected individuals (79.2%). The phagocytic activity of blood cells in infected snails was also significantly less (17.2%) compared to uninfected snails (27.8%). The hemocytes from the infected snails did not form a complete capsule around Sephadex beads in vitro. The protective reactions of the snail hosts were independent of the parasite load (daily cercariae production). In vitro, dead cercariae of P. mutationis were encapsulated by hemocytes from uninfected snails. The hemocytes of the infected snails formed a complete capsule around only 20% of dead cercariae in vitro, with remaining cercariae either unencapsulated (50% of cercariae) or incompletely encapsulated (30% of cercariae). The total number of hemocytes in the infected snails was twofold less than in uninfected individuals. The results of this study showed that the cellular response of snail host L. stagnalis to P. mutationis trematode infection is similar to the previously studied snail-trematode model systems.

Comparison of remote consequences in Taraxacum officinale seed progeny collected in radioactively or chemically contaminated areas.

We carried out a comparative study of seed progeny taken from the dandelion (Taraxacum officinale s.l.) coenopopulations exposed for a long time to radioactive or chemical contamination originated from the East-Ural radioactive trace zone (EURT) or Nizhniy Tagil metallurgical combine impact zone (NTMC), respectively. Coenopopulations from EURT, NTMC and background areas significantly differ from each other with respect to the qualitative and quantitative composition of allozyme phenes. An analysis of clonal diversity showed the uniqueness of all coenopopulations in terms of their phenogenetics. P-generation seed viability was found to decrease in a similar manner as all types of the industrial stress increased. Studies of F (1)-generation variability in radio- and metal resistance by family analysis showed that seed progeny from EURT impact zone possessed high viability that, however, was accompanied by development of latent injuries resulting in low resistance to additional man-caused impacts. In F (1)-generation originated from NTMC zone, high seed viability was combined with increased resistance to provocative heavy metal and radiation exposure. No significant differences in responses to 'habitual' and 'new' factors, i.e. pre-adaptation effect, were found in samples from the contaminated areas.