Transcriptome analysis reveals fundamentaldifferences in plant response to acute and
chronic exposure to ionizing radiation
Igor Kovalchuk a, Jean Molinier b, Youli Yao a,Andrey Arkhipov c, Olga Kovalchuk a,∗
a Department of Biological Sciences, University of Lethbridge, Lethbridge, Alta. T1K 3M4, Canadab Institut de Biologie Moleculaire des Plantes, French National Center for Scientific Research (CNRS),
12, rue du general Zimmer, 67000 Strasbourg, Francec International Radioecology Laboratory, ICC, Post Box 151, Slavutych, Kiev, Ukraine
Received 23 November 2006; received in revised form 7 April 2007; accepted 18 April 2007Available online 5 May 2007
bstract
We analyzed the influence of acute and chronic ionizing radiation (IR) on plant genome stability and global genome expression.lants from the “chronic” group were grown for 21 days on 137Cs-artificially contaminated soil, and received a cumulative dosef 1 Gy. The “acute” plant group was exposed to an equal dose of radiation delivered as a single pulse. Analysis of homologousecombination (HR) events revealed a significantly higher increase in HR frequency (HRF) in the “chronic” group as compared toacute” group.
To understand the observed difference we performed global genome expression analysis. RNA profiling at 2 h and 24 h aftercute irradiation showed two-third of up- and down-regulated genes to be similarly regulated at both time points. In contrast, lesshan 10% of the genes up- or down-regulated at 2 h or 24 h post-acute irradiation were similarly changed after chronic exposure.romoter analysis revealed substantial differences in the specific regulatory elements found in acute and chronic transcriptomes.
urther comparison of the data with existing profiles for several stresses, including UVC and heavy metals, showed substantial
ranscriptome similarities with the acute but not the chronic transcriptome. Plants exposed to chronic but not acute radiation showedarly flowering; transcriptome analysis also revealed induction of flowering genes in “chronic” group.
2007 Elsevier B.V. All rights reserved.
sis; Gen
eywords: Ionizing radiation; Acute and chronic exposure; Arabidop
. Introduction
The ionizing radiation (IR) causes a variety of DNAamages, including base and sugar alterations, formation
of DNA–DNA and DNA–protein cross-links, as well assingle-strand breaks (SSBs) and double-strand breaks(DSBs). It is, however, generally accepted that the DSBsare the main, if not the only type of damage that leads to
the cell death [1]. Generated strand breaks are repairedby two major mechanisms, non-homologous end-joining(NHEJ) and homologous recombination (HR). IR isknown to induce HR frequency (HRF), although it is not
clear whether it is due to the increase of strand breaksor due to the stimulation of the activity of the repairenzymes.
The response to IR varies dramatically among plants.The general effect on growth and development rangesfrom stimulatory effects at very low doses to increas-ingly harmful effects for vegetative and reproductivetissue at high radiation levels. The degree of the radiationeffects is dependent on the species, age, plant morphol-ogy and physiology, and genome size and composition[2]. Woody plant species, in general, tend to be less resis-tant to IR as compared to herbaceous species [2]. Thebest known example is the pine Pinus silvestris, wherean acute exposure to 60 Gy of IR resulted in the deathof pine stands near the Chernobyl Nuclear Power Plant[3]. However, the lethal dose for Arabidopsis thalianaexceeds 100 Gy [4]. Similar doses of IR obtained fromdifferent types of radiation also trigger substantiallydifferent effects on the same plants. Several types of radi-ation, including �-, �-, and fast neutrons have been testedon soybean seedlings. �- and �-irradiation caused simi-lar effects, while fission neutrons were 6–15 times moredamaging [2]. In comparison to X-rays, irradiation of A.thaliana seeds with an equal dose of fast neutrons wasshown to be nearly 10 times more efficient in causingsemi-sterility [5].
