Science of the Total Environment 496 (2014) 317–327

Contents lists available at ScienceDirect

Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Genetic diversity in Scots pine populations along a radiation exposure gradient Stanislav A. Geras'kin ⁎, Polina Yu. Volkova Russian Institute of Agricultural Radiology and Agroecology, Kievskoe shosse, 109 km, 249032 Obninsk, Russia

H I G H L I G H T S • • • • •

Polymorphism of antioxidant enzymes was studied in affected Scots pine populations. Genetic processes in affected Scots pine populations increase genetic diversity. Chronic exposure at dose rates from 0.8 μGy/h lead to increasing of mutation rates. Changes in population genetic structure were observed at dose rates from 10.4 μGy/h. The higher rate of mutations had no effect on antioxidant enzymes activities.

a r t i c l e

i n f o

Article history: Received 28 March 2014 Received in revised form 5 July 2014 Accepted 5 July 2014 Available online xxxx Editor: J. P. Bennett Keywords: Scots pine populations Enzymatic loci mutations Genetic diversity Antioxidant enzymes Chernobyl accident Chronic radiation exposure

a b s t r a c t 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. © 2014 Elsevier B.V. All rights reserved.

1. Introduction As a result of radiation accidents in the South Urals, Russia in 1957 and at the Chernobyl NPP in 1986, large forested areas were severely affected because atmospherically dispersed radionuclides were absorbed through leaf stomata, directly influencing the plants (Tikhomirov and Shcheglov, 1994). Conifers are particularly vulnerable to radiation exposure, due to their large chromosome size (Sparrow et al., 1968). After the Chernobyl accident, the area of lethal destruction of pine forests was 500–600 ha, with strong and moderate damage across 3000 and 12 000 ha, respectively (Kozubov and Taskaev, 2002). As a result of the accident in the South Urals, pines were completely lost in an area of 2000 ha (Alexakhin et al., 2004). Much larger forest areas were polluted with levels of radioactive contamination that were insufficient ⁎ Corresponding author. Tel.: +7 48439 96964; fax: +7 48439 68066. E-mail address: [email protected] (S.A. Geras'kin).

http://dx.doi.org/10.1016/j.scitotenv.2014.07.020 0048-9697/© 2014 Elsevier B.V. All rights reserved.

for the mass death of trees. Currently, little is known regarding the long-term consequences of the chronic radiation exposure of these forest ecosystems. Chronic radiation exposure can affect natural populations in many ways; genetic change is one of the more subtle effects with potentially large long-term consequences. Genetic variation is one of the three pillars of biodiversity recognized in the Rio Convention of 1993, because it contributes to phenotypic diversity and may facilitate adaptation to environmental change. This is especially important in long-lived trees, where adaptation to rapid changes in the environment must rely on existing variation within populations. Chronic radiation exposure can alter the structure of intra-population variability (Shevchenko et al., 1992; Theodorakis, 2001). However, to date, there is no complete understanding of the effects of increased frequencies of genetic and cytogenetic damage in somatic and germ cells on reproductive capacity, adaptive differentiation and the general fate of the populations (Geras'kin et al., 2013). Radiation is a form of stress that elicits community responses

318

S.A. Geras'kin, P.Y. Volkova / Science of the Total Environment 496 (2014) 317–327

often similar to those resulting from other forms of stress (Woodwell, 1967). Therefore, considerable insight into the basic nature of plant communities and their ability to withstand or to recover from stress can be obtained from observations of irradiated forests. Electrophoresis of isozymes can reveal more than 40% of the point mutations related to amino acid substitutions in isozymes as well as all mutations that affect isozyme functions (Altukhov, 2003). Therefore, this method can be used to quantify the genetic diversity and differentiation of populations in ecologically contrasting areas. Mutations in isozyme loci have co-dominant inheritance and manifest themselves in the seeds of the first generation (Verta et al., 2013). This is especially important in long-lived trees, where it is unfeasible to generate the inbred lines or crossed progeny that facilitate traditional analysis. Traditionally, reactive oxygen species (ROS) were considered toxic by-products of aerobic metabolism. Under normal growth conditions, the production of ROS in cells is low, whereas many stresses, including ionizing radiation, increase their production (Limon-Pacheco and Gonsebatt, 2009). Radiation can cause ROS production through radiolysis of water or as by-products of a hampered metabolism. Enhanced production of ROS during stress can harm cells through membrane lipid peroxidation, protein oxidation, enzyme inhibition, and DNA and RNA damage. ROS are also thought to act as signals for the activation of stress-response and defense pathways (Mittler, 2002; Foyer and Noctor, 2005). Thus, ROS can be considered both the cellular indicators of stress and the secondary messengers involved in the stress-response signal transduction pathway. In this study, we attempted to answer the following questions: (i) Do Scots pine populations growing under relatively low levels of chronic radiation exposure (0.8–14.8 μGy/h) show an increased rate of enzymatic loci mutations? (ii) Can chronic radiation exposure modify the genetic makeup of Scots pine populations in the dose rate range studied? and (iii) Does chronic radiation exposure in the dose rate range studied significantly modify the activity of antioxidant enzymes? 2. Materials and methods 2.1. Test organism Scots pine (Pinus sylvestris L.), the dominant tree species in North European and Asian boreal forest, was chosen as the test organism for

an assessment of the possible effects of the radioactive contamination. Scots pines are widespread in the area affected by the Chernobyl accident, and samples can be obtained from trees growing in different contamination levels. The reproductive organs of conifers are particularly susceptible to radiation exposure because of their complex organization and long generative cycle (Cairney and Pullman, 2007). The availability of a haploid endosperm (megagametophyte) and a diploid embryo in each seed enables the investigation of mutagenesis on the gametic level, which allows the direct determination of the haplotype and recessive mutations (Verta et al., 2013). Such special properties cause conifers to be a unique model for the study of the mutation processes in populations developing under chronic radiation exposure. 2.2. Study area and data collection In 1986, the Bryansk region was significantly contaminated by the fallout from Chernobyl, with an initial 137Cs ground deposition level greater than 1 MBq/m2 in some locations (Ramzaev et al., 2008). Currently, sites still exist in this area where radioactive contamination significantly exceeds background. Four radioactively contaminated sites, VIUA (52° 29′ N; 31° 50′ E), SB (52° 33′ N; 31° 44′ E), Z1 (53° 5′ N; 31° 42′ E), and Z2 (53° 5′ N; 31° 42′ E), were chosen approximately 200 km northeast of the Chernobyl nuclear power plant (Fig. 1). The reference site, Ref (53° 1′ N; 33° 55′ E), was selected based on its proximity to the impacted area and the similarity of its environmental properties. Latitude and longitude measurements were made using a geographical positioning system (GPS). The γ-radiation dose rates were measured at the study sites using a DRG-01T dose rate meter (“Leninez”, Russia) at a height of 1 m above the ground, 5–7 times under every tree from which cones were collected. All dose rate values were expressed in Roentgen units initially and were converted to Gray units (Gy) using a multiplication factor of 8.76 × 10− 3 (Mashkovich and Kudriavtseva, 2013). Dose rates at the study sites ranged from 0.37 to 1.21 μGy/h, compared with 0.10 μGy/h at the reference site (Table 1). Insofar as we could determine, all other readily measurable aspects of the microenvironments at these study sites, except the radiation level, were identical. Samples were collected at the same sites as in our previous study of the influence of chronic radiation exposure on cytogenetic effects and

Fig. 1. Location of the study sites.

S.A. Geras'kin, P.Y. Volkova / Science of the Total Environment 496 (2014) 317–327

319

Table 1 Radioactive contamination of the study sites and doses absorbed by reproductive organs of pine trees. Study site

Ref VIUA SB Z1 Z2

137

Cs, 0–5 cm, Bq/kg

73 ± 1.6 × 1.7 × 3.9 × 9.7 ×

21 103 104 104 104

± ± ± ±

450* 5 × 103* 1.2 × 104* 2.9 × 104*

Exposure dose rate, μGy/h

0.10 0.37 0.49 0.73 1.21

± ± ± ± ±

0.01 0.03 0.16 0.18 0.23

Activity concentrations of radionuclides in cones, Bq/kg

Estimated doses and dose rates to generative organs

137

Dose rate, μGy/h

Dose, mGy/y

0.015 0.8 2.6 10.4 14.8

0.13 7. 23. 91.2 130.

90

Cs

30.0 ± 3.7 9.5 × 102 ± 3.4 × 102 ± 3.2 × 103 ± 1.0 × 103 ±

Sr

1.3 16.8 20.8 64.3 83.0

13* 8* 32* 18*

± ± ± ± ±

0.7 1.9 1.3 2.2 2.2

*Significant difference from the Ref site, р b 0.05.

reproductive ability of Scots pine (Geras'kin et al., 2011). Soil samples were taken from each site at depths of 0–5, 5–10 and 10–15 cm at the locations with the highest γ-dose rate level. The soils of the study sites are sandy podzols with similar granulometric composition, chemical and physical properties, and nutrient availability. Pine cones were collected in December of 2009–2011 from 30 to 50 year old Scots pine trees. At each site, 30–50 cones were taken from each of 20–29 trees, at a height of 1.5–2.0 m above the soil surface. The cones were stored outside and allowed to mature through the end of February, then moved into the laboratory and stored at room temperature under reduced humidity until dehiscing and seed removal. Seeds were de-winged manually. Only freely released well-formed seeds were used for the electrophoretic analysis. 2.3. Measurements of radionuclides in soil and plant samples Soil samples were homogenized manually, air-dried and sieved at 1 mm. Basic chemical and physical properties of soils were measured using conventional techniques (Pansu and Gautheyrou, 2006). One randomly selected cone from each tree was taken to form a pooled sample of cones from each study site. Activity concentrations of 137 Cs in the cones as well as 137Cs, 40K, 226Ra, and 232Th in soil samples were determined with γ-spectrometry using a computercontrolled MCA InSpector-1270 with coaxial germanium detector and the spectroscopy software GENIE-2000 (Canberra Industries, USA). The lowest measurable activity concentrations for the 1 h measurement duration were 2, 10, 10, and 40 Bq/kg for 137Cs, 226Ra, 232Th, and 40K, respectively. To determine 90Sr activity concentrations, cones were ashed at 600 °С, and 90Sr was extracted from a double treatment with a boiling solution of 6 М HCl, with a quantification limit of 0.05 Bq for the 1800 s measurement duration.

γ

Di ¼

2πΓρs qiv ρi μ s

(

" 

E2

μs ρs

ρ0 Δh0 þ

A dosimetric model was developed to calculate the total (internal + external) radiation dose absorbed by the reproduction organs (cones) of the pine trees (Spiridonov et al., 2008; Geras'kin et al., 2011). Several layers were defined (crown, under crown, and three soil layers at depths of 0–5, 5–10 and 10–15 cm). Each layer was treated as an infinitely thick source. A uniform distribution of radionuclides within each layer was assumed. The upper layers were considered to shield and attenuate a portion of the gamma energy to calculate the absorbed dose in the cones from γ-ray emitting radionuclides in a particular soil layer. Doses were calculated for cones located within the “crown” layer. The Taylor form of an accumulation factor was applied to consider the multiple scattering of radiation by the upper layers (Mashkovich and Kudriavtseva, 2013): ð1Þ

where E0 is the energy of γ-quantum, μ is the linear attenuation factor, d is the layer thickness, and A, α1 and α2 are constants.

i−1 X k¼1

!# ρk hk

" 

−E2

μs ρs

ρ0 Δh0 þ

i X

!#) ρk hk

;

k¼1

ð2Þ



E2 ðzÞ ¼

A 1−A E ½ð1 þ α 1 Þz þ E ½ð1 þ α 2 Þz; 1 þ α1 2 1 þ α2 2

ð3Þ

where Г is the gamma constant, qiv is the 137Cs activity concentration in the i-layer, ρi is the medium density in the i-layer, ρs is the air density, μs is the linear attenuation factor for γ-radiation in air, hk is the thickness of the k-layer, Δh0 is the distance between the location of the reproductive organs of pine trees and the upper limit of zone 1, and E2 is the King function (Mashkovich and Kudriavtseva, 2013). To calculate the γ-ray dose rate from radionuclides located in the tree crown, Eq. (2) was transformed to: γ

D0 ¼

0

2πΓρs qv μ s ρ0

       A 1−A ρ ρ   −E2 μ s 0 ðh0 −Δh0 Þ −E2 μ s 0 Δh0 ; 2 þ 1 þ α1 1 þ α2 ρs ρs

ð4Þ where q0v is the radionuclide activity concentration in the crown, ρ0 is the medium density, and h0 is the thickness of the crown layer. The 137Cs and 90Sr (90Y) activity concentrations in the cones were utilized to estimate the β-radiation dose rate. Because the depth of penetration of β-particles into biological tissue is much less than the size of a cone, the equation for an infinite source was used: Dβ ¼ kEβ qβ ;

2.4. Dose assessment

BðE0 ; μdÞ ¼ A expð−α 1 μdÞ þ ð1−AÞ expð−α 2 μdÞ;

The following equations were used to calculate the γ-dose rate from radionuclides located in the i-layer of soil (Spiridonov et al., 2008):

ð5Þ

where Eβ is the average energy of the β-quantum, qβ is the concentration of β-emitters, and k is a constant. 2.5. Allozyme loci analysis Three antioxidant enzymes, superoxide dismutase (EC 1.15.1.1, SOD), glutathione reductase (EC 1.6.4.2, GR), and glutathione peroxidase (EC 1.11.4.2, GPX), were chosen for the investigation of genetic diversity in chronically irradiated Scots pine populations. The analyses were based on 15 endosperms and embryos per tree on average. Seeds from different sites were randomized and encoded. Identification was performed at the end of the experiment, after decoding and documenting of the zymograms. Each embryo and endosperm was individually homogenized in 100 μl of extraction buffer (1% triton Х-100 solution and 0.2% solution of β-mercaptoethanol). The supernatants were used for enzyme assays after homogenization and centrifugation (14 500 g for 10 min). Electrophoresis was performed for 1.5–2.0 h at 60–80 mA in Protean II xi Cell (USA) and Hoefer SE 600 Chroma (Finland) vertical chambers using a 7.5% polyacrylamide gel in a Tris–HCl buffer system, рН 8.0, with Tris-glycine, pH 8.9, as an electrode buffer. Gel polymerization

320

S.A. Geras'kin, P.Y. Volkova / Science of the Total Environment 496 (2014) 317–327

was performed for 30–60 min. Isozymes were stained using conventional techniques (Manchenko, 1994). Only those bands that could be scored without ambiguity were considered. SOD concentrations in the endosperm and embryos were analyzed in seeds collected in 2009, and GR and GPX in the endosperm were analyzed in seeds collected in 2010. In total, 12176 locus tests were performed. Allozymes were identified by gel mobility. The most frequent allozyme and its corresponding allele were designated as 1.00, and faster (F) and slower (S) migrating variants received designations according to their relative mobility with respect to the most common allele. An allele was considered rare if its frequency of occurrence in a population did not exceed 5%. Three types of mutations were revealed. Null mutations were identified as the absence of the corresponding allelic variant in the zymogram. Duplications were manifested by the appearance of two enzyme bands in the zymogram, one above the other. Changes in the electrophoretic mobility of the isozyme were identified based on the appearance of the enzyme bands outside the previously identified areas of activity. 2.6. Analysis of enzyme activity SOD, catalase (EC 1.11.1.6, CAT) and peroxidase (EC 1.11.1.7, POD) activities were assessed following H. Bisswanger (2004). Isozyme activities were evaluated using a “NanoDrop-2000” (USA) spectrophotometer. SOD activity was assayed based on its ability to inhibit photochemical reduction of nitro blue tetrazolium chloride (NTC) in the presence of hydroxylamine hydrochloride. CAT activity was measured by the decomposition rate of hydrogen peroxide. POD activity was measured by the transformation rate from guaiacol to tetraguaiacol. The absorption coefficient used was 26 600 for guaiacol and 19 000 for NTC. Analysis was performed in triplicate. Readings were converted to international units of enzymatic activity (МЕ): Unit ΔА= min  vol react:mixtureðmlÞ  ER  10000  ¼ ml enzyme solution εnm l ∗ mol−1 ∗ cm−1  vol enz:ðmlÞ where ΔА is the difference between the absorbance values А1 and А2, and εnm(l ∗ mol−1 ∗ cm−1) is the absorption factor. 2.7. Data analysis For each population, we calculated the allele frequencies and several indices of genetic diversity. The significance of the differences in the allele frequencies among the reference and impacted populations was evaluated using a modified χ2 test recommended in Zhivotovsky (1991) for small sample volumes and the presence of rare alleles. The test statistics and the number of degrees of freedom were calculated as χ2G = cχ2 and νG = cν, where с is the correction for the rarity of the expected occurrence of some alleles. Significance of differences among the means was determined using Student's test. The Zhivotovsky index of allelic diversity, μ, (Zhivotovsky, 1991) was calculated using pffiffiffiffiffi pffiffiffiffiffi pffiffiffiffiffiffi 2 p2ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi þ … þ pm Þ μ ¼ ð p1 þ r μ ðm−μ Þ and sμ ¼ : N

The effective number of alleles (ne) was determined by the formula 2

nе ¼ 1=∑pi ; where p1, p2, …pm are the frequencies of allele occurrence, and m is the number of alleles. Expected heterozygosity (He) was estimated as He ¼ 1−

m X

2

pi :

i ¼ 1

The observed heterozygosity (Ho) was calculated by dividing the number of heterozygous trees by the total number of trees analyzed for this locus. Genetic relationships among populations were evaluated by calculating genetic distances (D) (Nei, 1972) D ¼ − ln r ¼ − ln J pq þ

1 ln J p þ ln J q ; 2

where J pis the theoretical homozygosity in the first population averaged over all loci, J q is the theoretical homozygosity in the second population averaged over all loci, and J pq is the mutual identity of the two populations for all loci. The genetic distance matrix was analyzed using cluster analysis by applying the unweighted pair group method (UPGMA) to group the populations based on their genetic similarity. Statistical analyses were run using MS Office Excel 2007, Statistica 6.0 for Windows, PopGene 1.32 and F-stat. 3. Results 3.1. Radioactive contamination of the study sites and doses absorbed by the reproductive organs of pine trees The study sites did not differ substantially in soil properties or heavy metal content in the soil or pine cones (Geras’kin et al., 2011). Radionuclide concentrations, however, varied by orders of magnitude among the study sites (Table 1). The 137Cs activity concentrations in the soil at the contaminated sites ranged from 1.6 to 97 kBq/kg. These activity concentrations exceeded those at the Ref site by factors of 20–1320. In cones from the impacted populations, the radionuclide activity concentrations were significantly higher than in samples from the Ref site (Table 1). The maximum activity concentrations of 137Cs and 90Sr exceeded the reference level by factors of 108 and 63, respectively. The 137Cs content in the cones exceeded that of 90Sr by at least one order of magnitude. According to our estimates (Table 1), the doses absorbed by cones ranged from 7 to 130 mGy/y (absorbed dose rate 0.8– 14.8 μGy/h). The majority of the dose absorbed by cones was due to 137Cs located in the upper 10-cm soil layer; β-radiation contributed approximately 0.4–9.3% to the total dose at the impacted study sites. At the beginning of this study, the pine populations had grown under chronic radiation exposure for more than 20 years. According to our estimates, the dose accumulated from 1986 to 2008 in the crowns of the test trees amounted to 0.2–1.0 Gy, which is in good accordance with the results of an independent study (Ramzaev et al., 2008) at a site very close to one of our study sites. 3.2. Genetic diversity

The proportion of rare alleles (hμ) was represented by vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi u uh 1−h t μ μ : hμ ¼ 1 – ðμ=mÞ and sh ¼ N

To investigate the pattern of genetic variation, five Scots pine populations were compared based on the electrophoretic diversity of three enzymes: SOD, GR, and GPX. Among the 6 loci analyzed, 4 loci exhibited variability and 2 loci were monomorphic in all populations (Table 2). In the analysis of 1836 locus tests at the reference

S.A. Geras'kin, P.Y. Volkova / Science of the Total Environment 496 (2014) 317–327 Table 2 Allozyme allele frequencies at sod, gr, and gpx loci. Study site

Loci

Allele

Frequency

Loci

Allele

Frequency

Ref

gr

1.00 1.10 n 0.80 1.00 n 1.00 1.10 n 1.00 1.10 n 0.80 1.00 n 1.00 1.10 n 1.00 1.10 n 0.80 1.00 n 1.00 1.10 n 1.00 1.10 n 0.80 1.00 n 1.00 1.10 n 1.00 1.10 n 0.80 1.00 n 1.00 1.10 n

0.848 ± 0.024 0.152 ± 0.024 – 0 + 0.004 1 − 0.004 – 1 − 0.004 0 + 0.004 – 0.818 ± 0.026 0.173 ± 0.025 0.009 ± 0.006 0 + 0.004 0.982 ± 0.009 0.018 ± 0.009 0.995 ± 0.006 0 + 0.005 0.006 ± 0.006 0.797 ± 0.029 0.178 ± 0.027 0.025⁎ ± 0.011 0.088⁎⁎ ± 0.020 0.886⁎⁎ ± 0.023 0.026⁎ ± 0.011 0.986 ± 0.008 0 + 0.004 0.014 ± 0.008 0.764⁎ ± 0.028 0.173 ± 0.025 0.063⁎⁎ ± 0.015 0.102⁎⁎ ± 0.020 0.847⁎⁎ ± 0.023 0.051⁎⁎ ± 0.014 0.756⁎ ± 0.029 0.200⁎ ± 0.027 0.044⁎ ± 0.014 0.777 ± 0.026 0.159 ± 0.022 0.064⁎⁎ ± 0.015 0.095⁎⁎ ± 0.018 0.850⁎⁎ ± 0.023 0.055⁎⁎ ± 0.014 0.676⁎⁎ ± 0.031 0.271⁎⁎ ± 0.003 0.053⁎ ± 0.015

gpx-2

0.95 1.00 1.20 n 1.00 n 1.00 n

0 + 0.004 0.901 ± 0.020 0.094 ± 0.020 0.005 ± 0.004 1 − 0.004 – 1 − 0.004 –

0.95 1.00 1.20 n 1.00 n 1.00 n

0 + 0.004 0.901 ± 0.020 0.094 ± 0.020 0.005 ± 0.004 1 − 0.006 – 1 − 0.006 –

0.95 1.00 1.20 n 1.00 n 1.00 n

0 + 0.005 0.866 ± 0.025 0.118 ± 0.023 0.016 ± 0.009 1 − 0.005 – 1 − 0.005 –

0.95 1.00 1.20 n 1.00 n 1.00 n

0.072⁎⁎ ± 0.017 0.754⁎⁎ ± 0.028 0.114 ± 0.02 0.060⁎⁎ ± 0.015 0.987 ± 0.008 0.013 ± 0.008 0.996 ± 0.004 0.004 ± 0.004

0.95 1.00 1.20 n 1.00 n 1.00 n

0.063⁎⁎ ± 0.015 0.778⁎⁎ ± 0.026 0.108 ± 0.02 0.041⁎⁎ ± 0.012 0.987 ± 0.008 0.013 ± 0.008 0.991 ± 0.006 0.008 ± 0.006

gpx-1

sod-1

VIUA

gr

gpx-1

sod-1

SB

gr

gpx-1

sod-1

Z1

gr

gpx-1

sod-1

Z2

gr

gpx-1

sod-1

sod-2 sod-3

gpx-2

sod-2 sod-3

gpx-2

sod-2 sod-3

gpx-2

sod-2 sod-3

gpx-2

sod-2 sod-3

n — null mutation. Number of loci-tests — 8936. ⁎ Significant difference from the Ref site, р b 0.05. ⁎⁎ Significant difference from the Ref site, р b 0.01.

