Ž .Mutation Research 449 2000 47–56www.elsevier.comrlocatermolmut

Community address: www.elsevier.comrlocatermutres

Plants experiencing chronic internal exposure to ionizingradiation exhibit higher frequency of homologous recombination

than acutely irradiated plants

Olga Kovalchuk a, Andrey Arkhipov b, Igor Barylyak c, Ivan Karachov c,Victor Titov d, Barbara Hohn a, Igor Kovalchuk a,)

a Friedrich Miescher Institute, P.O. Box 2543, CH-4002 Basel, Switzerlandb Chernobyl Scientific and Technical Center of International Research, Shkolnaya Str. 6, 255620, Chernobyl, Ukraine

c Ukrainian Scientific Genetics Center, Popudrenko Str. 50, 253660 KieÕ-94, Ukrained IÕano-FrankiÕsk State Medical Academy, Galitska Str.2, 284000 IÕano-FrankiÕsk, Ukraine

Received 26 October 1999; received in revised form 13 December 1999; accepted 3 February 2000

Abstract

Ž .Ionizing radiation IR is a known mutagen responsible for causing DNA strand breaks in all living organisms. StrandŽ .breaks thus created can be repaired by different mechanisms, including homologous recombination HR , one of the key

wmechanisms maintaining genome stability A. Britt, DNA damage and repair in plants, Annu. Rev. Plant. Phys. Plant Mol.Ž .Biol., 45 1996 75–100; H. Puchta, B. Hohn, From centiMorgans to basepairs: homologous recombination in plants, Trends

Ž . xPlant Sci., 1 1996 340–348. . Acute or chronic exposure to IR may have different influences on the genome integrity.Although in a radioactively contaminated environment plants are mostly exposed to chronic pollution, evaluation of bothkinds of influences is important. Estimation of the frequency of HR in the exposed plants may serve as an indication ofgenome stability.

We used previously generated Arabidopsis thaliana and Nicotiana tabacum plants, transgenic for non-active versions ofŽ . wthe b-glucoronidase gene uidA P. Swoboda, S. Gal, B. Hohn, H. Puchta, Intrachromosomal homologous recombination in

Ž .whole plants, EMBO J., 13 1994 484–489; H. Puchta, P. Swoboda, B. Hohn, Induction of homologous DNAŽ . xrecombination in whole plants, Plant, 7 1995 203–210. serving as a recombination substrate, to study the influence of

acute and chronic exposure to IR on the level of HR as example of genome stability in plants. Exposure of seeds andseedlings to 0.1 to 10.0 Gy 60Co resulted in increased HR frequency, although the effect was more pronounced in seedlings.For the study of the influence of chronic exposure to IR, plants were grown on two chemically different types of soils, eachartificially contaminated with equal amounts of 137Cs. We observed a strong and significant correlation between the

Žfrequency of HR in plants, the radioactivity of the soil samples and the doses of radiation absorbed by plants in all cases.r)0.9, ns6, P-0.05 . In addition, we noted that plants grown in soils with different chemical composition, but equal

radioactivity, exhibited different levels of HR, dependent upon the absorbed dose of radiation. Remarkably, we observed a

) Corresponding author. Tel.: q41-61-697-7493; fax: q41-61-697-3976.Ž .E-mail address: igor@fmi.ch I. Kovalchuk .

0027-5107r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.Ž .PII: S0027-5107 00 00029-4

( )O. KoÕalchuk et al.rMutation Research 449 2000 47–5648

much higher frequency of HR in plants exposed to chronic irradiation when compared to acutely irradiated plants. Althoughacute application of 0.1–0.5 Gy did not lead to an increase of frequency of HR, the chronic exposure of the plants to severalorders of magnitude lower dose of 200 mGy led to a 5–6-fold induction of the frequency of HR as compared to the control.q 2000 Elsevier Science B.V. All rights reserved.

Keywords: Ionizing radiation; Acute exposure; Chronic exposure; Plants; Genome stability; Homologous recombination

1. Introduction

Ž .Despite the evidence that ionizing radiation IRcauses several types of DNA damage, alters basesand sugars, induces the formation of DNA–DNAand DNA–protein cross-links and causes single-Ž . Ž .SSBs and double-strand breaks DSBs , it is gener-ally accepted that the DSBs are the main, if not theonly type of damage that leads to the death of

w xirradiated cells 5 . IR may stimulate recombinationby inducing DNA-strand breaks, which are thoughtto be a critical step in initiating recombination. In-

Ž .duction of homologous recombination HR appearsto be a broad indicator of an agent’s mutagenecity,reflecting repair of induced DNA damage. Mitoticrecombination is stimulated by a wide spectrum ofmutagenic agents, including those which cause singlebase changes, deletions and frameshifts. Hence, itappears that recombination is a cellular response toDNA insults; the induction of recombination is par-ticularly suited as an indicator of mutagenecity.

IR is known to have a general effect on plantgrowth and development, ranging from stimulatoryeffects at very low doses to increasingly harmfuleffects for vegetative growth and to pronounceddecreases in reproductive effectiveness and yields athigh radiation levels. The degree of the effects variesin different plants and is dependent upon the species,age, plant morphology, physiology, and genome or-

w xganization 6 . So far studies have concentrated onthe gross effects of irradiation on plant viability andon the assessment of the radiosensitivity of differentplant species. Woody species seemed to be generally

w xmore sensitive to IR than herbaceous species 6 .Ž . ŽThus, acute irradiation 60 Gy of pine Pinus sil-

.Õestris stands resulted in death of pine trees near theŽ . w xChernobyl Nuclear Power Plant NPP 7 . However,

the lethal dose for Arabidopsis thaliana was esti-w xmated to be more than of 80 Gy 8 . Several types of

radiation, including b-, g-, and fast neutrons havebeen tested on soybean seedlings. b- and g-irradia-

tion caused similar effects, while fission neutronsw xwere 6 to 15 times more damaging 6 . In compari-

son to X-rays, irradiation of A. thaliana seeds withan equal dose of fast neutrons was shown to benearly 10 times more efficient in causing semi-steril-

w xity 9 .Radiation-induced DNA damage and the ability of

the cell to repair lesions are among the most impor-tant aspects of radiation genetics. Acute IR wasshown to have hazardous effects on different plantsspecies; the incidence of chromosome aberrations in

w xAllium cepa was found increased 10 ; the initial cellnumber in A. thaliana embryos was reported to

w xdecrease 11 ; the frequency of chlorophyll deficientembryonic mutation in A. thaliana was demon-

w xstrated to increase 12 ; the frequency of deletion ofthe dominant gene responsible for the blue color offlower petals, stamen filaments and stamen hairs inTradescantia species, so called ‘‘pink’’ mutation,

w xwas noted to increase 13 .However, the influence of chronic exposure of

plants to IR is not so well studied. A linear relation-ship between the level of chronic exposure to 60Cog-ray and the frequency of ‘‘pink’’ mutation in

w xTradescantia species was reported 14 . The effectscaused by chronic exposure to IR began to be moreextensively studied after the Chernobyl NPP acci-dent. An increased level of embryo lethal mutationswas documented for A. thaliana plants grown in the

w xChernobyl exclusion zone in 1986–1992 15 . Sev-eral morphogenetic changes were reported in 96plant species belonging to 28 families widespread

w xaround the Chernobyl NPP site 16 . An extremelyhigh frequency of chromosomal aberrations in ryeand wheat grown within the exclusion zone was

w xdocumented 17 . The incidence of DNA strandbreaks in natural A. thaliana plant populationschronically exposed to the IR of the Chernobyl

w xexclusion zone was also found to be enhanced 18 .Careful analysis of the effects of IR on the molec-

ular level was done in some studies via measurement

( )O. KoÕalchuk et al.rMutation Research 449 2000 47–56 49

of the frequency of HR. X-ray irradiation of tobaccoprotoplasts caused an increase in HR between re-peated sequences at the dose ranges of 1.25–2.5 Gyand a decrease, dropping to below the level ofuntreated samples, at the ranges from 5.0 to 15.0 Gyw x19 .

In previous studies, we could show a mutagenicinfluence of chronic exposure to IR, stemming frompolluted soils in the Chernobyl areas, on the genomeof two plant species: All. cepa and A. thalianaw x20,21 . We observed an increase of chromosomalaberration rates, mainly due to the increase of thefraction of the cells with chromosomal bridges and

Ž .fragments in the root-tip cells of All. cepa onion ,as well as a significantly enhanced frequency of HRin A. thaliana plants, cultivated in the soils fromChernobyl. However, so far no data are available onthe comparison of the influence of acute and chronicexposure to IR on the genome of the same plantspecies.

To address this question, we used the previouslydescribed A. thaliana and Nicotiana tabacum plantstransgenic for a truncated version of the b-gluco-

Ž .ronidase uidA gene as a recombination substratew x1,2,21 for comparative studies on the influence ofacute and chronic exposure to IR on the frequency ofintrachromosomal HR. We report on a pronouncedeffect of acute and chronic exposure to IR on thefrequency of HR in the tested plants. Surprisingly,chronic exposure to very low doses of IR has acomparatively stronger influence on recombinationthan an acute dose.

2. Materials and methods

2.1. Plants used for experiments

For both sets of experiments with acute andchronic exposure, previously generated and de-

scribed transgenic A. thaliana and N. tabacum plantsw xwere used 4 . The A. thaliana plant-line 651 carried

one copy per haploid genome of an overlapping,non-functional, truncated version of a chimeric b-

Ž .glucuronidase uidA marker gene as a recombina-tion substrate, with 566 bp overlapping sequences in

w xinverted orientation 3,4 . The N. tabacum plant-line9 carried four copies of the same overlapping se-

w xquences in one locus 4 .

2.2. Irradiation of plants

A. thaliana and N. tabacum plants were irradiatedwith 60Co at different development stages. Part ofthe plants was irradiated at the seed stage; the secondpart was treated at the stage of active growth, 10days after germination. The absorbed doses were 0.1,0.5, 2.5, 5.0 and 10.0 Gy. Irradiation was done using

60 Ž .a Co gun Agat-R1 , 0.025 Gyrs. Immediatelyafter the irradiation, seeds of the transgenic A.thaliana and N. tabacum plants were sown in soiland grown under 16 h light and 8 h dark at 248C.Ten-day-old plants germinated in soil were put ingrowth chambers immediately after irradiation andcontinued to grow in the same standard conditions.

Ž .Roughly 1200 plants "10% were used for eachexperiment.

2.3. Chronic irradiation of plants grown in soilsartificially contaminated with 137Cs

Two types of soil with different chemical compo-Ž . Ž .sition — soddy–podsolic s–p and peat–bogs p–b

were used for this experiment. The background activ-ities of the soil samples for 137Cs were 24.6 and 29.3Bqrkg, respectively. Chemical characterization ofthe soils was conducted on split samples. Soil sam-ples were analyzed for mobile forms of phosphorusand potassium, mobile nitrogen, pH of the soil saltextract, humus and Cu, Zn, Cd, Cr, Ni, Pb and Hg

Ž .content Table 1 . Soils were contaminated using a

Table 1Agrochemical characteristics of the soils used

Soil type N, P, K, Ca, Mg, Cu, Zn, Cr, Cd, Pb, Ni, pH Humus,mgrkg mgrkg mgrkg mg-eq.r mg-eq.r mgrkg mgrkg mgrkg mgrkg mgrkg mgrkg %

kg kg

Peat–bogs 117 127.0 48.2 67 3.1 19.5 54.8 1.7 1.7 13.0 1.3 7.15 6.24Soddy–podsolic 56 32.1 22.0 20 1.2 2.2 17.2 0.5 0.5 15.3 1.0 7.35 3.92

( )O. KoÕalchuk et al.rMutation Research 449 2000 47–5650

standard solution of 137CsCl and carefully homoge-nized. The activity of the soil samples were: 125,

Ž .250, 500, 1000, 2000, 4000 Bqrkg, "10% . Theseeds of A. thaliana and N. tabacum were sown instandard plastic pots and grown under the standardconditions of 16 h dayr8 h night, at 248C. Controlpots were separated from the contaminated ones toexclude the influence of IR originating from thecontaminated soils. Roughly 1200 plants were sownfor each experimental condition.

2.4. Estimation of the absorbed doses of radiation

The absorbed dose for plants was calculated asthe sum of the external and internal dose, as recom-

w xmended by Moiseev and Ivanov 22 . The gamma-Ž .irradiation exposure rate mRrh in the air at the soil

level was determined using a SRB9-1 ship beta-gamma radiometer, a FD-5 field dosimeter and a

DC9-04 dosimeter control signal. External dose astotal g-dose per day was calculated from readings.The internal dose is a consequence of uptake of 137Csby plants. 137Cs content in plant tissues was deter-mined by spectrometric and radiochemical methods.The radiation transfer factor for A. thaliana plantswas calculated as a relationship between the concen-

137 Ž .tration of Cs Bqrkg of dry weight in plants to137 Ž 2 .the concentration of Cs in soil kBqrm , taking

w xsoil properties into consideration, as described 23 .The two different radionuclides, 60Co and 137Cs,used for the experiment have comparable radiologi-cal characteristics. Both are b- and g-emitters andhave similar equivalent doses. Dose rate at a depth of

Ž .0.07 mm in tissue directional equivalent dose rateat a distance of 10 cm from a source of radiation

Ž 9.with an activity of 1 GBq 10 is 1000 mSvrh for60Co and 2000 mSvrh for 137Cs. A surface contami-nation of 1 kBqrcm2 leads to the dose of 1.1

Fig. 1. Detection of homologous recombination in transgenic plants. Recombination events are visualized as blue sectors in an A. thalianaplant.

( )O. KoÕalchuk et al.rMutation Research 449 2000 47–56 51

mSvrh for 60Co and 1.5 mSvrh for 137Cs. Thesesmall differences in dose rates are thus not relevantfor the observed influences of chronic and acuteirradiation on the stability of the plant genome.

2.5. Recombination assay

Ž .Homozygous transgenic A. thaliana line 651Ž .and N. tabacum line 9 plants were used to study

w xthe recombination frequency 3,4 . Upon HR, thegene was restored and upon histochemical staining,the recombination events were scored as blue sectorson the white plants, enabling us to conduct a quanti-

Ž .tative assay Fig. 1 . This transgenic system allowsthe detection of recombination events in all plantorgans at different developmental stages, from the

w xseed stage to the flowering stage 3 . Plants areusually examined at the stage of full rosette; at thisstage the plant size is small enough to allow theanalysis of large plant populations, yet the plants arelarge enough to present sufficient leaf-surface. For

Žstaining, plants at the full rosette stage 5 weeks after.germination were vacuum infiltrated for 15 min in

sterile staining buffer containing 100 mg 5-bromo-Ž .4chloro-3indolyl glucuronide X-Glu substrate

Ž .Jersey Labs, USA in 300 ml 100 mM phosphateŽ .buffer pH 7.0 , 0.05% NaN , 0.1% Triton X-1003

w xand incubated at 378C for 48 h 3,20 .Recombination frequencies were calculated by

Ž .counting recombination events sectors in each plantseparately, summing and relating these data to thenumber of plants in the population. HRFsXrN,where HRF is recombination frequency, X is num-

Žber of recombination events blue sectors on the.leaves in a plant population and N is the number of

plants in a population. For each experimental condi-tion, roughly 200 plants were scored.

2.6. Statistical treatment of the data

The main statistical procedures were described byw xSokal and Rohlf 24 . The mean values of recom-

bination frequencies for plant populations for eachexperimental condition were calculated. For the de-termination of the significance of the difference be-tween the means, the Student’s t-test for independentvariance was used. Correlation analysis was carriedout between doses of radiation absorbed by plants,

the activity of 137Cs in the soil samples and HRF inpopulations of A. thaliana and N. tabacum plants.The significance of the correlation coefficients was

w xtested using Fisher’s Z-transformation 24 . Statisti-cal treatment and plotting of the HRF in A. thalianaand N. tabacum populations were performed usingthe Sigma Plot 4.0 and Excel 5.0 for Windows 95program.

3. Results

3.1. Influence of acute exposure to IR on HR

Transgenic A. thaliana and N. tabacum plantswere used, which previously have been shown torespond with increased frequency of HR to different

w xgenotoxic agents 2 , including chronic IR stemmingw xfrom radioactively contaminated Chernobyl soils 20 .

These plants were irradiated with 60Co at differentdevelopmental stages: as seeds or 10-day-old plants.Absorbed doses ranged from 0.1 to 10.0 Gy. Underthe applied conditions the plants had no visibledamage such as lesions or necroses of the leaves.

The germination rates of both A. thaliana and N.tabacum plants exposed to acute irradiation with60Co at the seeds stage did not differ drastically fromthe control plants, at least up to 10.0 Gy. Applicationof 10.0 Gy resulted in a decrease of germination ofArabidopsis seeds from 84.0"6.3% in the control

Ž .population to 71.0"5.9% P-0.05 . Upon irradia-tion of tobacco seeds to the same 10.0 Gy dose, thegermination ratio decreased from 81.0"8.2% in

Ž .control population to 65.0"7.1% P-0.05 . In-crease of the doses of acute exposure of A. thalianaand N. tabacum plants irradiated at the seed stageand at the stage of young seedlings resulted in an

Žincrease of the frequency of HR r)0.9, ns6,. Ž .P-0.05 in all cases Table 2 . However, the effect

of the same doses of acute radiation exposure on thefrequency of HR was more pronounced in plantsirradiated during the period of active growth — atthe age of 10 days. Application of 0.5 Gy to seedlingsresulted in an almost twofold induction of HRF inA. thaliana and in 1.4-fold induction in N. tabacum,whereas a similar effect for the plants irradiated atthe seed stage was observed only after application of

( )O. KoÕalchuk et al.rMutation Research 449 2000 47–5652

Table 2HRF in A. thaliana and N. tabacum plants irradiated with 60Co at different developmental stages

Absorbed dose, Gy Seeds 10-day seedlings

A. thaliana N. tabacum A. thaliana N. tabacum

Control 0.45"0.09 0.59"0.05 0.41"0.05 0.63"0.060.1 0.41"0.11 0.62"0.07 0.47"0.09 0.58"0.08

U U U0.5 0.67"0.10 0.69"0.09 0.79"0.11 0.83"0.10UU2.5 0.78"0.08 0.78"0.04 1.23"0.09 0.95"0.16

U UU UU5.0 1.01"0.06 0.76"0.12 1.78"0.07 1.56"0.15U U U10.0 1.07"0.14 0.97"0.13 1.81"0.13 1.87"0.12

Ž .HRF events per plant "SD, 95% confidence limits.U

P-0.05, Student’s t-test for independent variance.UU

P-0.01, Student’s t-test for independent variance.

Ž .2.5 Gy Table 2 . The difference between the induc-tion of the HR in plants irradiated at the seed oryoung seedling stage became more drastic when theplants had absorbed doses ranging between 5.0 and10.0 Gy. We observed a 4.3- and 4.5-fold increase ofthe frequency of HR in irradiated A. thalianaseedlings and a 2.5-and 2.9-fold increase in N.tabacum. The effect of the same doses of radiationapplied at the seed stage was less pronounced: 2.2-and 2.4-fold increase in A. thaliana and 1.3 and 1.6

Ž .in N. tabacum Table 2 .An important observation was the distribution of

the recombination events in the stained plants. Plantsirradiated at the seed stage had almost equal distribu-tion of the recombination sectors between leaves,roots and stems, whereas the plants exposed at the10-day stage had higher frequency of the recombina-tion events in leaves and a lower frequency in roots,

Žas compared to the total number of HR events data.not shown . Thus, acute exposure to IR both at the

seed or 10-day-old seedling stage induced HR inplants. The effect was more pronounced when plantswere irradiated in the age of 10 days, during theperiod of active growth.

3.2. Influence of chronic exposure to IR on HRF inthe plant genome

To model chronic exposure of plants to IR, soilwas artificially contaminated with a standardizedsolution of radioactive 137Cs. The different soil types,soddy–podsolic and peat–bogs, were used in parallel

in order to check the influence of the chemicalproperties of the soils on absorption of radioactivesubstances and on HR in plants. Soil samples werepolluted with 137CsCl, amounting to soil activities of125, 250, 500, 1000, 2000 and 4000 Bqrkg, respec-

Ž .tively Table 3 .A strong significant correlation between the activ-

ity of soil samples for 137Cs and the frequencies ofHR in both transgenic plants, cultivated in radioac-tively contaminated soils of both types was deter-

Ž .mined r)0.9, ns6, P-0.05 in all cases . Gener-ally, plants grown in different types of soil withsimilar levels of radioactive contamination exhibitedsimilar increases of the HR frequency, dependent onthe activity of 137Cs in the soil. Interestingly, inplants grown in peat–bogs soil, the increase of HRFwas steeper than in plants grown in soddy–podsolic

Table 3Doses of radiation absorbed by A. thaliana plants grown during35 days in the soils artificially contaminated with 137Cs

Activity External Internal dose, Total absorbedof the soil dose, mGy mGy dose, mGysample, Soddy– Peat– Soddy– Peat–Bqrkg podsolic bogs podsolic bogs

125 1.51 2.66 4.59 4.17 6.1250 3.01 5.28 9.19 8.29 12.2500 5.99 9.84 17.27 15.83 23.26

1000 11.97 21.92 36.74 33.89 48.712000 23.94 40.63 65.08 64.57 89.024000 47.88 85.9 151.36 133.78 199.24

( )O. KoÕalchuk et al.rMutation Research 449 2000 47–56 53

soil. The observed difference in the frequency of HRfor plants grown in two different soils can mostprobably be explained by the different amounts ofthe radioactivity incorporated by the plants. This wasmainly due to the fact that the radiation dose ab-sorbed by the plants was dependent on soil proper-ties. Indeed, transfer of radioactive 137Cs from thesoil to meadow plants via root uptake was shown to

w xbe more efficient in peat–bogs soil 23,25 . Also, inour experiments plants grown on peat–bogs soilsincorporated a nearly 60% higher amount of radioac-tive Cs, in comparison to the ones grown in soddy–

Ž .podsolic soil Table 3 . Transfer of radionuclidesfrom soil to plant could be defined by soil properties,chemical properties of radionuclides and biologicalproperties of the plants. Cs is usually sorbed by clayminerals, such as montmorilonite, vivianite or others,

w xpresent in mineral soil 23 . Soddy–podsolic is amineral soil with low organic compound content.Peat–bogs soil is an organic soil that contains fewermineral components in comparison to mineral soilsand has lower sorption properties for Cs.

We observed a strong significant correlation be-tween the absorbed dose of radiation and the fre-quency of HR in the studied A. thaliana plantsŽ . Ž .r)0.9, ns6, P-0.05 Table 4 . The significantcorrelation between the doses of radiation chroni-cally incorporated by plants and the frequency of HRin the plant genome could most closely be approxi-mated by the exponential growth regression modelŽ .data not shown .

4. Discussion

DNA is subject to continuos alterations. Biochem-ical modifications of DNA bases and strand breakscan result in point mutations, insertions, deletions orgross rearrangements. The frequency of all thesemodifications is increased by environmental stimuli,such as IR. DSBs are critical lesions in genomes andeven a single genomic DSB is able to block cell

w xdivision in mammalian cells 26 . Efficient repair ofDBSs is therefore important for maintaining the sta-bility of the genome and survival of all organismsw x27 : it can be performed either via illegitimate or

w xhomologous recombination 5,28 . Thus, HR is oneof the mechanisms of maintaining the stability of theplant genome.

Plants transgenic for a marker gene revealing HRenabled us to study and compare the effects of acuteand chronic radiation exposure on the frequency ofHR and thus on the genome stability of plants. Ourresults revealed a monotonic dose-dependent in-crease of HR in A. thaliana and N. tabacum plantsafter acute and chronic exposure to IR.

We studied the effects of acute exposure to IR atdifferent stages of plant development. Acute irradia-tion of plants at the stage of 10-day seedlings hadmore pronounced effects on the stability of plantgenome than irradiation of seeds. For both A.thaliana and N. tabacum the frequency of HR washigher in plants exposed at the stage of 10-day oldseedlings than at the seed stage, while the absorbed

Table 4HRF in A. thaliana and N. tabacum plants grown in soils artificially contaminated with 137Cs

Activity of the soil sample, Bqrkg A. thaliana N. tabacum

Peat–bogs Soddy–podsolic Peat–bogs Soddy–podsolic

25 0.41"0.09 0.63"0.1129 0.39"0.04 0.59"0.15

125 0.42"0.01 0.35"0.05 0.65"0.06 0.61"0.07UU U250 0.61"0.09 0.48"0.11 0.75"0.07 0.71"0.04U U U500 0.82"0.07 0.69"0.07 0.89"0.09 0.78"0.09UU UU UU UU1000 1.86"0.18 1.53"0.28 1.65"0.13 1.64"0.13U2000 2.31"0.21 1.74"0.13 2.13"0.19 1.86"0.12U U UU U4000 2.67"0.17 2.13"0.18 2.47"0.20 2.01"0.28

Ž .HRF events per plant "SD, 95% confidence limits.UU

P-0.01, Student’s t-test for independent variance.U

P-0.05, Student’s t-test for independent variance.

( )O. KoÕalchuk et al.rMutation Research 449 2000 47–5654

doses of radiation were the same. Indeed, the abilityof plants to resist radiation was previously reportedto be dependent on the growth stage during irradia-tion as well as on plant parts or organs that were

w xirradiated 29 . Cereals such as barley, wheat andoats exhibited a lower survival rate after irradiationduring vegetative stage than under a similar radiation

w xduring the embryogenic stage 30 . The higher activ-ity of HR of dividing cells contrasts to lower HRactivity in seeds, the cells of which do not undergodivision. It is also known that the most frequentprimary target of IR in actively metabolizing plantcells is water, and the majority of DNA damageinduced by IR probably results from interaction of

w xDNA with hydroxyl radicals 6,28 . Due to muchhigher metabolism and water content, plants in thestage of 10-day-old seedlings are more sensitive toDNA damaging agents and thereby have higher lev-els of recombinational repair.

To compare the effects of acute and chronicexposure to IR on the plant genome, we calculatedand compared the doses of radiation absorbed byplants with the frequencies of HR. A. thaliana andN. tabacum plants were constantly exposed for 35days to different levels of radiation stemming fromcontaminated soil. The theoretically calculated ab-sorbed dose for these plants consisted in a combina-tion of internal and external exposure. The internalexposure is defined by the uptake of radioactive137Cs, whereas the external dose stems from theouter exposure originating from polluted soil and is

w xbelieved to be fully absorbed 22 .Quite unexpectedly, the effect of acute and chronic

exposure to IR on HRF and thus on the stability ofthe genome differed drastically. A. thaliana plantsgrown in uncontaminated soil accumulated about 1mGy. Arabidopsis plants cultivated in peat–bogs

Žsoil with the highest level of contamination 4000.Bqrkg accumulated a dose of about 200 mGy; in

soddy–podsolic soil the radiation dose absorbed byŽ .the plants constituted about 134 mGy Table 3 . This

led to a 5- and 6-fold increase of HRF in plantsgrown in peat–bogs and soddy–podsolic soils, re-

Ž .spectively Table 4 . In contrast after acute applica-tion of 0.1 Gy, no increase of the HRF was notedŽ .Table 2 . The most important result is that afteracute application of doses several orders of magni-tude higher than estimated for the accumulated

chronic irradiation, the induction of HRF in plantswas much lower. The frequency of HR in plants that

Žchronically accumulated a dose of 200 mGy Table.4 was 1.5–2-fold higher than in plants that have

Ž .received an acute dose of 10 Gy Table 2 . Ourresults thus show that the acute application of lowdoses of IR causes relatively little genome instabil-ity, whereas chronic accumulation of even severalorders of magnitude lower doses of it may poten-tially destabilize the plant genome. However, this isnot consistent with the data of van Gastel and deNettancourt, who have previously shown that chronicirradiation is less effective in inducing mutations

w xthan acute irradiation 31 .During acute exposure to IR and immediately

after it, plants probably mobilize mechanisms ofprotection and DNA repair to combat any damage.About 65% of DSBs are created by free radicals

w xstemming from ionized water 32 . As a response tothe creation of reactive oxygen species, scavengermechanisms are activated, which can inactivate mostof produced radicals by converting them into non-

w xmutagenic forms 32 . However, part of the radicalscan be converted into toxic forms such as OHP –,which can directly damage DNA, causing strandbreaks. Some of these breaks are apparently repairedby HR shortly after irradiation. In contrast to acutelyirradiated plants, plants grown in the polluted soilhave to deal with the constant exposure to IR andhence, with the constant production of free radicals.Although the amount of radicals generated duringchronic exposure to low doses of radiation is signifi-cantly lower in comparison to acute irradiation, thepermanent exposure may induce many more DSBs,which are in part repaired by HR. Another explana-tion could be that after acute exposure, plants utilizethe fastest possible mechanism of DNA repair, and arelatively higher percentage of the damage is re-

Žpaired via illegitimate recombination end-to-end. w xjoining 28,33 . In this case, an increase of the

frequency of HR would not be observed, since therepair by illegitimate recombination cannot be de-tected by the system used.

Acknowledgements

We are grateful to O. Mittelsten Scheid, J.M.Lucht, P. Pelczar and A. Ziemienowicz for critical

( )O. KoÕalchuk et al.rMutation Research 449 2000 47–56 55

reading of the manuscript. We are thankful to L.Kovalchuk for help and support, to N. Semenyuk forexcellent technical assistance in contaminating thesoils with 137Cs solution and to H. Moser and V.Panchuk for help with the statistical analysis. Thework was supported by the Swiss National ScienceFoundation in the terms of the program of collabora-tion with the Former Soviet Union Countries andNewly Independent States to O.K. and I.K. and bythe European Science Foundation ‘‘Plant Adapta-tion’’ fellowship to O.K.

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