A number of different plant-based assays exist tostudy DNA damage by radiation. Acute IR led toincreased incidences of chromosome aberrations inAllium cepa [6], decreases in the initial cell numberin A. thaliana embryos [7], increases in the frequencyof chlorophyll deficient embryonic mutations [8] andof the frequency of homologous recombination eventsin Arabidopsis plants [9,10], as well as in the increasein appearance of recessive phenotypes in Tradescantiaspecies [11]. Similarly, the exposure to chronic radiationwas also shown to have a significant influence on plantmorphology and genome integrity. For example, 60Co�-rays increased the frequency of “pink” mutations inTradescantia [12]. Further, ionizing radiation from Cher-nobyl exclusion zone increased the level of embryo lethalmutations in Arabidopsis [13], increased the recom-bination frequency and number of strand breaks inArabidopsis plants [14], caused a number of morpho-genetic changes in 96 different plant species [15], as wellas induced an extremely high frequency of chromosomalaberrations in rye and wheat plants [16].
There is substantial amount of information accumu-
lated on the influence of stress on plant transcriptomeprofile [17–22]. We recently reported the whole genomeexpression of plants exposed to Cd and Pb as well asto such mutagens as bleomycin, UVC, and xylanase
arch 624 (2007) 101–113
[17,18]. However, a comparison of acute and chronicionizing radiation stress on plant transcriptome is miss-ing.
We have previously found that the chronic expo-sure to the low dose of IR had a more pronouncedeffect on recombination than the acute exposure. Tounderstand the changes occurring in plants exposed tochronic or acute IR, we repeated the analysis of HRFand profiled the global genome expression. Our data con-firmed the stronger HRF increase in chronically exposedplants and showed drastic differences in their transcrip-tome. The comparison of the “acute” and “chronic”IR-induced transcriptomes with transcriptomes of theother stresses revealed substantial similarities between“acute” IR transcriptome and transcriptomes of otherstresses. In contrast, the “chronic” transcriptome wasrather unique and had no similarities to any other tran-scriptome.
2. Methods
2.1. Plant growth, radiation exposure and sampling
Plants of transgenic line #11 that carried in the genome sin-gle copy of truncated �-glucuronidase gene [23] were plantedon clean soil or on soil artificially polluted with 137Cs. Plantswere grown at 22 ◦C at a 16 h/8 h day/night regime. Plantsfrom acute group were irradiated with 60Co at 21st day (forthe microchip experiment) or at 14th day (for recombinationmeasurement) after germination. Irradiation was done using60Co gun (Agat-R1), 0.025 Gy/s to the total dose of 1 Gy. Plantsfrom chronic group were grown on soddy-podsolic soil artifi-cially polluted with a standard solution of 137CsCl to the finalsample activity of 34 MBq/kg (±10%). The sample activitywas calculated based on our previous experiments with thissoil. Previous data revealed that Arabidopsis plants grownfor 35 days on the soil with the activity of 4 kBq/kg absorbsthe total dose of 199.2 �Gy, with the contribution of inter-nal dose being −24% [10]. To obtain the total absorbed doseof 1 Gy within shorter period of time (21 days), one wouldneed to grow plants on soil with the activity of −34 MBq/kg(to obtain 5000-fold higher dose in −1.7-fold faster time;5000 × 1.7 × 4 kBq/kg = 34 MBq/kg). The control group wasmock treated. Approximately 300 plants were planted foreach treatment, and the experiment was performed threetimes.
The actual absorbed dose for plants was calculated asthe sum of the external and internal dose [24]. The gamma-irradiation exposure rate (R/h) in the air at the soil level wasdetermined using a SRB9-1 ship beta–gamma radiometer, a
FD-5 field dosimeter and a DC9-04 dosimeter control signal.External dose as total �-dose per day was calculated from read-ings. The internal dose is a consequence of uptake of 137Csby plants. 137Cs content in plant tissues was determined byspectrometric and radiochemical methods. The radiation trans-
on Rese
fbtercedao1
o
2
[14I4
2
2rRcwNPatoMoptsvFsb
2
pctifiwUs
I. Kovalchuk et al. / Mutati
er factor for A. thaliana plants was calculated as relationshipetween concentration of 137Cs (Bq/kg of dry weight) in plantso the concentration of 137Cs in soil (kBq/m2), taking soil prop-rties into consideration, as described [25]. The two differentadionuclide used for the experiment, 60Co and 137Cs, haveomparable radiological characteristics. Both are �- and �-mitters and have similar equivalent doses. The dose rate at aepth of 0.07 mm in tissue (directional equivalent dose rate) atdistance of 10 cm from a source of radiation with an activityf 1 GBq (109) is 1000 mSv/h for 60Co and 2000 mSv/h for
37Cs. A surface contamination of 1 kBq/cm2 leads to the dosef 1.1 mSv/h for 60Co and 1.5 mSv/h for 137Cs.
.2. Histochemical staining procedure
Histochemical staining was done according to Jefferson26]. For destructive staining plants were vacuum infiltrated 2×0 min in sterile staining buffer containing 100 mg 5-bromo--chloro-3-indolyl glucuronide (X-glu) substrate (Jersey Labsnc., USA). Afterwards plants were incubated at 37 ◦C during8 h and bleached with ethanol.
.3. RNA preparation and microchip hybridization
Total RNA was isolated from control, acutely (2 h and4 h) and chronically exposed Arabidopsis tissue using Trizoleagent (Life Technologies) following the supplier’s protocol.NA was then further purified using the RNeasy total RNAlean up protocol (Qiagen). The integrity of the RNA samplesas assessed by running an aliquot of samples on RNA 6000ano LabChip (Agilent) using the 2100 bioanalyzer (Agilent).robe synthesis, hybridization to Affymetrix ATH1-121501rrays and scanning were done according to Affymetrix pro-ocol. Statistical analyses of the scans were done with the helpf the Kensington Discovery Edition version 1.8 (Inforsense).ean values of gene expression were calculated for each group
f three RNA samples (three independent RNA hybridizations)repared from 20 plants each. The expression values of thereatment groups were related to the respective controls andignificance of the differences between the mean expressionalues was assessed using a Student’s t-test, two-tailed, paired.or further in depth analysis we selected those genes that hadignificantly (p < 0.05) increased or decreased their expressiony more than 3-fold.
.4. RNA preparation and reverse transcription
The total RNA samples used for the real-time PCR wererepared exactly the same way, from control, acutely andhronically treated Arabidopsis tissue. The samples werereated with DNase I (Invitrogen) according to manufacturer’s
nstructions. After DNase inactivation the samples were puri-ed with RNeasy Mini-columns (Qiagen). The RNA yieldsere measured using RiboGreen assay (Molecular Probes).sing 1.0 �g of the purified RNA as a template, reverse tran-
cription was performed in a total volume of 33 �L for 1 h at
arch 624 (2007) 101–113 103
37 ◦C according to manufacturer protocol (You-Prime-First-Strand Kit, Amersham, UK).
2.5. Real-time PCR analysis
The following genes and primers were used for real-time PCR: cor15a precursor (At2g42540) (+5′-atggcgat-gtctttctcagg-3′; −5′-acgacgaactgagttttctgg-3′), MYB-relatedtranscription factor CCA1, LHY (At2g46830), alternativeNADH-dehydrogenase (256057 at), DNA-binding pro-tein similar to CCA1 (At1g01060) (+5′-gaagaattattagc-taaggc-3′; −5′-atgttcttcaattcgttgcc-3′), putative protein(At3g54500) (+5′-tttggaacaagatgattctgg-3′; −5′-ctgcactacc-agccaaccgg-3′), DNAJ protein (At5g23240) (+5′-tctcc-gacgactcttcctcc-3′; −5′-atcaaagtcagtgatagacg-3′), DREB2A(At5g05410) (+5′-atggcagtttatgatcagag-3′; −5′-ctcgttatactctt-tccatc-3′), transcriptional activator CBF1, similar DREB1A(At1g12610) (+5′-aacgccgcatttggctcggg-3′; −5′-tccggatcattg-gattccgg-3′), At14a-1 (At3g28290) (+5′-atatggagaagtagt-gtggg-3′; −5′-tttttcaaactgtgccacgg-3′), putative proteinethylene-responsive element binding protein homolog(At4g34410) (+5′-aacagaaccgaattcgtcgg-3′; −5′-ctggaaaa-accctgacacgg-3′). Real-time PCR was performed accord-ing to previously published protocols [17]. The actinRNA was used as a control, the actin primers sequencewas: sense 5′-ACTGGCATGGCCTTCCG-3′, antisense5′-CAGGCGGCACGTCAGATC-3′.
3. Results and discussion
3.1. Experimental set-up
First, we analyzed the homologous recombinationfrequency in plants exposed to acute or chronic IR.Three groups of line #11 plants, “control”, “acute”and “chronic” (250 each), were planted in the soil in25 cm × 50 cm × 5 cm trays (Fig. 1A). The “chronic”group was germinated and grown in the same typeof soil, but artificially polluted with 137Cs. The totaldose absorbed by chronically exposed plants was0.93 ± 0.22 Gy and consisted of internal dose of0.71 ± 0.21 Gy and external dose of 0.22 ± 0.03 Gy.The plants of the “acute” group received 1.0 Gy at14 days post-germination, whereas “control” groupwere mock-irradiated. HRF was measured at 21 dayspost-germination.
Second, we performed a global genome expressionanalysis of the “control”, “acute” and “chronic” tis-sue. For this experiment, three similar groups, “control”,“acute” and “chronic” were germinated and grown in
pots with clean or contaminated soil. Each group con-sisted of 50 plants (Fig. 1B). Plants from “chronic” groupwere grown in polluted soil for 21 days, whereas plantsfrom “acute” and “control” groups were grown in clean
104 I. Kovalchuk et al. / Mutation Research 624 (2007) 101–113
Fig. 1. Experimental set-up. (A) Treatment for recombinationanalysis. “Control” group was mock treated; “chronic” group wasgrown on contaminated soil and received 0.93 Gy; “acute” groupwas irradiated at day 14 after germination, obtaining 1 Gy. All plantswere harvested for recombination analysis at day 21. Recombinationsubstrate consists of two non-functional truncated overlapping copiesof uidA transgene serving as a homologous recombination substrate.Repair of the damage that occurs to either region of the homology (U)via homologous recombination results in activation of the transgene.(B) Treatment for microchip analysis. “Control” and “chronic”groups were treated the same way as described in (A); “acute”
Fig. 2. Plants exposed to chronic radiation exhibited more pronouncedincrease in the frequency of homologous recombination. Y-axis showsthe number of spots per plant, and X-axis shows the treatment group.
group was irradiated at day 21 post-germination. Twenty plants fromeach experimental group were pooled together for RNA preparationand microchip analysis. Plants from acutely irradiated group werecollected twice, at 2 h at 24 h after irradiation.
soil for 21 days and then were �-irradiated or mock-irradiated, respectively. RNA samples were collectedfrom all groups, pooling 20 plants together. “Acute” sam-ples were collected at two different time points, 2 h and24 h post-irradiation (Fig. 1B).
3.2. Chronic exposure to IR resulted in asubstantially higher HRF increase than acuteexposure
HRF was found to be increased in both groupsof plants, acutely and chronically treated. On aver-age, the control plants had 1.25 ± 0.13 recombinationevents per plant, whereas acutely and chronically treated2.57 ± 0.19 and 4.33 ± 0.36, respectively (Fig. 2). Fur-ther, the differences between HRFs in all groups were allstatistically significant (single factor ANOVA, α = 0.01,p < 0.01 in all cases).
Several papers suggest that the majority of DNA dam-age events are the result of the non-targeted influenceof radiation on DNA [9,10,27–29]. The non-targetedevents represent the variety of activities in the cell that
were triggered by the response of irradiated cells. Theseactivities could include the generation of free radicals,the production of small molecules, metabolites, shortpeptides or small regulatory RNAs capable of traveling
All data points (diamonds), the mean (small black box inside of thewhite box), the maximum and minimum values (− sign), the 5–95%confidence interval (bars), 1–99% confidence interval (×) as well asS.D. (boxes) were calculated from three independent experiments.
from treated tissue and leading to the generation of non-targeted events such as DNA damage or activation ofcertain DNA repair mechanisms. On the other hand, it issimply possible that chronic radiation results in constantproduction of radicals that are under threshold of theactivation of scavenging enzymes. In this case it wouldlead to more frequent and constant DNA damage, leadingto regular formation of DSBs. We measured the level ofDSBs and found most of the breaks to be repaired alreadyat 6 h after acute radiation. In contrast, plants grown oncontaminated soil exhibited higher level of DSBs as com-pared to control plants at any given time during the plantgrowth (data not shown). This implies that radiation trig-gers a series of events resulting in increased genomeinstability. Since a substantial and statistical differencein the level of HR events in “acute” and “chronic” groupswas found, it was possible that there was also a substan-tial difference on the level of global genome expression.
3.3. Transcriptome analysis reveals significantoverlap between 2 h and 24 h acute groups andalmost no overlap with chronic group
The RNA expression profiling consisted of three“control”, three “chronic”, six “acute” (2 h and 24 htime points) samples. The average data for plant tissue
exposed to acute (2 h and 24 h) or chronic radiation wererelated to the average data for control plants. After thetwo cut-offs, a 3-fold change in activity (up- or down-regulation) and a statistical significance (p < 0.05), the
I. Kovalchuk et al. / Mutation Research 624 (2007) 101–113 105
Fig. 3. Diagram showing the genes commonly and differently regulated by acute and chronic stress. Numbers inside of each circle show then g part)s “2h 3fo“ mber od
nwaare1ctaafaorheg
tddf“maaf
3ed
a
umber of the genes that are uniquely (inside of the non-overlappinhows the genes that change their activity by more than 3-fold, where24 3fold D” and “ch 3fold D” were down-regulated genes. Total nuown-regulated, respectively.
umber of up- and down-regulated genes for each groupas obtained. Our analysis revealed 449 up-regulated
nd 423 down-regulated genes in plants harvested 2 hfter acute exposure and 237 up-regulated and 256 down-egulated genes in plants harvested 24 h after acutexposure. Chronic radiation resulted in up-regulation of38 and down-regulation of 139 genes (Fig. 3). Crossomparison between these three groups showed substan-ial similarity between “2 h” and ‘24 h” groups of plantsnd very little between “chronic” group and either ofcute. It was found that 60–70% of all genes that wereound to be up- or down-regulated 2 h after acute irradi-tion remained up- or down-regulated at 24 h (166 outf 237 and 161 out of 254 up- or down-regulated genes,espectively). However, plants from the “chronic” groupad only about 10% of genes regulated commonly withither of “acute” groups (19 and 7 out of 138 up-regulatedenes and 9 out of 139 down-regulated genes; Fig. 3).
These data suggest that most of the genes that changeheir expression 2 h after acute exposure stay up- orown-regulated even 24 h after exposure; however, theegree of the regulation may change (see below). Theact that there was only a minor overlap between thechronic” and “acute” groups suggest that differentechanisms are involved in the response to chronic or
cute exposure to radiation. To test this hypothesis, up-nd down-regulated genes were grouped into severalunctional categories/protein groups.
.4. Grouping the genes that changed their
xpression by the pathways revealed substantialifferences
To identify the genes/pathways influenced by acutend chronic exposure to radiation, we have grouped
or commonly (inside of overlapping part) regulated. Left diagramld I”, “24 3fold I” and “ch 3fold I” were induced and “2h 3fold D”,f genes showing “response” were 20,683 and 21,225 for induced and
up- and down-regulated genes into the following cate-gories: “unknowns”, “cell wall associated”, “pathogenresistance”, “signal transduction”, “oxidative stress”,“hormone response”, “nucleic acid metabolism”, “tran-scription factors”, “general stress”, “sugar and lipidmetabolism”, “calmodulins”, “protein and amino acidmetabolism”, “transport”, “development and morpho-genesis” and “not determined” (Fig. 4).
The chronic group had the largest percentage ofgenes associated with “nucleic acid metabolism”, 6%and 11% for up- and down-regulated genes, respec-tively. In contrast the acute group had 3% up-regulatedand 4% down-regulated genes in the 2 h group, and 1%up-regulated and 2% down-regulated genes in the 24 hgroup. The “nucleic acid metabolism” group includedgenes dealing with DNA repair, DNA binding (excludingthe transcription factors), and DNA or RNA metabolism.The correlation between the higher frequency of HR andthe substantially higher percentage of genes involvedin nucleic acid metabolism in the chronic group couldpotentially suggest more frequent modification of theirnucleic acids (DNA/RNA). Indeed, several putativetransposons, retrotransposon, and reverse transcriptaseswere found to be down-regulated in the “chronic” group,while not one such gene changed expression upon acuteexposure.
The most pronounced difference was observed inthe regulation of genes involved in signal transduction,where 10% and 7% of up- and down-regulated genes inthe “acute 2 h” group and 20% and 12% in the “acute24 h” group, compared to 5% and 3% in the chronic
group were observed, respectively. This is not surpris-ing as the acute exposure is a severe stress that changesthe regulation of multiple pathways, and thus elaboratesmultiple signal transduction cascades.
106 I. Kovalchuk et al. / Mutation Research 624 (2007) 101–113
ys. Upnes wit
Fig. 4. Up- and down-regulated genes were grouped to various pathwaand Ch D) genes were distributed to 15 different groups, including geoxidative stress response, transport, etc.
The most well represented group of genes was thegroup of oxidative stress-related genes. There were 12%,11% and 14% up-regulated genes, and 15%, 34%, 9%down-regulated genes in “acute 2 h”, “acute 24 h” and“chronic” groups, respectively. We observed differencesin regulation of several specific genes. For example,20 different cytochrome P450 genes were up- anddown-regulated in 2 h group, and 16 of them remainedsimilarly changed at 24 h. In contrast, only three suchgenes changed their expression upon chronic exposure.Cytochrome P450 monooxygenases are involved in thebiosynthesis of various compounds in plants such asphenylpropanoids, lipids, and phytohormones [30]. TheCYP86A and CYP94B cytochrome P450 monooxy-genase subfamilies are fatty acid omega-hydroxylases
involved in the synthesis of cutin, production of signal-ing molecules, and prevention of accumulation of toxiclevels of free fatty acids in plant cells liberated by phos-pholipases in early response to stress [31]. The fact that
-regulated (A2 I, A24 I and Ch I) and down-regulated (A2 D, A24 Dh unknown function as well as genes involved in signal transduction,
we found changes in the expression of the monooxyge-nases primarily at 2 h and 24 h after the exposure supportsthe idea that they are mostly important in an immediatestress response. Kitahata et al. also showed the involve-ment of monooxygenases in the chemical regulation ofabscisic acid catabolism [32], furthering this notion.
Another group of oxidative stress-related genes thatchanged their expression were the peroxidases. Wefound pr10, ATP8a, ATP3a, ATP12a, ATP13a, ATP14a,ATP17a, ATP20a, ATP21a, and ATP23a to be down-regulated at 2 h or 24 h after the acute exposure. However,in the chronic group, a single putative ATP2a-like per-oxidase was down-regulated. It has previously beenshown that the exposure of Arabidopsis to salt stressresulted in the down-regulation of several key per-
oxidases [33]. Moreover, it was found that antisensesuppression of cytosolic tobacco ascorbate peroxidaseresulted in higher tolerance to salt and heat stress [34],while the overexpression of ascorbate peroxidase in
on Rese
taoissdc
fgG2cgdernat
acaarraoob
Ft(d
I. Kovalchuk et al. / Mutati
obacco chloroplasts enhanced tolerance to salt stressnd water deficit [35]. Further still, Arabidopsis plantsverexpressing thylakoidal ascorbate peroxidase showedncreased resistance to paraquat-induced photooxidativetress and to nitric oxide-induced cell death [36]. Thesetudies all demonstrate that the regulation of peroxi-ases and other oxidative stress-related enzymes are keyomponents of the response variety of stresses.
Another group of oxidative stress-related genes dif-erentially regulated by acute and chronic stress are thelutathione-S-transferases (GSTs). Ten and 11 differentSTs were up- and down-regulated at both the 2 h and4 h post-acute exposure, respectively. At the same time,hronic exposure resulted in the change of a single GSTene. GSTs are known to be induced by various ROS-ependent stresses [37], drought [38], and influence ofthylene and auxin [39]. Again, it is possible that theesponse that is triggered in plants by chronic stress doesot reach a ‘threshold level’ for the regulation of theforementioned oxidative stress-related genes, and thushey are not present in the microchip profile.
The comparison of the “hormone response” grouplso revealed substantial differences. The acute 2 h groupontained 8 ethylene-responsive binding factors and 5uxin-responsive factors out of 17 up-regulated genesnd as well as 6 auxin-responsive and 1 ethylene-esponsive genes out of 14 down-regulated “hormoneesponse” genes. The acute 24 h group contained 4
uxin-responsive and 9 ethylene-responsive genes outf 13 “hormone-related” genes. In contrast, sevenut of eight “hormone response” genes up-regulatedy chronic stress were auxin-related. Recent research
ig. 5. Plants exposed to chronic radiation flowered earlier. For the pilot expero acute or chronic radiation or mock-treated. Flowering time (Y-axis, in daX-axis) and averages were calculated. Similar letters show statistically idenifferent results.
arch 624 (2007) 101–113 107
papers describe the close link between the exposureto abiotic stress and auxin/ethylene regulation. Tran-scription factors operating downstream of ethylene andauxin have been shown to be responsive to salt stress[40]. Further, Mishra et al. has shown that stress sig-naling occurs not only through MAP kinases, but alsothrough auxin and ethylene pathways [41], and it waspreviously shown that mechanical wounding regulates anumber of auxin- and ethylene-responsive genes [42]. Aschronic exposure to radiation induces vegetation growth,this might be in part due the (in)direct stimulation ofauxin-responsive genes. In our experiments, we foundthat the exposure to chronic, but not to acute radiation,induced the putative flowering-time gene CONSTANS(At3g02380) by 31-fold. Chronic stress also inducedseveral genes involved in photosynthesis and carbohy-drate metabolism, starch synthase (11-fold; At1g32900),chlorophyll a/b binding protein 151 precursor (10-fold;At3g27690), and a putative chlorophyll a/b bindingprotein (3.4-fold; At2g05070). In contrast, acute stressdown-regulated this group of genes at both 2 h and24 h: sucrose synthase (56.0- and 26.6-fold; At3g43190),6-phosphogluconolactonase-like protein (4.2- and 3.5-fold; At1g13700), and glucose fermentation (3.1- and3.2-fold; 255924 at). This further supports the idea thatchronic stress induces vegetative growth, and that this islikely regulated via auxin-responsive genes. In contrast,acute exposures to stress are perceived as more immedi-
ate and severe, and thus result in an abrupt inhibition ofmost of the growth-related pathways. This is, however, atransient reaction that is normally reversible after severaldays.
iment, three groups of plants (10 plants per each group) were exposedys) for individual plants (10 per group) was recorded in each grouptical results, whereas different letters show statistically significantly
on Rese
108 I. Kovalchuk et al. / Mutati
As a support of the microchip findings, we found thatplants exposed to chronic stress flowered on average 3days earlier than plants belonging to either “acute” or“control” groups (Fig. 5). Several early reports have sug-gested that the chronic exposure to radiation stimulateflowering [43,44]. However, one report showed no sig-nificant changes in the flowering time of Arabidopsischronically exposed to gamma-radiation [45]. Despitethis, other works have shown the regulation of flower-ing time upon stress exposure. Daly and Thompson [45]showed that exposure of plants to drought stress pos-itively influences the flowering time [46], while, morerecently, Martinez et al. suggested that various stresses,such as light, drought, high temperatures, pathogeninfection and exposure to UVC can influence the transi-tion to flowering [47].
Several other stress-induced genes were influencedby exposure to radiation. Cold regulated proteins cor15aand cor15b were induced by more than 20-fold by bothchronic and acute stress. DREB2A were induced by14-, 12.1-, and 3.4-fold by acute (2 h and 24 h) andchronic stress, respectively. DREB2A was shown to beinduced by water stress and regulating the expression ofmany water stress-inducible genes [48]. Similarly, genesDREB2B and DREB1C, commonly induced by variousstresses, were also induced by acute radiation exposureby 3.3- and 11.4-fold for DREB2B and 3.9- and 16.9-foldfor DREB1C at 2 h and 24 h, respectively.
Another substantial difference observed betweenacute and chronic groups was the regulation ofchromatin. CCR4-associated factor 1-like (CAF1,At3g44260) was induced only by acute stress (14.2-and 21.3-fold at 2 h and 24 h, respectively). CCR4 andCAF1 are two catalytic subunits with deadenylase activ-ity believed to be involved in DNA damage responsein yeast [49]. Mulder et al. suggested that the inductionof CAF1 by ionizing radiation in yeast is part of thegeneral stress response [50]. Further, Westmoreland etal. showed that the yeast cell cycle transition throughG1 and S phases is CCR4-dependent upon the expo-sure to stress [51]. Also regulated was GCN5-relatedN-acetyltransferase (GNAT, At2g39030). It was inducedby 5.5-fold at 2 h post-acute exposure. GCN5 was asso-ciated with UV-radiation response and response to highlight in yeast models [52,53].
3.5. