monitoring homologous recombination in rice (oryza sativa l.)

9
Mutation Research 691 (2010) 55–63 Contents lists available at ScienceDirect Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis journal homepage: www.elsevier.com/locate/molmut Community address: www.elsevier.com/locate/mutres Monitoring homologous recombination in rice (Oryza sativa L.) Zhuanying Yang a,1 , Li Tang a,1 , Meiru Li b , Lei Chen a , Jie Xu a , Goujiang Wu b , Hongqing Li a,a Guangdong Provincial Key Lab of Biotechnology for Plant Development, College of Life Sciences, South China Normal University, Guangzhou 510631, China b South China Botanic Garden, Chinese Academy of Sciences, Guangzhou 510650, China article info Article history: Received 21 June 2009 Received in revised form 11 April 2010 Accepted 9 July 2010 Available online 27 July 2010 Key-words: Oryza sativa L. Homologous recombination GUS staining Organ specific Rad51 abstract Here we describe a system to assay homologous recombination during the complete life cycle of rice (Oryza sativa L.). Rice plants were transformed with two copies of non-functional GUS reporter overlap fragments as recombination substrate. Recombination was observed in all plant organs examined, from the seed stage until the flowering stage of somatic plant development. Embryogenic cells exhibited the highest recombination ability with an average of 3 × 10 5 recombination events per genome, which is about 10-fold of that observed in root cells, and two orders of that observed in leaf cells. Histological analysis revealed that recombination events occurred in diverse cell types, but preferentially in cells with small size. Examples of this included embryogenic cells in callus, phloem cells in the leaf vein, and cells located in the root apical meristem. Steady state RNA analysis revealed that the expression levels of rice Rad51 homologs are positively correlated with increased recombination rates in embryogenic calli, roots and anthers. Finally, radiation treatment of plantlets from distinct recombination lines increased the recombination frequency to different extents. These results showed that homologous recombination frequency can be effectively measured in rice using a transgene reporter assay. This system will facilitate the study of DNA damage signaling and homologous recombination in rice, a model monocot. © 2010 Elsevier B.V. All rights reserved. 1. Introduction In all living organisms, DNA damage is caused by numerous exogenous (abiotic and biotic) and endogenous (cell metabolic activity, polymerase error.) factors [1–8]. In plant cells, UV-B radi- ation can induce the accumulation of various DNA lesions such as cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6–4) pyrimidinone dimers, and also minor oxidized or hydrated bases and single-strand breaks [9–12]. Exposure to chemicals and radi- ation often causes DNA crosslinks and strand breaks [13–17,2]. DNA recombination can be stimulated by pathogen stress [18]. Fur- thermore, growth conditions and the developmental stage of the plant also influence DNA damage and homologous recombination [19]. In view of the multiple types of DNA lesions, no single repair process can cope with all kinds of damage. To maintain genomic integrity, cells have evolved different DNA repair pathways. The major mechanisms of DNA repair include excision, recombinational and mismatch repair [20–22]. Because DNA damage occurs so frequently during the life cycle of different organisms, the study of factors influencing its occur- rence, its effects on cell growth and development and its repair are Corresponding author. Tel.: +86-20-85211275-8514. E-mail address: [email protected] (H. Li). 1 These authors contributed equally to this work. of significant importance. First, DNA damage caused by environ- mental conditions may be a signal which influences cell division and differentiation. Research in mammalian cells and in yeast cell demonstrated that DNA damage response pathways were linked to cell proliferation, cell-cycle arrest, cellular senescence and apopto- sis [23–25]. Even the single double-stranded break (DSB) would stop the division of yeast cell [26]. Characterization of Arabidop- sis mutants in genes encoding DNA damage sensors, such as ATM (ataxia-telangiectasia mutated) and ATR (ATM- and Rad3-related) kinases, suggested that DNA damage signaling is also conserved in plants [27]. However, the cellular components involved in the downstream cascade are still largely unknown. Second, DNA dam- age may stimulate the repair system inside the cell, and thus a method to detect DNA damage could be used to study DNA repair systems in different organisms [28,29]. Generally, various DNA lesions might be experienced during the life of a cell. For those lesions affecting only one of the two DNA strands, they can be repaired using the intact, complementary strand as a template [22]. For DSBs, they can be repaired through two pathways called homologous recombination (HR) and non-homologous end-joining (NHEJ) [30–33]. The detection of DNA damage in vivo can be performed using different methods, including comet assay, TUNEL (Terminal deoxynucleotidyl Transferase-mediated dUTP nick end labeling), and synthetic nucleotide probe etc. [34–37]. However, these meth- ods are not suitable for tracing DNA damage during the life cycle of 0027-5107/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2010.07.005

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Page 1: Monitoring homologous recombination in rice (Oryza sativa L.)

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Mutation Research 691 (2010) 55–63

Contents lists available at ScienceDirect

Mutation Research/Fundamental and MolecularMechanisms of Mutagenesis

journa l homepage: www.e lsev ier .com/ locate /molmutCommuni ty address : www.e lsev ier .com/ locate /mutres

onitoring homologous recombination in rice (Oryza sativa L.)

huanying Yanga,1, Li Tanga,1, Meiru Lib, Lei Chena, Jie Xua, Goujiang Wub, Hongqing Lia,∗

Guangdong Provincial Key Lab of Biotechnology for Plant Development, College of Life Sciences, South China Normal University, Guangzhou 510631, ChinaSouth China Botanic Garden, Chinese Academy of Sciences, Guangzhou 510650, China

r t i c l e i n f o

rticle history:eceived 21 June 2009eceived in revised form 11 April 2010ccepted 9 July 2010vailable online 27 July 2010

ey-words:ryza sativa L.

a b s t r a c t

Here we describe a system to assay homologous recombination during the complete life cycle of rice(Oryza sativa L.). Rice plants were transformed with two copies of non-functional GUS reporter overlapfragments as recombination substrate. Recombination was observed in all plant organs examined, fromthe seed stage until the flowering stage of somatic plant development. Embryogenic cells exhibited thehighest recombination ability with an average of 3 × 10−5 recombination events per genome, which isabout 10-fold of that observed in root cells, and two orders of that observed in leaf cells. Histologicalanalysis revealed that recombination events occurred in diverse cell types, but preferentially in cells

omologous recombinationUS stainingrgan specificad51

with small size. Examples of this included embryogenic cells in callus, phloem cells in the leaf vein, andcells located in the root apical meristem. Steady state RNA analysis revealed that the expression levels ofrice Rad51 homologs are positively correlated with increased recombination rates in embryogenic calli,roots and anthers. Finally, radiation treatment of plantlets from distinct recombination lines increasedthe recombination frequency to different extents. These results showed that homologous recombinationfrequency can be effectively measured in rice using a transgene reporter assay. This system will facilitate

sign

the study of DNA damage

. Introduction

In all living organisms, DNA damage is caused by numerousxogenous (abiotic and biotic) and endogenous (cell metabolicctivity, polymerase error.) factors [1–8]. In plant cells, UV-B radi-tion can induce the accumulation of various DNA lesions suchs cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6–4)yrimidinone dimers, and also minor oxidized or hydrated basesnd single-strand breaks [9–12]. Exposure to chemicals and radi-tion often causes DNA crosslinks and strand breaks [13–17,2].NA recombination can be stimulated by pathogen stress [18]. Fur-

hermore, growth conditions and the developmental stage of thelant also influence DNA damage and homologous recombination19]. In view of the multiple types of DNA lesions, no single repairrocess can cope with all kinds of damage. To maintain genomic

ntegrity, cells have evolved different DNA repair pathways. Theajor mechanisms of DNA repair include excision, recombinational

nd mismatch repair [20–22].Because DNA damage occurs so frequently during the life cycle

f different organisms, the study of factors influencing its occur-ence, its effects on cell growth and development and its repair are

∗ Corresponding author. Tel.: +86-20-85211275-8514.E-mail address: [email protected] (H. Li).

1 These authors contributed equally to this work.

027-5107/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.mrfmmm.2010.07.005

aling and homologous recombination in rice, a model monocot.© 2010 Elsevier B.V. All rights reserved.

of significant importance. First, DNA damage caused by environ-mental conditions may be a signal which influences cell divisionand differentiation. Research in mammalian cells and in yeast celldemonstrated that DNA damage response pathways were linked tocell proliferation, cell-cycle arrest, cellular senescence and apopto-sis [23–25]. Even the single double-stranded break (DSB) wouldstop the division of yeast cell [26]. Characterization of Arabidop-sis mutants in genes encoding DNA damage sensors, such as ATM(ataxia-telangiectasia mutated) and ATR (ATM- and Rad3-related)kinases, suggested that DNA damage signaling is also conservedin plants [27]. However, the cellular components involved in thedownstream cascade are still largely unknown. Second, DNA dam-age may stimulate the repair system inside the cell, and thus amethod to detect DNA damage could be used to study DNA repairsystems in different organisms [28,29]. Generally, various DNAlesions might be experienced during the life of a cell. For thoselesions affecting only one of the two DNA strands, they can berepaired using the intact, complementary strand as a template[22]. For DSBs, they can be repaired through two pathways calledhomologous recombination (HR) and non-homologous end-joining(NHEJ) [30–33].

The detection of DNA damage in vivo can be performedusing different methods, including comet assay, TUNEL (Terminaldeoxynucleotidyl Transferase-mediated dUTP nick end labeling),and synthetic nucleotide probe etc. [34–37]. However, these meth-ods are not suitable for tracing DNA damage during the life cycle of

Page 2: Monitoring homologous recombination in rice (Oryza sativa L.)

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n organism. To monitor DNA damage and repair inside plant cells,n intrachromosomal recombination system has been establishedy inserting two copies of non-functional GUS reporter overlapragments into the Arabidopsis genome [38]. DNA damage repairetween the repeats through homologous recombination recoverseporter gene expression, and thus the damage and repair eventsan be easily scored. This non-selective method allowed the local-zed analysis of recombination events in the whole life cycle ofrabidopsis. As the reporter construct contains two direct repeats ofhe GUS region, the recombination events detected by this systemnclude both intrachromosomal and interchromosomal ones.

By using this method, DNA damage due to different environ-ental factors and the recombination frequencies, as well as the

unction of genes involved in DNA repair, were analyzed [39,40].hese studies in Arabidopsis demonstrated that homologous recom-ination occurred during different stages of plant developmentnd in different organs, with higher recombination frequencies inotyledon than in leaf and root [41]. Additionally, integration ofhe recombination construct into the genome also exhibited posi-ion effects [42]. Treatment of plants with DNA mutagens suchs UV, MMS (Methyl methanesulfonate), bleomycin and radiationffectively enhanced homologous recombination through stimu-ation of the expression of repair-related genes [6,42–45]. Heavy

etals such as cobalt and cadmium which causes DNA damagelso promoted homologous recombination. Transcriptome pro-ling revealed similarities and differences in plant responses toadmium and lead [46]. Exposure of Arabidopsis seedlings to 50 mMalt resulted in a 3.0-fold increase in recombination frequency [47].ther factors such as temperature and day-length all influencedomologous recombination events [39]. Interestingly, pathogen

nfection and application of xylanase also increased recombina-ion frequency [18]. The same system had also been used to studyhe function of the recombination repair proteins in plants suchs Rad51C, Rad54, RecQ, and KU80, through introducing of theeporter to the mutants of these genes [48–51]. Furthermore,olinier et al. [52] used the reporter system to monitor genomics

hanges (hyper-recombination) after treatment with stress factors,nd they found that recombination occurred in the somatic tissuef not only the treated plants but also their progeny, a phenomenonalled transgenerational stress imprint.

Rice is a model of monocot grass species and also the stapleood source for a large proportion of people in the world [53,54].ice is different from the dicots with respect to leaf veins, vas-ular arrangement, root development, secondary growth, flowerrgans ([55–57], http://www.ucmp.berkeley.edu/glossary/gloss8/onocotdicot.html). Sequencing of the rice genome indicated thatost Arabidopsis genes have homologs in rice, whereas a large

umber of rice genes do not have homologs in Arabidopsis [58].hus, the genetic networks controlling rice development might beore complicated than in Arabidopsis. Given that differences in HR

ave been described in the dicot models Arabidopsis and tobacco59–61], it is reasonable to hypothesize that large differences in HRrequencies and mechanisms exist will be discovered in monocotsnd dicots.

In this study, we characterized a homologous recombinationystem in rice, which enabled us to visualize homologous recom-ination events throughout the whole life cycle and in all organs ofhe plant. We studied HR in different organs and cell types, as wells the factors influencing HR rates in this plant.

. Materials and methods

.1. Recombination substrate

The homologous recombination construct pINTRA7 consists of two truncated,on-functional, overlapping copies of the ˇ-glucuronidase (GUS) reporter gene sep-rated by the hygromycin selectable marker in the T-DNA region of pCAMBIA1300.

rch 691 (2010) 55–63

The transcription of HPT is driven by the CaMV 35S promoter and terminated by thepolyadenylation signal from the nopaline synthase gene (NOS-ter). The expressionof GUS gene was driven by the maize ubiquitin promoter and the translation of thetruncated GUS was stopped by an in-frame stop codon in the anti-sense sequenceof NOS-ter. The size of the overlapping sequence is 550 bp. Two 4.2 kb and 2.7 kbfragments can be obtained by digesting the construct with Sac I and Hind III (Fig. 1A).

2.2. Plant transformation and Southern blot analysis

The plant binary expression vector (Intra7) was mobilized into Agrobacteriumtumefaciens EHA105 using the freeze–thaw method. Rice “Zhonghua 11”(Oryzasativa L. japonica) was transformed via A. tumefaciens-mediated transformation [62],and 69 transgenic lines were obtained.

For Southern blot analysis, 10 �g of genomic DNA per sample was digestedwith Hind III restriction enzyme at 37 ◦C overnight, separated on a 0.8%(w/v) agarose gel, and transferred to Hybond N+ membranes. A digoxygenin(DIG) labeled GUS cDNA fragment was synthesised by PCR and used as theprobe. Prehybridization, washing, and chemiluminescent detection of the blotswas performed according to the manufacturer’s instructions (Roche Diagnos-tics GmbH, Mannheim, Germany). The primers used for amplification of theGUS fragment were GUS forward 5′-CGGCAAAGTGTGGGTCAATAATC-3′; reverse 5′-ACCAAAGCCAGTAAAGTAGAACGG-3′ .

2.3. Callus induction

Transgenic plants used in experiments were germinated and grown on 1/2 MS[63] medium, at 25 ◦C, with 16/8 h day/night light regime. Embryogenic calli wereobtained after six weeks of cultivation (mature seeds of the transgenic lines weredehusked, surface sterilized and cultured on NMB callus induction medium (N6

macro + MS micro + B5 organic compounds + 2 mg L−1 2,4-D + 3% sucrose + 0.8% agar,pH 5.8)).

2.4. Histochemical staining procedure

Histochemical staining of plant materials was performed as described by Jef-ferson [64]. To facilitate the penetration of staining buffer, leaves were cut intosegments of approximately 2 cm, calli were divided into pieces, and seeds were dis-sected in half. These tissues were then vacuum infiltrated in sterile staining buffer for15 min and incubated at 37 ◦C overnight. Plant tissues were subsequently bleachedwith 75% ethanol, then observed and imaged with a Nikon Microscope at 100× mag-nification. Recombination events were scored as separated blue sectors within thetissues, one sector represents one event.

2.5. Calculation of number of genomes

Total DNA of transgenic callus, leaves and roots was isolated from the respec-tive transgenic lines. The yield of total DNA was compared with the mean DNAcontent (0.55 pg) in a single Oryza sativa cell, to give an estimate of the number ofgenomes present. The DNA extraction method had no significant influence on theratio between the amounts of DNA in different plant organs.

2.6. Semi-quantitative reverse transcription PCR (RT-PCR)

Total RNA was extracted from 200 mg tissue samples using Trizol reagent. Allsamples were treated with DNase I. First-strand cDNA syntheses were performedusing 5 �g of total RNA and Moloney Murine Leukemia Virus reverse transcriptase(Promega). Expression of target genes was compared to the housekeeping geneActin, amplified for 20 to 25 cycles staring with 1 �L of the cDNA solution. Cyclingconditions were as follows: 5 min at 94 ◦C; and 20 cycles of 30 s at 94 ◦C, 30 s at 55 ◦C,and 40 s at 72 ◦C; and 10 min at 72 ◦C. The resulting cDNAs were subjected to PCRwith primers designed to amplify Rad51 families and Ku70 families. The primers areDMC 1 (Os11g0146800) forward 5′-GTGAGCAGATGGCGCCGTCCAAGC-3′ , reverse5′-TGTCAGGCTCTTCTTTGTATGC-3′; DMC 1 (Os12g0143800) forward 5′-GGCGCCG-TCCAAGCAGTACA-3′ , reverse 5′-TTCAATCCCTCCGCCAAGCA -3′; Rad51(Os11g0615-800) forward 5′-CGGGTTGAATGGTGCTGATGTA-3′ , reverse 5′-GGCAGAACCATC-CACTTGTG-3′; Rad51 (Os12g0497300) forward 5′-GGATGGTGGAATAGAAAC-TGGGT-3′ , reverse 5′-AACATCTGTGACGCCTTCCG-3′; XRcc3 (Os02g0562100) for-ward 5′-TCCACCAACAGCCAGCAAGA-3′ , reverse 5′-CGTATCACAAACTCGCACG-3′;Rad51D (Os09g0104200) forward 5′-AGGATGCGACGGAGAACAA-3′ , reverse 5′-GTTTACCTTGCTCTTCAGTGAC-3′; Rad51B (Os05g0121700) forward 5′-ATCTCTCT-CTCCCCCCACACA-3′ , reverse 5′-TGGTCATTGAGCCAGGACAT-3′; Rad51C(Os01g05-78000) forward 5′-ATCCTCAACCTCCCAAGTTCG-3′ , reverse 5′-CCATCTGCTCCCTTG-GACTT-3′; KU70 (Os07g0184900) forward 5′-GTCTGGAGGGAGATGAAATAGT-3′ ,

reverse 5′-GCGTAGATGATGACTTGAAACC-3′; KU70 (Os03g0856200) forward5′-ATGGCTCGCAACAAGGAAG-3′ , reverse 5′-TTAGTCACCAGGGGAAGTTC-3′ . Theexpression of Actin was used as an internal control, Primers for rice Actin were5′-AGGAATGGAAGCTGCGGGTAT-3′ and 5′-GCAGGAGGACGGCGATAACA-3′ . TheRT-PCR product was analyzed by electrophoresis on a 1% agarose gel. The relativeexpression level was calculated as the ratio of the absorbance peak value of the
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Z. Yang et al. / Mutation Research 691 (2010) 55–63 57

Fig. 1. Schematic map of the T-DNA region of recombination substrate (pINTRA7), and analysis of transgenic plants by Southern blot analysis. (A) Maps of pINTRA7 and itsproduct after homologous recombination. The homologous recombination substrate contained two homologous regions (G) in identical orientation used for recombinationanalysis. A recombination event restored the active GUS gene. A resistance marker HPT gene (hygromycin phosphotransferase) between the GUS sequence overlaps was lostduring this process. The transcription of HPT is driven by the CaMV 35S promoter and terminated by the polyadenylation signal from the nopaline synthase gene (NOS-ter).The expression of GUS gene was driven by the maize ubiquitin promoter and the translation of the truncated GUS was stopped by an in-frame stop codon in the anti-sensesequence of NOS-ter. Restriction enzymes used for Southern blot analysis were Sac I and Hind III and two fragments of 4.2 kb and 2.7 kb could be obtained after double digestw motei probe( US-sps

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ith these enzymes. LB, left border; RB, right border; Pubi, the maize ubiquitin prondividual recombinant lines. (B) Hind III digested DNA blotted with a GUS-specificC) Hind III and Sac I digested DNA from lines with single integration loci with a Gubstrate’s integrity were present in lines listed. Fragment sizes are given in kb.

ene of interest to the value of Actin using Gene Tools of Gene Company Ltd. Allxperiments were performed in triplicate under the same conditions.

.7. Plant sectioning

GUS stained tissues were removed from 70% ethanol (the final step of his-ochemical staining, see above), fixed overnight in FAA (acetic acid, 5 mL; 4%araformaldehyde, 5 mL; 70%ethanol, 90 mL). These were then dehydrated usingthanol/Citrisolv (Diamed) series and embedded in Paraffin for sectioning similar toethods described by Lozano-Baena et al. [65]. Each plant was sectioned at 8 �m

sing Leitz microtome and fixed to glass slides at 40 ◦C. Paraffin was removed withitrisolv and sections were rehydrated through graded series of ethanol followedy 10 min incubation in PBS prior to applying very dilute Safranin O (0.5% in water)or 2–3 min to more clearly differentiate GUS producing cells. Tissue was mountedn 10% glycerol for microscopy.

. Results

.1. Establishment of a system to detect homologousecombination in rice

To establish a homologous recombination system in rice, wetilized a GUS expression cassette under the direction of theaize ubiquitin promoter which directs strong gene expression inonocots [66]. The T-DNA region of the recombination construct

ncludes three parts: two truncated, overlapping fragments fromGUS expression cassette separated by the hygromycin selectablearker. The homologous sequence shared by the overlap fragments

s 550 bp long (Fig. 1A). The recombination substrate plasmid (pIN-RA7) was mobilized into A. tumefaciens EHA105 and the resulting

r; T: Nopaline synthase terminator; (B and C) Representative Southern analysis of. 1–14: DNA from transgenic lines. WT: DNA from untransformed “zhonghua 11”.

ecific probe shown in the figure. The 4.2 kb and 2.7 kb fragments indicative of the

strain was used to transform rice “Zhonghua 11”. In total, 69 inde-pendent transgenic lines were generated, and their T2 homozygousprogeny were grown in a greenhouse. To evaluate the numberof integration loci of transgenes in different lines, genomic DNAwas isolated and digested with Hind III which cuts once withinthe T-DNA region, and the resulting digested DNA was subject toSouthern blot analysis with a GUS probe. Most of the transgeniclines contained from one to five integration loci (Fig. 1B), whichis similar to transgenic lines produced with other constructs [62].To confirm the integrity of the recombination construct within therice genome, 15 lines with only one single hybridization band wereselected for further analysis. Further Southern blots of DNA iso-lated from these lines and digested with both Hind III and Sac Idemonstrated the expected 4.2 kb and 2.7 kb bands (Fig. 1C). Wealso observed that some of the lines exhibited additional hybridiza-tion bands which are probably caused by the truncated T-DNA orthe integration of more than one copies of the transgene in onesingle locus during the transformation process.

To test whether the recombination system functions in rice, westained various transgenic lines grown to the 3-leaf stage for detec-tion of GUS expression. We determined that the level of stainingintensity was sufficient for detection of recombination events, andwe did not find constitutive expression of GUS in all the transgenic

lines. GUS staining regions contained one to several cells, and some-times cell clumps in callus, cell lineage in leaf, root and other organswere found (Fig. 2B–N). As the occurrence of recombination is ran-dom, the number of GUS positive cells could be used to judge thetiming and location of the recombination events. For instance, an
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58 Z. Yang et al. / Mutation Research 691 (2010) 55–63

Fig. 2. Histochemical GUS staining of recombination events in whole plants. (A) A plantlet containing a functional GUS gene driven by the maize ubiquitin promoter, as positivec pINTRe ly reco stain( binatt referr

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ontrol. (B) Calli induced from mature seed containing the recombination substratevents during leaf growth giving rise to different sizes of stained sectors. (E) An earccurred in anther wall and pollen. (H) An early recombination event resulting largeK and L) Recombination events distributed in seed coat and endosperm. (M) Recomext (For interpretation of the references to color in this figure legend, the reader is

vent in a meristematic cell of the developing shoot apex yieldedsector in which more than one leaf was stained; if only parts ofne leaf are stained, this suggests that the sector originated from anvent early during leaf development (Fig. 2C). The large root sec-

A7, blue cell clumps represent the recombination events. (C and D) Recombinationombination event resulting full staining of a husk. (F and G) Recombination eventsed sectors in root cylindal. (I and J) Recombination events in root and root hair tips.ion events in coleoptile. (N) A recombination event in seed embryo. For details seeed to the web version of the article.).

tor in Fig. 2H can be explained by a recombination event duringan early stage of root development. Small sectors are formed byrecombination events late in development, e.g. in root cells (Fig. 2Ior J), which do not divide further or only divide once or twice more.

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Z. Yang et al. / Mutation Research 691 (2010) 55–63 59

Table 1The relative homologous recombination frequencies and rates in transgenic plant organs.

Lines Roots Leaves Callus

RF Genomes RR RF Genomes RR RF Genomes RR

IT14 2.6 ± 1.3 1.9E+06 1.4E−06 1.3 ± 0.3 7.2E+06 1.7E−07 863 ± 86 2.6E+07 3.32E−05T19 0.8 ± 0.1 2.0E+06 3.8E−07 0.3 ± 0.0 8.0E+06 3.1E−08 204 ± 29 2.6E+07 7.85E−06T21 2.3 ± 0.7 1.6E+06 1.4E−06 1.1 ± 0.2 7.3E+06 1.5E−07 504 ± 67 2.6E+07 1.9E−05T29 1.8 ± 0.3 1.8E+06 1.0E−06 0.2 ± 0.0 6.9E+06 2.4E−08 377 ± 31 2.6E+07 1.45E−05

IIT5 5.3 ± 0.4 1.9E+06 2.8E−06 1.7 ± 0.4 6.0E+06 2.8E−07 442 ± 64 2.6E+07 1.7E−05T8 16.5 ± 1.2 2.0E+06 8.2E−06 1.5 ± 0.4 1.1E+07 1.4E−07 690 ± 94 2.6E+07 2.7 E−05T15 23.4 ± 5.5 1.9E+06 1.2E−05 4.6 ± 1.7 8.0E+06 5.8E−07 1431 ± 163 2.6E+07 5.5E−05T31 2.0 ± 0.6 1.0E+06 2.0E−06 0.3 ± 0.0 3.9E+06 6.5E−08 256 ± 38 2.6E+07 9.8E−05

IIIT24 16.2 ± 4.1 2.0E+06 7.9E−06 2.1 ± 0.5 7.2E+06 2.9E−07 840 ± 84 2.6E+07 3.3 E−05T34 25.4 ± 3.4 3.7E+06 6.7E−06 4.6 ± 0.9 4.7E+06 9.9E−07 579 ± 68 2.6E+07 2.2 E−05T39 9.0 ± 1.2 1.7E+06 5.2E−06 3.8 ± 0.7 7.6E+06 5.0E−07 357 ± 45 2.6E+07 1.4 E−05T41 15.8 ± 2.1 2.1E+06 7.5E−06 5.3 ± 1.2 8.9E+06 5.9E−07 344 ± 46 2.6E+07 1.3 E−05

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ifty plantlets in each line were used for the analysis. “RF” stands for average recombalculated by relating RF to the total number of genomes present. The genome conhe means ±standard deviations (SD), n = 50. I, II, III transgenic lines containing 1, 2

omatic recombination also took place during flower developmentFig. 2E–G) and in the seed coat (Fig. 2K). In summary, we demon-trated that recombination occurred in all plant organs tested asell as throughout the somatic life cycle of the plant.

.2. Homologous recombination in different transgenic lines

To estimate the influence of the integration loci of transformedecombination substrate on recombination rates, we randomlyelected 12 lines which included four lines with single locus, fourines with two loci, and four lines with three loci of transgene inte-rated in the genome. Somatic recombination frequencies wereetermined by counting recombination events (sectors) in popu-

ations of different transgenic lines and relating these data to thepproximate number of genomes present per plant or per partic-lar organ at the indicated stages. The number of genomes wasetermined as described by Boyko et al. [67]. Representative GUStaining results for these experiments were presented in Fig. 2.

In all transgenic lines, we observed that embryogenic cal-us exhibited the highest recombination rates of approximately× 10−5 events per genome, which is about 10 times of thatbserved in root, and 100 times of that observed in leaf tissue. Linesith two or three loci generally had higher relative homologous

ecombination rates than those with a single locus, especially inoot and leaf tissue (Table 1).

For transgenic lines with equal number of integration loci, largeifference in recombination rates were also observed. Both T14 and19 contained a single integration locus, while their recombinationates could be 5- to 10-fold difference in either root or leaf. Thearge difference in recombination rates also existed between T15nd T31, which had two loci of recombination substrate integrated.owever, in callus derived from the above four lines, the difference

n recombination rates was much smaller as in the two to four timesange (Table 1).

In addition, we found that recombination rate for T15 was siximes of that for T31 in root; While in callus and leaf, the recom-ination rate for T15 was even lower than that for T31 (Table 1).his indicated that recombination rates were influenced by both

ell types and the chromatin structure near the recombination sub-trate.

Based on the above results, we suggest that organ or cellype is the major factor influencing recombination frequency, andecombination rates are positively correlated with the number of

n frequency per single plant organ or per gram of calli. “RR” is a recombination ratef the different organs was determined according to Boyko et al. [39]. Values were

i of the recombination substrate integrated, respectively.

integrated recombination substrates. The large influence of organtype on recombination rates can be explained by the observationthat different cell types exhibit different recombinogenic capac-ities. This has been previously described in Arabidopsis [67]. Thehigher recombination rates in lines with multiple integration locican be explained by the additive effects of independent recombi-nation events in different loci. Although the higher recombinationrates generally appeared in lines with more than one integrationloci, exceptions to this trend were observed. Lines T8 and T15, bothcontaining two integration loci showed higher recombination ratesthan the lines with three loci. The reason for this phenomenonmight be the complicated integration patterns of the construct,which had been revealed in the Southern blot (Fig. 1). And moreimportantly, integration sites of the construct in the genome, theso-called position effects, may also influence the recombinationfrequency.

3.3. Recombination frequency in various plant organs anddifferent cell types

The above experiments demonstrated that large differencesexist in HR rates among organs. As each organ is composed of var-ious cell types, and the cells undergoing HR might preferentiallyoccur in certain cells, thus, the localized analysis of cells which areactively involved in HR is necessary. We first performed GUS stain-ing in each organ to detect cells that are prone to recombination,and then made sections of the GUS stained explants (Fig. 3).

Recombination events were observed in the leaf blade and theleaf sheath (Fig. 3A and B). Interestingly, regions staining positivefor GUS activity in leaf tissue were mostly round spots, which indi-cated that recombination in leaf cells happened at later stages ofleaf development. We also found that about 2% of the seedlingsexamined showed GUS staining cell linage in at least one leaf(Fig. 3B). When counting the recombination events in leaves of lineT15, we noticed that recombination events located in epidermal,mesophyll and vascular cells at the frequencies of about 15%, 10%and 75%, respectively. A similar trend was also found in leaves ofline T14 (Table 2). The GUS stained region generally contains five

cells or more (Fig. 3C and D).

In root tissue, recombination could be detected in the mainroots, lateral roots and root hairs (Fig. 3E and F). Besides the bluespots, we observed much more cell lineages (about 8% of theseedlings examined showed GUS staining cell linage in at least

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60 Z. Yang et al. / Mutation Research 691 (2010) 55–63

F sentata (D). (Er ex (G)i to the

orrrcAldtTa

gsntcbc

i

TT

Tgt

ig. 3. Histology analysis of recombination events in leaf and root. (A and B) Reprend D) Sections showing the HR events in leaf mesophyll (C) and leaf sheath cellsoot tip (F). (G and H) Sections showing the HR events in root epidermis and cortnterpretation of the references to color in this figure legend, the reader is referred

ne root or one lateral root) compared with leaf tissue. Histologyevealed that recombination events occurred in all tissues of theoot (Fig. 3G and H). In the root explants of line T15, we found thatecombination events distributed in epidermal, cortex and cylinderells at the frequencies of about 56%, 30% and 14%, respectively.similar percentage of events were also detected in the roots of

ine T14 (Table 2). Recombination sectors in epidermal cells wereetected frequently but contained few cells. Recombination sec-ors in the cortex and cylinder generally contained multiple cells.he GUS stained lateral root tips were apparently derived from rootpical meristem cells (Fig. 2H–J).

We also detected high frequencies of recombination in embryo-enic callus, floral organs and seed organs including young husk,eed coat, embryo and endosperm. In embryogenic calli, recombi-ation occurred mainly on the surface, where cells are smaller andhe events in callus were evenly distributed. GUS staining region

ontained single cell or multiple cells, which indicates the recom-ination occurred continuously during the time course of tissueulture (Fig. 2B).

Homologous recombination was also detected in rice husk andn the anther (Fig. 2E and F). Recombination in pollen, however,

able 2he relative homologous recombination frequency in particular leaf and root tissue.

Transgenic line Number of spots in leaf

Epidermis (%) Mesophyll (%) Vascular bundl

T14 0.2 ± 0.1 (17) 0.1 ± 0.0 (8) 0.9 ± 0.1(75)T15 0.8 ± 0.1 (15) 0.5 ± 0.1 (10) 3.9 ± 0.6(75)

ransgenic line T14 (with recombination substrate integrated in a single locus) and T15rown to 3-leaf stage. Recombination events were examined by GUS staining as in the texhree replications. Data listed are presented as mean ±SD. Percentage of recombination e

ive GUS staining of HR events in epidermis of leaf blade (A) and leaf sheath (B). (Cand F) Representative GUS staining of HR events in root epidermis (E) and lateral, and central cylindal cells (H). Arrows show the location of the blue sectors (Forweb version of the article.).

occurred at high frequency. In line T34, about 70% of all antherscontained pollen at least one of which was stained GUS positive(Fig. 2G). In some anthers, numerous pollen grains were stained,which indicated that recombination occurred in the pollen mothercells or during the process of microsporogenesis. As DNA recom-bination occurs actively during meiosis stage, the high level ofrecombination in pollen was expected in our experiments. In seedtissues, recombination was observed in seed coat, endosperm,embryo and coleoptile (Fig. 2K–N). Rice endosperm is made up oftriploid cells, therefore the higher rates of recombination observedin this tissue is probably related to an additional copy of the recom-bination substrate. The numerous GUS stained cells in the coleoptilerevealed the active recombination of DNA in this organ during seedgermination.

3.4. Expression of genes involved in DNA recombination

Rad51 and its paralogs are essential for homology search dur-ing the recombination process and for DSB repair in mitosisand meiosis [68–70], while Ku70 functions in the NHEJ path-way. As recombination rates varied widely among different organs

Number of spots in root

e (%) Epidermis (%) Cortex (%) Central cylinder (%)

1.3 ± 0.3 (57) 0.7 ± 0.1 (30) 0.3 ± 0.1 (13)15.3 ± 3.5 (56) 8.2 ± 1.9(30) 3.8 ± 0.6(14)

(with recombination substrate integrated in two loci) seeds were germinated andt. Recombination events were counted from 50 to 60 plants in each treatment, withvents in a specific tissue per organ (leaf or root) were given in brackets.

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Z. Yang et al. / Mutation Research 691 (2010) 55–63 61

F n of Ri contre ard d

aRNhBtKawiapt(silblotgsRfliiilhRa

ig. 4. Transcripts of Rad51-like and KU-like genes in different tissues. (A) Expressiosolated from calli, seedling, root, flag leaf and anther. Actin was used as an internalxpression levels are relative to the Actin mRNA. The average values (means ±stand

nd cell types, we further analyzed the differential expression ofad51-like genes and the KU70-like genes in rice. Searches of theCBI (National Center for Biotechnology Information databases,ttp://www.ncbi.nlm.nih.gov/) with Rad51 and KU70 proteins asLAST queries, we identified at least eight Rad51-like proteins andwo KU70 proteins in rice (Os03g0856200 is a truncated version ofU70). Phylogenic analysis demonstrated that there are two DMC1nd two Rad51 in rice, which exhibited high amino acid homologyith their Arabidopsis counterparts. For other Rad51-like proteins

n rice, each has only one homologous protein in Arabidopsis. Tonalyze the contribution of Rad51-like genes to recombination, weerformed a semi-quantitative RT-PCR analysis of the steady stateranscripts of these genes in different organs (Fig. 4). The two DMC1Os11g0146800 and Os12g0143800) showed the highest expres-ion level in anther, which correlates well with its important rolen meiosis. Interestingly, these two genes also had a medium RNAevel in flag leaf and young root. Of the two rice Rad51 homologs,oth Os11g0615800 and Os12g497300 exhibited the highest mRNA

evel in callus, which is more than three times of their transcripts inther organs. Compared with Os11g0615800, more Os12g497300ranscripts were accumulated in anthers. For other Rad51-likeenes, Rad51B (Os05g0121700) and Rad51C (Os01g578000) tran-cripts were detected in different organ types except in callus, whilead51D (Os09g0104200) showed a higher expression level in callus,ag leaf and anther. XRcc3 (Os02g0562100) RNA level was very low

n all organs examined. Finally, the two KU70-like genes gave a sim-lar expression pattern, with the highest level in callus and lowest

n anther (Fig. 4). From these results, it is clear that the expressionevel of the two Rad5-like genes in rice correlated well with theigher recombination rates in callus, root and anther. And the otherad51-like genes might function in homologous recombination inn organ-specific way.

AD51-like genes and KU-70 like genes by semi-quantitative RT-PCR. Total RNA wasol. (B) Quantified expression levels of Rad51-like and KU-like genes by RT-PCR. Theeviation) are based on three separate RNA extractions.

3.5. Ionizing radiation (IR) stimulates homologous recombinationin different organs

IR is a known mutagen responsible for causing DNA strandbreaks in all living organisms [71]. To investigate the effects of DNAstrand breaks on homologous recombination, we treated seven-dayold seedlings of the recombination lines T14 (with recombinationsubstrate integrated in a single locus) and T15 (with recombinationsubstrate integrated in two loci) with different doses of Co60-� radi-ation. Following irradiation, the seedlings were grown to the 3-leafstage. Recombination rates were then examined by GUS stainingas before. We found that � radiation dosage had a significant influ-ence on recombination events in leaf and root tissue in both lines.Recombination rates increased in seedlings treated with � radia-tion from 0 mR to 0.75 mR. Higher doses of above 1 mR showed aninhibitory effect (Table 3). Treatment of line T14 with � radiationat 0.75 mR resulted in 3.4 events in leaf and 5.8 events in root perseedling, which are 2.8- and 2.3-fold of that in untreated control.The same treatment of line T15 caused 1.8- and 1.5-fold increaseof HR events in leaves and roots, respectively, as compared withuntreated control. These results demonstrated that DNA breakscaused by � radiation can stimulate homologous recombination,and thus support a strong and significant correlation between DNAbreaks and HR frequency in rice.

4. Discussion

In this study we have established and characterized a homolo-gous recombination system in the monocot model plant rice, andanalyzed some of the factors influencing recombination in thisplant. Specifically we found that: (i) homologous recombinationoccurred in all the organs in rice and the frequency ranged from

Page 8: Monitoring homologous recombination in rice (Oryza sativa L.)

62 Z. Yang et al. / Mutation Resea

Table 3The recombination frequency after treatment with Co60-� radiation.

Radiation Number of spots (Line T14) Number of spots (Line T15)

dose (mR) Leaf Root Leaf Root

0 1.2 ± 0.1 2.5 ± 0.1 5.2 ± 0.1 27.4 ± 0.80.12 1.7 ± 0.1 3.3 ± 0.1 6.1 ± 0.2 30.9 ± 0.90.25 2.1 ± 0.1 4.1 ± 0.2 7.4 ± 0.3 36.2 ± 1.00.50 2.6 ± 0.1 5.6 ± 0.2 8.6 ± 0.2 37.8 ± 1.20.75 3.4 ± 0.2 5.8 ± 0.2 9.4 ± 0.3 40.2 ± 1.71.00 3.0 ± 0.2 4.9 ± 0.2 7.6 ± 0.2 32.6 ± 1.0

Seeds of recombination lines T14 (with recombination substrate integrated in asingle locus) and T15 (with recombination substrate integrated in two loci) weregerminated for seven days in distilled water, and treated with different doses ofCo60-� radiation. Following irradiation, the seedlings were grown to 3-leaf stage.Rbp

1liitrm

HebitliTnAprh l).Bhrtrbmetmc( l).Td

tbbfkWsapHwt

[

[11] J. Narbutt, B. Cebula, A. Lesiak, A. Sysa-Jedrzejowska, M. Norval, T. Robak, P.

ecombination rates were then examined by GUS staining as in the text. Recom-ination events were counted from 20 plants in each treatment. Data listed areresented as mean ±SD.

0−5 to 10−7, with the highest frequency in callus tissue and theowest in leaves; (ii) recombination events preferentially occurredn embryogenic cells, phloem cells in the leaf vein, and cells locatedn the root apical meristem. (iii) DNA damage caused by � radia-ion can significantly enhance the recombination frequency; (vi) HRates in different organs are correlated well with the steady stateRNA level of the Rad51-like genes.A similar recombination system has been used for analyzing

R in Arabidopsis. The studies in Arabidopsis revealed that HRvents in leaves are about two times of that in roots (recom-ination events per single genome), and recombination events

n floral tissue are very rare [59,67]. Interestingly, we foundhat HR events in rice roots are 10 times more than that ineaves. In addition, we observed a higher HR rate occurredn rice anthers and seed organs (endosperm and coleoptile).hese findings indicate that significant difference in recombi-ation ability exists between the monocot rice and the dicotrabidopsis. Although both rice and Arabidopsis are floweringlants, they have major difference in vascular architecture,oot development, secondary growth and floral organs ([55–57],ttp://www.ucmp.berkeley.edu/glossary/gloss8/monocotdicot.htmecause these tissues are composed of different cell types whichave different cell growth and division activity, this might beesponsible for the different recombination rates observed inhe same organs of the two plants. For example, the higherecombination rates observed in rice roots could be explainedy the different modes of the root origin. In most dicots (and inost seed plants) the root develops from the lower end of the

mbryo, from a region known as the radicle. The radicle gives riseo an apical meristem which continues to produce root tissue for

uch of the plant’s life. In contrast, the radicle aborts in mono-ots, and new roots arise adventitiously from nodes in the stemhttp://www.ucmp.berkeley.edu/glossary/gloss8/monocotdicot.htmhese differences might influence the mode of cell growth andivision, as well as lateral root development.

Homologous recombination is also influenced by recombina-ion repair proteins in plant cells. The repair of DSBs in plantsy both NHEJ and HR pathways is essential for chromosomal sta-ility. Although high fidelity DSB repair via HR in plants is lessrequent than NHEJ, there is apparently a competition between theey enzymes of these pathways concerning strand availability [21].orks in Arabidopsis illustrated that tissue with a higher level of

trand breaks correspondingly had higher production of AtKU70nd AtRAD51, and the high ratio of available AtRAD51 RNA (as com-

ared to AtKU70) in leaves could be one of the reasons for higherR rates observed in this organ [67]. In rice, eight Rad51-like genesere identified and phylogenetic analysis revealed the conserva-

ion of these genes between Arabidopsis and rice. Interestingly, we[

rch 691 (2010) 55–63

found that AtRad51 and AtDMC1 each have two homologs withinthe rice genome. The expression patterns of these genes in rice aredifferent from that reported in Arabidopsis. For instance, AtDMC1 ismainly expressed during meiosis, and has weak expression in roots[73,74], while we found that rice DMC1 transcripts were presentnot only in anther but also in seedlings, leaves and roots. Thetwo rice Rad51 homologs exhibited the highest expression levelin callus, which correlated well with the highest recombinationevents in these cells. In anther, the transcripts for Os12g497300were two times of that for Os11g0615800, this might indicate thatOs12g497300 plays an important role in the HR in anther. As one ofthe key player in NHEJ, the KU70 transcripts are quite constant invarious rice organs except in anther. This might be one of the rea-sons that a higher HR event in anther was observed. Based on ourresults in rice and the former work in Arabidopsis [67], we suggestthat the expression level and organ-specific pattern of the Rad51-like genes are strongly correlated to the HR rates in different riceorgans.

In this study we have established a homologous recombinationsystem in rice, which is efficient in detecting DNA damage in differ-ent cell types and during the life cycle of this plant. Our system willfacilitate studies of DNA homologous recombination, DNA damagesignaling and the characterization of proteins involved in these pro-cesses. Furthermore, this system will benefit comparative studiesof homologous recombination between monocot and dicot plants.

Conflicts of interest

None.

Acknowledgments

We thank Dr. R.A. Jefferson, CAMBIA, Canberra, Australia, forkindly providing pCAMBIA vectors. We are grateful to HirofumiUchimiya (Tokyo University, Japan) for pAHC27. This research wassupported by the National Natural Science Foundation of China(No.: 30370137) and the Joint Funds of NSFC-Guangdong (GrantNo.: U0731006).

References

[1] A.B. Britt, DNA damage and repair in plants, Ann. Rev. Plant Physiol. Plant Mol.Biol. 45 (1996) 75–100.

[2] S.S. Wallace, Enzymatic processing of radiation-induced free radical damage inDNA, Radiat. Res. 150 (1998) S60–S79.

[3] Y. Kovtun, W.L. Chiu, G. Tena, J. Sheen, Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants, Proc. Natl. Acad.Sci. U.S.A. 7 (1999) 2940–2945.

[4] M. Orozco-Cardenas, C.A. Ryan, Hydrogen peroxide is generated systemicallyin plant leaves by wounding and systemin via the octadecanoid pathway, Proc.Natl. Acad. Sci. U.S.A. 96 (1999) 6553–6557.

[5] I. Kovalchuk, J. Filkowski, K. Smith, O. Kovalchuk, The dualistic nature of radi-cals: the high–low phenomenon, Plant Cell Environ. 26 (2003) 1531–1539.

[6] I. Kovalchuk, O. Kovalchuk, V. Kalck, V. Boyko, J. Fikowski, M. Heinlein, B. Hohn,Pathogen induced systemic plant signal triggers DNA rearrangements, Nature423 (2003) 760–762.

[7] O. Kovalchuk, I. Kovalchuk, A. Arkhipov, B. Hohn, Y.E. Dubrova, Extremely com-plex pattern of microsatellite mutation in the germline of wheat exposed to thepost-Chernobyl radioactive contamination, Mutat. Res. 525 (2003) 93–101.

[8] A.B. Britt, J.J. Chen, D. Wykoff, A UV-sensitive mutant of Arabidopsis defectivein the repair of pyrimidine–pyrimidinone(6–4) dimmers, Science 261 (1993)1571–1574.

[9] K. Modos, S. Gaspar, T. Kerekgyarto, A.A. Vink, L. Roza, A. Fekete, The role of thespectral sensitivity curve in the selection of relevant biological dosimeters forsolar UV monitoring, J. Photochem. Photobiol. 53 (1999) 20–25.

10] G. Ries, W. Heller, H. Puchta, H. Sandermann, H.K. Seidlitz, B. Hohn, ElevatedUV-B radiation reduces genome stability in plants, Nature 406 (2000) 98–101.

Smolewski, The effect of repeated exposures to low-dose UV radiation on theapoptosis of peripheral blood mononuclear cells, Arch. Dermatol. 145 (2009)133–138.

12] L.A. Van, M. Garmyn, P. Agostinis, Starting and propagating apoptotic signals inUVB irradiated keratinocytes, Photochem. Photobiol. Sci. 1 (2009) 299–308.

Page 9: Monitoring homologous recombination in rice (Oryza sativa L.)

Resea

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[[

[

[

[

[

[

[

[

[

[

[

[

[

[

Z. Yang et al. / Mutation

13] L.F. Povirk, W. Wübter, W. Köhnlein, F. Hutchinson, DNA double-strand breaksand alkali-labile bonds produced by bleomycin, Nucleic Acids Res. 4 (1977)3573–3580.

14] L.F. Povirk, DNA damage and mutagenesis by radiomimetic DNA-cleavingagents: bleomycin, Mutat. Res. 355 (1996) 71–89.

15] E. Chlebowicz, W.J. Jachymczyk, Repair of MMS-induced DNA double-strandbreaks in haploid cells of Saccharomyces cerevisiae, which requires the presenceof a duplicate genome, Mol. Gene. Genet. 167 (1979) 279–286.

16] P.A. Jeggo, DNA breakage and repair, in: J.C. Hall, T. Friedmann, J.C. Dunlap, F.Giannelli (Eds.), Advances in Genetics, 38, Academic Press, San Diego, 1998, pp.185–218.

17] P.L. Olive, The role of DNA single- and double-strand breaks in cell killing byionizing radiation, Radiat. Res. 150 (1998) S42–S51.

18] J.M. Lucht, M.M. Brigitte, H.Y. Steiner, Pathogen stress increases somatic recom-bination frequency in Arabidopsis, Nat. Genet. 30 (2002) 311–314.

19] O. Kovalchuk, V. Titov, B. Hohn, I. Kovalchuk, A sensitive transgenic plant systemto detect toxic inorganic compounds in the environment, Nat. Biotechnol. 19(2001) 568–572.

20] D.B. Roth, J.H. Wilson, Nonhomologous recombination in mammalian cells: rolefor short sequence homologies in the joining reaction, Mol. Cell Biol. 6 (1986)4295–4304.

21] V.V. Gorbunova, A.A. Levy, Non-homologous DNA end-joining in plant cells isassociated with deletions and filler DNA insertions, Nucleic Acids Res. 25 (1997)4650–4657.

22] J.H.J. Hoeijmakers, Genome maintenance mechanisms for preventing cancer,Nature 411 (2001) 366–374.

23] B. Kaina, DNA damage-triggered apoptosis: critical role of DNA repair, double-strand breaks, cell proliferation and signaling, Biochem. Pharm. 66 (2003)1547–1554.

24] Y. Aylon, M. Kupiec, Cell cycle-dependent regulation of double-strand breakrepair, Cell Cycle 4 (2005) 259–261.

25] M. Simonatto, L. Latellam, P.L. Puri, DNA damage and cellular differentiation:more questions than responses, J. Cell. Physiol. 213 (2007) 642–648.

26] C.B. Bennett, A.L. Lewis, K.K. Baldwin, M.A. Resnick, Lethality induced by a singlesite-specific double-strand break in a dispensable yeast plasmid, Proc. Natl.Acad. Sci. U.S.A. 90 (1993) 5613–5617.

27] M.K. Culligan, C.E. Robertson, J. Foreman, P. Doerner, A.B. Britt, ATR and ATMplay both distinct and additive roles in response to ionizing radiation, Plant J.48 (2006) 947–961.

28] L. Aravind, D.R. Walker, E.V. Koonin, Conserved domains in DNA repair proteinsand evolution of repair systems, Nucleic Acids Res. 27 (1999) 1223–1242.

29] H. Puchta, Double-strand break-induced recombination between ectopichomologous sequences in somatic plant cells, Genetics 152 (1999) 1173–1181.

30] R. Sargent, M. Brenneman, J. Wilson, Repair of site-specific double-strandbreaks in a mammalian chromosome by homologous and illegitimate recom-bination, Mol. Cell Biol. 17 (1997) 267–277.

31] F. Liang, M. Han, P.J. Romanienko, M. Jasin, Homology-directed repair is majordouble-strand break repair pathway in mammalian cells, Proc. Natl. Acad. Sci.U.S.A. 95 (1998) 5172–5177.

32] D.O. Ferguson, J.M. Sekiguchi, S. Chang, K.M. Frank, Y.J. Gao, R.A. DePinho, F.W.Alt, The nonhomologous end-joining pathway of DNA repair is required forgenomic stability and the suppression of translocations, Proc. Natl. Acad. Sci.U.S.A. 97 (2000) 6630–6633.

33] R.D. Kolodner, C.D. Putnam, K. Myung, Maintenance of genome stability inSaccharomyces cerevisiae, Science 297 (2002) 552–557.

34] P. Poli, A. Buschini, F.M. Restivo, A. Ficarelli, F. Cassoni, I. Ferrero, C. Rossi, Cometassay application in environmental monitoring: DNA damage in human leuko-cytes and plant cells in comparison with bacterial and yeast tests, Mutagenesis14 (1999) 547–556.

35] H.E. Poulsen, A. Weimann, S. Loft, Methods to detect DNA damage by freeradicals: relation to exercise, Proc. Nutr. Soc. 58 (1999) 1007–1014.

36] P.L. Olive, J.P. Banáth, The comet assay: a method to measure DNA damage inindividual cells, Nat. Protocols 1 (2006) 23–29.

37] J. Gong, S.J. Sturla, A synthetic nucleoside probe that discerns a DNA adductfrom unmodified DNA, J. Am. Chem. Soc. 129 (2007) 4882–4883.

38] P. Swoboda, S. Gal, B. Hohn, H. Puchta, Intrachromosomal homologous recom-bination in whole plants, EMBO J. 13 (1994) 484–489.

39] A. Boyko, J. Filkowski, I. Kovalchuk, Homologous recombination in plants istemperature and day-length dependent, Mutat. Res. 572 (2005) 73–83.

40] J. Molinier, E.J. Oakeley, O. Niederhauser, I. Kovalchuk, B. Hohn, Dynamicresponse of plant genome to ultraviolet radiation and other genotoxic stresses,Mutat. Res. 571 (2005) 235–247.

41] A. Boyko, F. Zemp, J. Filkowski, Double-strand break repair in plants is devel-

opmentally regulated, Plant Physiol. 141 (2006) 488–497.

42] J. Molinier, G. Ries, S. Bonhoeffe, B. Hohn, Interchromatid and interhomologrecombination in Arabidopsis thaliana, Plant Cell 16 (2004) 342–352.

43] G. Ries, G. Buchholz, H. Frohnmeyer, B. Hohn, UV-damagemediated induction ofhomologous recombination in Arabidopsis is dependent on photosyntheticallyactive radiation, Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 13425–13429.

[

[

rch 691 (2010) 55–63 63

44] A. Boyko, M. Greer, I. Kovalchuk, Acute exposure to UVB has more pronouncedeffect on plant genome stability than chronic exposure, Mutat. Res. 602 (2006)100–109.

45] A. Boyko, P. Kathiria, F. Zemp, F.J. Zemp, Y.L. Yao, I. Pogribny, I. Kovalchuk,Transgenerational changes in the genome stability and methylation inpathogen-infected plants, Nucleic Acids Res. 35 (2007) 1417–1725.

46] I. Kovalchuk, V. Titov, B. Hohn, O. Kovalchuk, Transcriptome profiling revealssimilarities and differences in plant responses to cadmium and lead, Mutat.Res. 579 (2005) 149–161.

47] A. Boyko, D. Hudson, P. Bhomkar, Increase of homologous recombination fre-quency in vascular tissue of Arabidopsis plants exposed to salt stress, Plant CellPhysiol. 47 (2006) 736–742.

48] K. Abe, K. Osakabe, S. Nakayama, M. Endo, A. Tagiri, S. Todoriki, H. Ichikawa, S.Toki, Arabidopsis RAD51C gene is important for homologous recombination inmeiosis and mitosis, Plant Physiol. 139 (2005) 896–908.

49] K. Osakabe, K. Abe, T. Yoshioka, Y. Osakabe, S. Todoriki, H. Ichikawa, B. Hohn,S. Toki, Isolation and characterization of the RAD54 gene from Arabidopsisthaliana, Plant J. 48 (2006) 827–842.

50] F. Hartung, S. Suer, H. Puchta, Two closely related RecQ helicases have antag-onistic roles in homologous recombination and DNA repair in Arabidopsisthaliana, Proc. Natl. Acad. Sci. U.S.A. 104 (2007) 18836–18841.

51] M.E. Gallego, J.Y. Bleuyard, S. Daoudal-Cotterell, N. Jallut, C.I. White, Ku80 playsa role in non-homologous recombination but is not required for T-DNA inte-gration in Arabidopsis, Plant J. 35 (2003) 557–565.

52] J. Molinier, G. Ries, C. Zipfel, B. Hohn, Transgeneration memory of stress inplants, Nature 442 (2006) 1046–1049.

53] T. Izawa, K. Shimamoto, Becoming a model plant: the importance of rice toplant science, Trends Plant Sci. 1 (1996) 95–99.

54] T. Sasaki, Rice genomics to understand rice plant as an assembly of geneticcodes, Curr. Sci. 83 (2002) 834–839.

55] T. Nelson, N. Dengler, Leaf vascular pattern formation, Plant Cell 9 (1997)1121–1135.

56] J. Kyozuka, K. Takeshi, M. Masakazu, T. Kobayashi, M. Morita, K. Shimamoto,Spatially and temporally regulated ex-pression of rice MADS-box gene withsimilarity to Arabidopsis class A, B and C genes, Plant Cell Physiol. 41 (2000)710–718.

57] R. Flavell, Role of model plant species, Methods Mol. Biol. 513 (2009) 1–18.58] J. Yu, J. Wang, W. Lin, S. Li, H. Li, J. Zhou, P. Ni, W. Dong, S. Hu, C. Zeng, et al., The

genomes of Oryza sativa: a history of duplications, PLoS Biol. 3 (2) (2005), e38,doi:10.1371/journal.pbio.0030038.

59] H. Puchta, P. Swoboda, B. Hohn, Induction of homologous DNA recombinationin whole plants, Plant J. 7 (1995) 203–210.

60] N. Orel, H. Puchta, Differences in the processing of DNA ends in Arabidopsisthaliana and tobacco: possible implications for genome evolution, Plant Mol.Biol. 51 (2003) 523–531.

61] J. Filkowski, O. Kovalchuk, I. Kovalchuk, Dissimilar mutation and recombinationrates in Arabidopsis and tobacco, Plant Sci. 166 (2004) 265–272.

62] M.R. Li, H.Q. Li, A simple and highly efficient Agrobacterium-mediated rice trans-formation system, Acta Biol. Exp. Sin. 36 (2003) 289–294.

63] T. Murashige, F. Skoog, A revised medium for rapid growth and bioassay withtobacco tissue cultures, Physiol. Plant 15 (1962) 473–497.

64] R.A. Jefferson, Assaying chimeric genes in plants: the GUS gene fusion system,Plant Mol. Biol. Rep. 5 (1987) 387–405.

65] M.D. Lozano-Baena, E. Prats, M.T. Moreno, D. Rubiales, A. Perez-de-Luque, Med-icago truncatula as a model for nonhost resistance in legume-parasitic plantinteractions, Plant Physiol. 145 (2007) 437–449.

66] A.H. Christensen, P.H. Quail, Ubiquitin promoter-based vectors for high-levelexpression of selectable and/or screenable marker genes in monocotyledonousplants, Trans. Res. 5 (1996) 213–218.

67] A. Boyko, J. Filkowski, D. Hudson, I. Kovalchuk, Homologous recombination inplants is organ specific, Mutat. Res. 595 (2006) 145–155.

68] K. Osakabe, T. Yoshioka, H. Ichikawa, S. Toki, Molecular cloning and characteri-zation of RAD51-like genes from Arabidopsis thaliana, Plant Mol. Biol. 50 (2002)71–81.

69] J.Y. Bleuyard, C.I. White, The Arabidopsis homologue of Xrcc3 plays an essentialrole in meiosis, EMBO J. 23 (2004) 439–449.

70] W. Li, C. Chen, U. Markmann-Mulisch, L. Timofejeva, E. Schmelzer, H. Ma, B.Reiss, The Arabidopsis AtRAD51 gene is dispensable for vegetative developmentbut required for meiosis, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 10596–10601.

71] O. Kovalchuk, A. Arkhipov, I. Barylyak, I. Karachov, V. Titov, B. Hohn, I.Kovalchuk, Plants experiencing chronic internal exposure to ionizing radiationexhibit higher frequency of homologous recombination than acutely irradiatedplants, Mutat. Res. 449 (2000) 47–56.

73] V.I. Klimyuk, J.D. Jones, AtDMC1, the Arabidopsis homologue of the yeast DMC1gene: characterization, transposon-induced allelic variation and meiosis-associated expression, Plant J. 11 (1997) 1–14.

74] M.P. Doutriaux, F. Couteau, C. Bergounioux, C. White, Isolation and characteri-sation of the RAD51 and DMC1 homologs from Arabidopsis thaliana, Mol. Gene.Genet. 257 (1998) 283–291.