321

characterized by increased frequencies of duplications and changes in electrophoretic mobility (Table 3). The total mutation frequency significantly increased along with the level of radioactive contamination. Therefore, long-term chronic radiation exposure at dose rates greater than 0.8 μGy/h led to significant increases in enzymatic loci mutations in Scots pine populations. Genetic diversity statistics for Scots pine populations are presented in Table 4. Increased mutation rates resulting from radiation exposure could be manifested as a greater number of alleles at a given locus. Indeed, the Zhivotovsky index of allelic diversity (μ) markedly exceeded the reference level and increased with the absorbed dose (r = 0.99; p b 0.01) for all pine populations growing under chronic exposure. The most polymorphic populations were SB, Z1 and Z2 (P 95 ≥ 50%). Thus, the average number of genotypes in a population increased under chronic exposure conditions. The appearance of new alleles leads to a significant increase in the relative proportions of rare alleles in Scots pine populations growing under chronic exposure conditions (Table 4). The frequency of rare alleles increases along with the level of radioactive contamination, whereas their numbers remain the same. Therefore, their proportions at impacted sites do not differ significantly from each other. The effective number of alleles increases (Table 4) along with the level of radiation exposure because rare alleles contribute significantly to the allelic diversity in chronically irradiated populations. The values of observed (Ho) and expected (He) heterozygosity for the reference population are similar to the estimates obtained previously for different pine species (Guries and Ledig, 1982; Krutovsky et al., 1989). However, both the observed and the expected heterozygosity in exposed Scots pine populations increase with the level of radioactive contamination (r = 99%, p b 0.01), and in all exposed populations, both values significantly exceeded the reference level (Table 4). Inheritance of the allozymes has been determined for the polymorphic loci gr, gpx-1 and sod-1 (Table 5). For each locus and population, 58–120 haploid megagametophytes collected from 4 to 9 trees were used for calculations. Segregation ratios of 1:1 are expected for codominant alleles at heterozygous loci (Rubanovich and Kalchenko, 1994). We did not observe reliable deviations for the gr and sod-1 loci, whereas the gpx-1 locus had significant deviation from uniform segregation. The greatest deviation from the expected segregation was recorded for the population inhabiting the most contaminated site (Z2).

3.3. Evaluation of selection pressure against the rare electrophoretic variants site, only one null mutation in the gpx-2 gene was revealed. The null mutation frequency is significantly higher in the exposed populations (Table 3), and despite substantial variation among loci and populations, it strongly correlates with the absorbed dose (r = 0.99; p b 0.01). In addition, populations SB, Z1 and Z2 are

SOD polymorphism was evaluated simultaneously in endosperm and in embryos to assess the power of the selection pressure against the rare electrophoretic variants in the early stages of ontogenesis. We analyzed 3240 locus tests and did not observe significant elimination of rare electrophoretic variants in embryos,

Table 3 Enzymatic mutation frequency and spectrum in pine seed endosperm. Study site

Number of loci-tests

Null mutation frequency

Duplications frequency

Changes in electrophoretic mobility frequency

Total frequency of mutations

Ref VIUA SB Z1 Z2

1836 1657 1603 1956 1884

0.001 ± 0.001 0.007⁎ ± 0.002 0.014⁎⁎ ± 0.003 0.039⁎⁎ ± 0.005 0.041⁎⁎ ± 0.005

0 0 0.005⁎⁎ ± 0.002 0.012⁎⁎ ± 0.003 0.015⁎⁎ ± 0.003

0 0.001 ± 0.001 0.005⁎ ± 0.002 0.011⁎⁎ ± 0.003 0.011⁎⁎ ± 0.003

0.001 ± 0.001 0.008⁎ ± 0.003 0.024⁎⁎ ± 0.004 0.062⁎⁎ ± 0.006 0.067⁎⁎ ± 0.007

⁎ Significant difference from the Ref site, р b 0.05. ⁎⁎ Significant difference from the Ref site, р b 0.01.

322

S.A. Geras'kin, P.Y. Volkova / Science of the Total Environment 496 (2014) 317–327

Table 4 Estimates of genetic diversity parameters. Genetic diversity parameters

Ref

VIUA

SB

Z1

Z2

Number of trees Number of seeds Number of loci-tests P95 μ ne The relative contributions of rare alleles Ho He Fis

15 464 1836 33.3 1.36 ± 0.03 1.12 ± 0.01 0.12 ± 0.04 0.17 ± 0.01 0.11 ± 0.01 −0.167

15 405 1657 33.3 1.50⁎ ± 0.04 1.16⁎ ± 0.01 0.30⁎ ± 0.06 0.18 ± 0.01 0.13 ± 0.01 −0.194

14 407 1603 50.0 1.68⁎⁎ ± 0.04 1.25⁎⁎ ± 0.02 0.27⁎ ± 0.05 0.27⁎⁎ ± 0.01 0.19⁎⁎ ± 0.01 −0.183

16 462 1884 66.6 2.18⁎⁎ 1.54⁎⁎ 0.27⁎⁎ 0.48⁎⁎ 0.36⁎⁎

18 494 1936 66.6 2.16⁎⁎ 1.61⁎⁎ 0.28⁎⁎ 0.48⁎⁎ 0.35⁎⁎

± ± ± ± ± −0.264

0.04 0.08 0.03 0.04 0.03

± ± ± ± ± −0.236

0.04 0.04 0.03 0.04 0.02

P95 — polymorphism index; μ — Zhivotovsky index of the allele diversity; ne — effective number of alleles; Ho — observed heterozygosity; He — expected heterozygosity; Fis — fixation index. ⁎ Significant difference from the Ref site, р b 0.05. ⁎⁎ Significant difference from the Ref site, р b 0.01.

Table 5 The ratios of F and S allele numbers in the endosperm of heterozygotes and χ2 statistics for deviations from 1:1. Loci

Ref

VIUA

SB

Z1

Z2

gr gpx-1

40:34 0.486 (5) –

50:39 1.111 (6) –

42:35 0.461 (5) 41:17 9.931⁎⁎⁎ (4)

63:42 3.774 (8) 63:24 16.409⁎⁎⁎ (6)

sod-1







56:41 2.0 (7) 57:24 12.489⁎⁎⁎ (6) 59:61 0.034 (9)

52:45 0.367 (7)

Enclosed in brackets is the number of trees used for calculations. ⁎⁎⁎ Significant deviations from 1:1, р b 0.001.

although there was a steady downward trend in the frequency of mutations (Fig. 2).

contribution to the inter-population variability (0.145) was made by the sod-1 locus. Thus, Scots pine contains appreciable genetic variation, but this variation is distributed in a manner that suggests that little differentiation has occurred among populations. The genetic distances were calculated for each pair of populations (Table 7) and used to construct a tree of genetic relationships. Cluster analysis revealed significant differences between the heavily affected populations, Z1 and Z2, and the reference population, Ref. However, we have failed to detect significant differences among VIUA, SB and the reference population (Table 7). As a result, the analysis of the genetic differences showed two well-defined groups (Fig. 3): the first included the populations inhabiting heavily contaminated sites (Z1 and Z2), and the second included much less contaminated sites (Ref, VIUA, and SB). To some extent, these results indicate that chronic radiation exposure at dose rates from 10.4 μGy/h might play an important role in the genetic differentiation of Scots pine populations.

3.4. Analysis of population genetic structure

3.5. Enzymatic activities

The organization of the genetic variation in Scots pine populations was examined using F-statistics (Wright, 1965). A slight excess of heterozygotes was indicated by negative values of Fit (Table 6). The extent of genetic differentiation among the populations was measured using Fst with a mean across all loci of 0.048. The largest and most significant

SOD activity was found to be the lowest among the three enzymes studied; POD activity was much higher, and CAT activity was characterized by intermediate values (Table 8). The data obtained suggest that radiation exposure did not have any significant effect on SOD and CAT activities, whereas POD activity decreased significantly with increasing levels of radioactive contamination. 4. Discussion Environmental pollution underpins the dynamics and diversity of many ecosystems, yet its influence on the patterns and distribution of genetic diversity is poorly appreciated. This is an important knowledge gap because genetic diversity influences the fitness of individuals, the viability of populations, and the adaptability of species

Table 6 Estimates of Fit, Fis, and Fst for 4 polymorphic loci.

Fig. 2. Frequency of mutations in coded SOD loci in endosperm and embryos of pine seeds.

Loci

Fit

Fis

Fst

sod-1 gr gpx-1 gpx-2 Mean

−0.071 −0.261⁎⁎ −0.095 −0.224⁎⁎ −0.163⁎

−0.252⁎⁎ −0.237⁎⁎ −0.174⁎ −0.223⁎⁎ −0.222⁎⁎

0.145⁎ −0.019 0.067 −0.001 0.048

⁎ Significant difference from 0 (p b 0.05). ⁎⁎ Significant difference from 0 (p b 0.01).

S.A. Geras'kin, P.Y. Volkova / Science of the Total Environment 496 (2014) 317–327

323

Table 7 Genetic distance among Scots pine populations inhabiting study sites. Population

Ref

VIUA

SB

Z1

Z2

Ref VIUA SB Z1 Z2

0 0.0002 0.0064 0.0248⁎⁎ 0.0137⁎

0.0002 0 0.0020 0.0253⁎⁎ 0.0139⁎

0.0064 0.0020 0 0.0256⁎ 0.0164⁎

0.0248 0.0253 0.0256 0 0.0027

0.0137 0.0139 0.0164 0.0027 0

⁎ Significant difference from the Ref site, р b 0.05. ⁎⁎ Significant difference from the Ref site, р b 0.01.

to environmental change (Hughes et al., 2008; Banks et al., 2013; Verta et al., 2013). To fill this knowledge gap, we test if mutation rates, genetic diversity and activity of antioxidant enzymes are different in pine trees growing in sites contaminated with radionuclides versus a reference site. Natural sources of ionizing radiation play a very minor role among the selective processes of evolution as usually considered. However, the enhanced levels of background radiation (whether artificial or natural) represent a long-standing factor having a specific influence on all life (Geras'kin et al., 2007). The Chernobyl accident dramatically changed the conditions for the existence of plants and animals inhabiting contaminated areas. These areas may be considered ecological islands, providing the opportunity to investigate the first steps in the establishment of genetic differentiation in natural populations under severe stress. However, currently, there is a clear lack of quantitative data regarding the long-term biological consequences of chronic radiation exposure (Garnier-Laplace et al., 2013). Therefore, investigations in the territories affected by the Chernobyl accident may provide crucial information for understanding ecological and evolutionary questions regarding the long-term effects of anthropogenic pollution on natural populations. 4.1. Enzymatic mutation frequencies One of the most surprising results of this study is the demonstration of a significant increase in the mutation frequencies of enzymatic loci in Scots pine populations inhabiting sites with relatively low levels of radioactive contamination (Table 3). In the first years after the accident, the mutation frequencies of enzymatic loci in pine populations from the Chernobyl exclusion zone were 4–17 times higher than in the reference population (Fedotov et al., 2006). According to Fedotov et al. (2006), the rarest type of mutation was duplications. This type of mutation was observed only with high levels of radiation exposure. In contrast, in our case, the rarest type of mutations was changes in electrophoretic mobility. In general, the frequencies of changes in electrophoretic mobility and duplications in our study were comparable (Table 3). However, in populations inhabiting the most contaminated sites (SB, Z1, and Z2), changes in electrophoretic mobility are less common than duplications. The different origins of mutations can be considered by the analysis of isozyme polymorphism. The mutations induced in the mother cell of the megaspore may move to the archegonia cell during the meiosis (Verta et al., 2013). This mutation will appear in the endosperm as well as in the embryo. However, if a mutation was induced in the megaspore, which generates a multi-nuclear endosperm, this type of mutation will be found only in the endosperm. In contrast, in the analysis of embryos, mutations induced in the gametes of the mother as well as brought by pollen are recorded. These varying origins explain why, in different ecological situations, when different sets of genes are analyzed, different ratios between the mutation frequency in the maternal gametes and embryos were observed, even under strong selection against spontaneously occurring rare alleles (Altukhov et al., 1983). Thus, the mutation frequencies at loci got-2 and got-3 in embryos of pine seeds from the 30-km Chernobyl NPP zone were slightly greater than in the endosperm (Kalchenko et al., 1993). Adding an additional

Fig. 3. Hierarchical classification of the Scots pine populations by cluster analysis.

locus (got-1) to the analysis and increasing the number of locus tests analyzed allowed the same authors to conclude that there is strong selection against rare alleles under conditions of chronic exposure to radiation (Fedotov et al., 2006). In our study, no significant difference in the enzymatic mutation frequencies in maternal gametes and embryos was found (Fig. 2).

4.2. Genetic diversity Investigation of genetic diversity forms the logical core for the appraisal of the effects of any change in the environment, especially a change that has far-reaching and basic implications for life. High levels of radiation exposure can lead to rapid changes in the genetic structure of plant and animal populations inhabiting contaminated areas (Shevchenko et al., 1992; Theodorakis, 2001). For example, the decline of sensitive trees may result in the gradual genetic depletion of exposed populations through the loss of less frequent alleles with potential adaptive significance to altered stressor regimes in the future (van Straalen and Timmermans, 2002; Longauer et al., 2004). The information gathered in our study allows us to infer that, even under much less levels of chronic radiation exposure (0.8–14.8 μGy/h), the main genetic variability parameters of the affected Scots pine populations had considerably higher values than those from the reference site (Table 4). The estimates of observed and expected heterozygosity (Ho = 0.152 ± 0.043, He = 0.145 ± 0.016) (Krutovsky et al., 1989) based on numerous studies performed on 26 species of the genus Pinus are in good agreement with those obtained in our study for the reference population (Table 4). In contrast, the heterozygosity of the populations inhabiting the radioactively contaminated sites exceeds the reference level and increases along with the level of radiation exposure. Similar results were obtained in our previous study of the enzymes of the Krebs cycle, performed on the same Scots pine populations (Geras'kin et al., 2010). Many authors have reported that heterozygotes are more adapted to ecological stress (Kalchenko et al., 1991; Bush and Smouse, 1992; Theodorakis, 2001; Fedotov et al., 2006). Populations developing under unfavorable conditions (e.g., harsh climate or anthropogenic pollution) generally exhibit higher heterozygosity than those in the optimal conditions. Heterozygous individuals could be endowed with greater plasticity in the changing environment through the combination of two or more variants of the same enzyme. A decrease in the heterozygosity of individuals could be associated (Theodorakis, 2001; Altukhov, 2003) with decreased resistance to diseases, decreased growth rates, and decreased fertility. This would suggest that variations in individual heterozygosity could affect population growth and recruitment. The observed heterozygosity in pine populations inhabiting sites polluted with industrial chemicals was higher than expected from

324

S.A. Geras'kin, P.Y. Volkova / Science of the Total Environment 496 (2014) 317–327

Table 8 Activity of antioxidant enzymes in pine trees seeds from chronically exposed populations, МЕ. Enzyme

Ref

VIUA

SB

Z1

Z2

CAT SOD POD

0.053 ± 0.017 0.007 ± 0.006 0.409 ± 0.038

0.093 ± 0.022 0.012 ± 0.008 0.34 ± 0.035

0.115⁎ ± 0.028 0.007 ± 0.007 0.39 ± 0.043

0.080 ± 0.025 0.006 ± 0.006 0.31⁎ ± 0.042

0.100 ± 0.025 0.005 ± 0.005 0.15⁎⁎⁎ ± 0.03

⁎ Significant difference from the Ref site, р b 0.05. ⁎⁎⁎ Significant difference from the Ref site, р b 0.001.

Hardy–Weinberg equilibrium and increased with the anthropogenic load (Dukharev et al., 1992). In contrast, fish populations from basins with high level of acid stress had lower heterozygosity than the populations of the control sites (Kopp et al., 1992). It appears that the positive association between the heterozygosity of individuals and fitness could become stronger under moderate levels of stress. In general, uneven segregation in natural conifer populations is rarely observed (Krutovsky et al., 1987; Oreshkova, 2008). Indeed, in the present study as well as in our previous work (Geras'kin et al., 2010), no reliable deviations from uniform segregation in the reference population were observed. In contrast, in populations inhabiting sites contaminated with radionuclides, such deviations in some loci (gpx-1 in the present study and 6-pgd, gdh, and mdh in the previous study) were revealed. Note that for the gpx-1 locus, the extent of deviation from uniform segregation increases with increases in the dose rate of chronic exposure (Table 5). However, in our previous study, the relationship between the extent of deviation from the uniform segregation of alleles and the level of radiation exposure was not revealed (Geras'kin et al., 2010). A significant increase in the extent of deviation from the uniform segregation of alleles with dose was found in the first years after the Chernobyl accident at doses more than 1 Gy (Rubanovich and Kalchenko, 1994). Thus, doses much higher than those in our study are needed for sustainable gametic selection, expressed as irregular allele segregation in Scots pine populations. The structure of genetic diversity in our study is quite comparable to that observed for other conifers. Approximately 95% of the genetic variation in Scots pine resides within stands (Table 6), compared with 97% for pitch pine, 88% for ponderosa pine, 96% for lodgepole pine, and 97% for Douglas fir (Guries and Ledig, 1982). The results obtained in our study suggest that genetic processes in Scots pine populations developing under chronic radiation exposure lead to increased genetic diversity. Indeed, the intra-population variability at all impacted sites reliably exceeds the reference level (Р b 0.01), increasing with level of radioactive contamination (Table 4). Thus, one of the most important reactions of a population to moderate stress is an increase in the genetic and phenotypic variability (Mengoni et al., 2000; Slomka et al., 2011; Geras'kin et al., 2013). However, severe stress may cause a loss of genetic diversity when population size is reduced or due to a bottleneck effect (Deng et al., 2007; Kozyrenko et al., 2007). In contrast with our data, during the first years after the accident (1986–1989), no relationship between the Zhivotovsky index of allelic diversity and ionizing radiation dose rate was found in Arabidopsis populations from the 30-km Chernobyl NPP zone (Abramov et al., 1992). Yet, the proportion of polymorphic loci and the Zhivotovsky index decreased gradually along with decreases in the level of radiation exposure with time. The extent of genetic diversity in plant populations inhabiting contaminated territories is largely determined by the species sensitivity as well as the nature and strength of the exposure. Thus, a study of the allozyme structure of dandelion (Taraxacum officinale L.) populations from the floodplain of the Techa river in the South Urals revealed no significant differences in the Zhivotovsky index between the reference and affected populations (Ulianova et al., 2004). Although the absorbed doses both in Ulianova et al. (2004) and in our study were comparable, pines are much more sensitive to radiation than are dandelions (Sarapult'zev and Geras'kin, 1993); i.e., much higher doses are required to increase the intra-population diversity in dandelions.

Indeed, in a study of dandelion populations from the East-Ural radioactive trace (EURT) territory, where the level of exposure was much higher, the same authors have shown that the Zhivotovsky index significantly exceeds the reference level (Pozolotina et al., 2012). The contribution of rare alleles to genetic diversity is nearly identical at all contaminated sites (Table 4); however, it is significantly different from the reference level. Thus, the comparison of the allele frequency in the reference and chronically exposed populations indicates shifts in their distribution due to the sharp increase in the level of chronic radiation exposure (the dose rates at the reference and the least polluted VIUA site differ by more than 50 times), which appears in the increased number of rare alleles. A further increase in the level of radioactive contamination leads to an increase in the frequency of these alleles, while their numbers remain practically the same. Increased genetic diversity due to the growing proportion of rare alleles was shown for knapweed grungy (Centaurea scabiosa L.) (Kalchenko et al., 1991; 1996) and dandelion (Pozolotina et al., 2012) populations in the EURT zone as well as in stands from the Carpathian Mountains that were heavily damaged by air pollution (Longauer et al., 2004). A question arises regarding the biological relevance of the high variability observed and the mechanisms involved in its maintenance. It is still unclear whether the increased levels of genetic diversity in antioxidant enzymes observed in our study contribute to the adaptation of populations to chronic radiation exposure. Considering that selection acts primarily on phenotypes without regard to genetic underpinnings, there seems little a priori reason to expect enzymatic loci to be hotspots for selectively important polymorphisms. However, positive correlations between allozyme heterozygosity and fitness measures, primarily growth and fecundity, have been reported for a number of forest tree species (Bush and Smouse, 1992), i.e., allozymes may not be selectively neutral markers. Several studies, particularly in bacteria, have shown that isozyme polymorphisms appear to have no fitness consequences under optimal conditions, whereas fitness differences among genotypes become evident under stress (Hoffmann and Hercus, 2000). Indeed, a significant increase in the frequency of the S allele, encoding the SOD isozyme that more effectively eliminates ROS, was revealed in populations of knapweed grungy growing for 12 years under chronic radiation exposure in the EURT territory (Kalchenko et al., 1996). The same authors found a strong selection pressure against the F allele in populations of Scots pine growing in the 30 km Chernobyl NPP zone (Fedotov et al., 2006). It appears that enzymes often have a strong role in determining phenotypes, fitness and ecological outcomes (Marden, 2013). 4.3. Analysis of population genetic structure Evolutionary adaptations through changes in genetic structures associated with selection are believed to be one of the main responses of populations to environmental stress. The cluster analysis revealed significant changes in the genetic distances between the populations inhabiting the heavily contaminated and less contaminated sites (Fig. 3). Large differences in allele frequency between populations can be interpreted as evidence of adaptive genetic differentiation (Kuchma and Finkeldey, 2011). In general, the genetic differentiation of plant populations is affected by geographic location, plant species, type and intensity of anthropogenic impact, and other environmental

S.A. Geras'kin, P.Y. Volkova / Science of the Total Environment 496 (2014) 317–327

factors (Mengoni et al., 2000; Banks et al., 2013). The populations inhabiting the most contaminated sites, Z1 and Z2, are geographically much closer to each other compared with the other populations. Therefore, their integration into a single cluster could also be connected to their location and the constant exchange of genes. In contrast, the exchange of pollen and seeds among the Ref, VIUA and SB populations is impossible because of the considerable distances between them. Nevertheless, they are joined into one cluster due to the low values of between-population differentiation and genetic distance. Thus, our results suggest that despite their low values, dose rates greater than 10.4 μGy/h can be considered an ecological factor able to modify the genetic makeup of Scots pine populations. Similarly, Kuchma and Finkeldey (2011) found strong evidence that selection in response to radiation exposure changed the genetic structures of Scots pine populations inhabiting the Chernobyl exclusion zone. 4.4. Activity of antioxidant enzymes Long-lived trees, as sessile organisms, cannot escape from contaminated areas and must adapt to survive in a context where environmental changes can outpace their generation time. Therefore, they must develop an effective system of protection against adverse impacts. In particular, plants rely on a buffered and redundant antioxidant defense system that is sufficient to cope with an adversely changing environment. Antioxidant defense mechanisms keep the routinely formed ROS at low levels and prevent them from exceeding toxic thresholds. Abiotic stresses disrupt the equilibrium between ROS and their detoxification by SOD, CAT and POD (Sharma and Dietz, 2008). However, the relationship between antioxidant concentrations and oxidative stress is complex. The acute effects of many environmental factors on the enzymatic activities of plants are well known, but the effects of long-term, low-level exposure to environmental pollutants are much less understood. Thus, Babchi plants (Psoralea corylifolia L.) grown from seeds exposed to higher doses of γ-radiation exhibit greater activity of antioxidants such as SOD, GR and ascorbate peroxidase (Jan et al., 2012). In contrast, CAT activity in these plants declined in a dose-dependent manner. The increased radioresistance of alder (Duschekia fruticosa Rupr.) seeds from sites with high natural radioactivity in the Sakha Republic in northern Russia is at least partly related to SOD activation (Zuravskaya et al., 1995). Vornam et al. (2012) have shown that cat expression is strongly up-regulated in the irradiated pines from the Chernobyl exclusion zone (where the dose absorbed by an entire tree is approximately 5.3 Gy/y), and gp is down-regulated. Zaka et al. (2002) have shown that chronic radiation exposure at a dose rate of 25 μSv/h has a significant effect on the activities of SOD, CAT, and POD in the progeny of feather plants (Stipa capillata L.) originating from the Semipalatinsk nuclear test site, Kazakhstan. The absorbed dose during the growing season was 196 mSv, which greatly exceeds the maximum annual dose observed in our study. Significant, continuous dose-related increases of the activities of SOD and CAT were observed for five plant species inhabiting the former coke-factory site in northeastern France (Dazy et al., 2009). Enhanced zinc concentrations caused an increase in the activities of SOD, CAT and POD in the roots and trunks of Scots pine seedlings (Ivanov et al., 2012). However, a significant change in SOD activity within the aerial portion of seedlings was observed only at high concentrations of zinc. Cellular oxidizing stress plays an important role in cell damage from ionizing radiation at high dose rates, and antioxidant enzymes may have a protective effect (Jan et al., 2012). However, the quantity of radicals generated during chronic exposure to low doses of radiation is significantly lower in comparison with the response to acute irradiation. Thus, the generation of radiolysis products did not significantly impact the cellular concentrations of ROS or the cellular redox potential at

325

a dose rate less than 417 μGy/h (Smith et al., 2012). Moreover, no significant effect of dose rates in the range of 81–2336 μGy/h on enzyme capacity in the radioresistant plant Arabidopsis thaliana was observed (Vandenhove et al., 2010). These studies have shown that the activity of antioxidant enzymes frequently does not change substantially at low stress intensity, whereas high stress intensity can either decrease or increase those activities. In general, antioxidant enzyme activities in plants demonstrate stimulation, no effect, or suppression depending on the species, the type of exposure and the genetic characteristics of the object (Schutzendubel and Polle, 2002). In our study, at relatively low levels of radiation exposure, we did not find a consistent difference in CAT and SOD activities from the reference level (Table 8). In contrast, the POD activity decreased with increasing levels of radionuclide contamination. Similarly, the POD activity in tobacco leaves decreased under the influence of high concentrations of nitrobenzene (Si et al., 2012). Overall, the results obtained in our study have shown that Scots pine is well equipped in enzymatic antioxidant systems because normal antioxidant activity is largely sufficient to eliminate the different types of ROS produced by chronic radiation exposure in the range from 0.8 to 14.8 μGy/h. In addition, the isoforms of the antioxidant enzymes produced and the balance between different isoforms can also be important. Isoforms of SOD and POD are localized in several subcellular compartments, whereas CAT is mostly localized in the peroxisome (Sharma and Dietz, 2008). They strongly differ in their substrate affinities and provide tight control over ROS concentrations at very low levels. Increased isozyme polymorphisms in the absence of significant changes in total enzyme activity can be associated with the contributions of different isoforms to the overall activity of the enzymes studied. Thus, in Scots pine seedlings grown under chronic zinc exposure, the activity of some SOD isoforms decreased, whereas others increased (Ivanov et al., 2012). It is possible that radiation exposure changed the ratio of activity of individual isoforms of enzymes in our study. Therefore, further studies are needed to determine the differential activity of each isoform under the conditions of our field study. 5. Conclusion A population is an actual form of plant or animal existence in nature. Thus, the currently developing principles of protecting biota from ionizing radiation (Garnier-Laplace et al., 2010) must be based on a clear understanding of the processes induced by chronic radiation exposure in populations. The present study is a portion of a continuing longterm (2003–2013) investigation of the biological effects in Scots pine populations developing under chronic exposure conditions. Chronic radiation exposure at dose rates from 0.8 μGy/h has been demonstrated to cause a significant increase in cytogenetic effects (Geras'kin et al., 2011) as well as mutations at enzymatic loci in Scots pine populations (present paper). Populations at contaminated sites contain novel combinations of alleles that are different from those at the reference site, and the levels of genetic diversity at contaminated sites are notably dissimilar, with exposed populations exhibiting a greater range of variation. Changes in the genetic makeup of Scots pine populations were observed at dose rates greater than 10.4 μGy/h. Therefore, chronic radiation exposure may be regarded as an ecological factor that changes the genetic makeup of Scots pine populations. However, the higher mutation rate had no effect on the antioxidant enzyme activities (present paper) or the reproductive ability of Scots pines (Geras'kin et al., 2011). Under the EC-ERICA project, a screening dose-rate value of 10 μGy/h was derived, applicable as an incremental dose rate in addition to the background (Garnier-Laplace et al., 2010). At this dose rate, no effect at the population or ecosystem level is expected. Our results agree nicely with this benchmark value and indicate that response to radiation exposure is observable in pines even long after the Chernobyl accident. These trees are ideal models for further analysis of the response of longlived plants to extreme environmental change. Overall, much empirical

326

S.A. Geras'kin, P.Y. Volkova / Science of the Total Environment 496 (2014) 317–327

work remains to be completed before we will be able to objectively and comprehensively assess the biological consequences to natural plant and animal populations of chronic low-level radiation exposure. Only through the willingness to look at such difficult questions will we gain further insight into both the effects of radiation and the patterns of nature. Acknowledgments This work was supported by the Russian Foundation for Basic Research (grants N 11-04-00670 and 11-04-97524) and the Russian Scientific Foundation (grant 14-14-00666). We thank four anonymous reviewers for improving the English language of the paper as well as numerous constructive suggestions and comments. References Abramov VI, Fedorenko OM, Shevchenko VA. Genetic consequences of radioactive contamination for populations of Arabidopsis. Sci Total Environ 1992;112:19–28. Alexakhin RM, Buldakov LA, Gubanov VA, Drozhko YeG, Ilyin LA, Kryshev II, et al. Large radiation accidents: consequences and protective countermeasures. Moscow, Russia: IzdAT Publisher; 2004. Altukhov YuP. Genetic processes in populations. Moscow: Akademkniga; 2003 [in Russian]. Altukhov YuP, Dukharev VA, Zhivotovsky LA. Spontaneous mutation rate in populations under selection pressure against rare protein variants. Russ J Genet 1983;19:264–76. Banks SC, Cary GJ, Smith AL, Davies ID, Driscoll DA, Malcolm Gill A, et al. How does ecological disturbance influence genetic diversity? Trends Ecol Evol 2013;28:670–9. Bisswanger H. Practical enzymology. Weinheim: Willey-VCH Verlag GmbH & KGaA; 2004. Bush RM, Smouse PE. Evidence for the adaptive significance of allozymes in forest trees. New For 1992;6:179–96. Cairney J, Pullman GS. The cellular and molecular biology of conifer embryogenesis. New Phytol 2007;176:511–36. Dazy M, Beraud E, Cotelle S, Grevilliot F, Ferard J-F, Masfaraud J-F. Changes in plant communities along soil pollution gradients: responses of leaf antioxidant enzyme activities and phytochelatin contents. Chemosphere 2009;77:376–83. Deng J, Liao B, Ye M, Deng D, Lan C, Shu W. The effects of heavy metal pollution on genetic diversity in zinc/cadmium hyperaccumulator Sedum alfredii populations. Plant Soil 2007;297:83–92. Dukharev VA, Korshikov II, Riabokon SM, Kotova AA. Genetic differentiation of Scots pine populations under technogenic pollution. Cytol Genet 1992;26(3):7–11. Fedotov IS, Kalchenko VA, Igonina EV, Rubanovich AV. Radiation and genetic consequences of ionizing irradiation on population of Pinus sylvestris L. within the zone of the Chernobyl NPP. Radiat Biol Radioecology 2006;46:268–78. [in Russian]. Foyer CH, Noctor G. Oxidant and antioxidant signaling in plants: a re-evaluation of the concept of oxidative stress in a physiological context. Plant Cell Environ 2005;28: 1056–71. Garnier-Laplace J, Della-Vedova C, Andersson P, Copplestone D, Cailes C, Beresford N, et al. A multi-criteria weight of evidence approach for deriving ecological benchmarks for radioactive substances. J Radiol Prot 2010;30:215–33. Garnier-Laplace J, Geras'kin S, Della-Vedova C, Beaugelin-Seiller K, Hinton TG, Real A, et al. Are radiosensitivity data derived from natural field conditions consistent with data from controlled exposures? A case study of Chernobyl wildlife chronically exposed to low dose rates. J Environ Radioact 2013;121:12–21. Geras'kin SA, Vanina JC, Dikarev VG, Novikova TA, Oudalova AA, Spiridonov SI. Genetic variability in Scotch pine populations of the Bryansk region radioactively contaminated in the Chernobyl accident. Biophysics 2010;55:324–31. Geras'kin SA, Oudalova AA, Dikareva NS, Spiridonov SI, Hinton T, Chernonog EV, et al. Effects of radioactive contamination on Scots pine populations in the remote period after the Chernobyl accident. Ecotoxicology 2011;20:1195–208. Geras'kin SA, Evseeva TI, Belykh ES, Majstrenko TA, Michalik B, Taskaev AI. Effects on nonhuman species inhabiting areas with enhanced level of natural radioactivity in the north of Russia: a review. J Environ Radioact 2007;94:151–82. Geras'kin SA, Evseeva TI, Oudalova AA. Effects of long-term chronic exposure to radionuclides in plant populations. J Environ Radioact 2013;121:22–32. Guries RP, Ledig FT. Genetic diversity and population structure in Pitch pine (Pinus rigida Mill.). Evolution 1982;36:387–402. Hoffmann AA, Hercus MJ. Environmental stress as an evolutionary force. Bioscience 2000; 50:217–26. Hughes AR, Inouye BD, Johnson MTJ, Underwood N, Vellend M. Ecological consequences of genetic diversity. Ecol Lett 2008;11:609–23. Ivanov YuV, Savochkin YuV, Kuznetsov VV. Scots pine as a model for studying the mechanisms of coniferous adaptation to heavy metals. 2. Antioxidant enzymes activity in pine seedlings under chronic zinc exposure. Russ J Plant Physiol 2012;59:57–66. Jan S, Parween T, Siddiqi TO, Mahmooduzzafar. Anti-oxidant modulation in response to gamma radiation induced oxidative stress in developing seedlings of Psoralea corylifolia L. J Environ Radioact 2012;113:142–9. Kalchenko VA, Kalabushkin VA, Rubanovich AV. Chronic irradiation as an ecological factor affecting the genetic structure of populations. Russ J Genet 1991;27:676–83.

Kalchenko VA, Rubanovich AV, Fedotov IS, Arkhipov NP. Genetic effects in gametes of Pinus sylvestris L. induced in the Chernobyl accident. Russ J Genet 1993;29:1205–12. Kalchenko VA, Rubanovich AV, Shevchenko VA. Adaptive nature of the polymorphism for the superoxide dismutase locus in chronically irradiated natural populations of Centaurea scabiosa L. Russ J Genet 1996;32:1509–12. Kopp RL, Guttman SI, Wissing TE. Genetic indicators of environmental stress in central mudminnow (Umbra limi) populations exposed to acid deposition in the Adirondack Mountains. Environ Toxicol Chem 1992;11:665–76. Kozubov GM, Taskaev AI. Radiobiology investigations of conifers in region of the Chernobyl disaster. Moscow, Russia: PPC “Design. Information. Cartography”; 2002 [in Russian]. Kozyrenko MM, Artyukova EV, Shmakov VN, Konstantinov YuM. Effect of fluoride pollution on genetic variability of Larix gmelinii (Pinaceae) in East Siberia. J For Res 2007; 12:388–92. Krutovsky KV, Politov DV, Altukhov YuP. Genetic variability in Siberian cedar pine, Pinus Sibirica Du TOUR. I. Inheritance of isoenzyme systems. Russ J Genet 1987;23: 2216–28. Krutovsky KV, Politov DV, Altukhov YuP, et al. Genetic variability in Siberian cedar pine, Pinus Sibirica Du TOUR. IV. Genetic diversity and differentiation between populations. Russ J Genet 1989;25:2009–32. Kuchma O, Finkeldey R. Evidence for selection in response to radiation exposure: Pinus sylvestris in the Chernobyl exclusion zone. Environ Pollut 2011;159:1606–12. Limon-Pacheco J, Gonsebatt ME. The role of antioxidants and antioxidant-related enzymes in protective responses to environmentally induced oxidative stress. Mutat Res 2009;674:137–47. Longauer R, Gomory D, Paule L, et al. Genetic effects of air pollution on forest tree species of the Carpathian mountains. Environ Pollut 2004;130:85–92. Manchenko GP. Handbook of detection of enzymes on electrophoretic gels. CRC Press; 1994. Marden JH. Nature's inordinate fondness for metabolic enzymes: why metabolic enzyme loci are so frequently targets of selection. Mol Ecol 2013;22:5743–64. Mashkovich VP, Kudriavtseva AV. Shield from ionizing radiation. Moscow, Russia: Stolitsa; 2013 [in Russian]. Mengoni A, Connelli C, Galardi F, Gabbrielli R, Bazzicalupo M. Genetic diversity and heavy metal tolerance in populations of Silene paradoxa L. (Caryophyllaceae): a random amplified polymorphic DNA analysis. Mol Ecol 2000;9:1319–24. Mittler R. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 2002;7: 405–10. Nei M. Genetic distance between populations. Am Nat 1972;106:283–92. Oreshkova NV. Allozyme polymorphism in Siberian larch (Larix sibirica Ledeb.) and Cajanderi larch (Larix cajanderi Mayr). Coniferous Boreal 2008;25:160–8. [in Russian]. Pansu M, Gautheyrou J. Handbook of soil analysis. Mineralogical, organic and inorganic methods. The Netherlands: Springer; 2006. Pozolotina VN, Antonova EV, Bezel VS. Comparison of remote consequences in Taraxacum officinale seed progeny collected in radioactively or chemically contaminated areas. Ecotoxicology 2012;21:1979–88. Ramzaev V, Botter-Jensen L, Thompsen KJ, Andersson KG, Murray AS. An assessment of cumulative external doses from Chernobyl fallout for a forested area in Russia using optically stimulated luminescence from quartz inclusions in bricks. J Environ Radioact 2008;99:1154–64. Rubanovich AV, Kalchenko VA. Segregation distortion in chronically irradiated populations of Pinus sylvestris L. growing in the area of Chernobyl nuclear meltdown. Russ J Genet 1994;30:126–8. Sarapult'zev BI, Geras'kin SA. Genetic basis of radioresistance and evolution. Moscow, Russia: Energoatomizdat Publishers; 1993 [in Russian]. Schutzendubel A, Polle A. Plant responses to abiotic stresses: heavy metal-induced oxidative stress and protection by micorrhization. J Exp Bot 2002;53:1351–65. Sharma SS, Dietz K-J. The relationship between metal toxicity and cellular redox imbalance. Trends Plant Sci 2008;14:43–50. Shevchenko VA, Pechkurenkov VL, Abramov VI. Radiation genetics of natural populations: genetic consequences of the Kyshtym accident. Moscow, Russia: Nauka; 1992 [in Russian]. Si L, Guo C, Cao Y, Cong W, Yuan Z. The effect of nitrobenzene on antioxidative enzyme activity and DNA damage in tobacco seedling leaf cells. Environ Toxicol Chem 2012;31:2078–84. Slomka A, Sutkowska A, Szczepaniak M, Malec P, Mitka J, Kuta E. Increased genetic diversity of Viola tricolor L. in metal-polluted environments. Chemosphere 2011;83: 435–42. Smith JT, Willey NJ, Hancock JT. Low dose ionizing radiation produces too few reactive oxygen species to directly affect antioxidant concentrations in cells. Biol Lett 2012;8: 594–7. Sparrow AH, Rogers AF, Schwemmer SS. Radiosensitivity studies with woody plants. Radiat Bot 1968;8:149–86. Spiridonov SI, Fesenko SV, Geras'kin SA, Solomatin VM, Karpenko YeI. The dose estimation of woody plants in the long-term after the Chernobyl accident. Radiat Biol Radioecology 2008;48:432–8. [in Russian]. Theodorakis CW. Integration of genotoxic and population genetic endpoints in biomonitoring and risk assessment. Ecotoxicology 2001;10:245–56. Tikhomirov FA, Shcheglov AI. Main investigation results on the forest radioecology in the Kyshtym and Chernobyl accident zones. Sci Total Environ 1994;157:45–57. Ulianova EV, Pozolotina VN, Sarapult'sev IE. Ecological-genetic characteristics of coenopopulations of Taraxacum officinale sl from the flood lands of the Techa river. Russ J Ecol 2004;35:349–57. van Straalen NM, Timmermans MJTN. Genetic variation in toxicant-stressed populations: an evaluation of the “genetic erosion” hypothesis. Hum Ecol Risk Assess 2002;8:983–1002. Vandenhove H, Vanhoudt N, Cuypers A, van Hees M, Wannijn J, Horemans N. Life-cycle chronic exposure of Arabidopsis thaliana induces growth effects but discernable effects on oxidative stress pathways. Plant Physiol Biochem 2010;48:778–86.

S.A. Geras'kin, P.Y. Volkova / Science of the Total Environment 496 (2014) 317–327 Verta J-P, Landry CR, Mackay JJ. Are long-lived trees poised for evolutionary change? Single locus effects in the evolution of gene expression networks in spruce. Mol Ecol 2013;22:2369–79. Vornam B, Arkhipov A, Finkeldey R. Nucleotide diversity and gene expression of catalase and glutathione peroxidase in irradiated Scots pine (Pinus sylvestris L.) from the Chernobyl exclusion zone. J Environ Radioact 2012;106:20–6. Woodwell GM. Radiation and the patterns of nature. Science 1967;156:461–70. Wright S. The interpretation of population structure by F-statistics with special regard to system of mating. Evolution 1965;19:395–420.

327

Zaka R, Vandecasteele CM, Misset MT. Effects of low chronic doses of ionizing radiation on antioxidant enzymes and G6PDH activities in Stipa capillata (Poaceae). J Exp Bot 2002;53:1979–87. Zhivotovsky LA. Population biometrics. Moscow: Nauka; 1991 [in Russian]. Zuravskaya AN, Kertsengolts BM, Kuliluk ТТ, Tserbakova ТМ. Enzymological mechanisms of plant adaptation to the conditions of higher natural radiation background. Radiat Biol Radioecology 1995;35:349–55. [in Russian].

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 re...
597KB Sizes 3 Downloads 5 Views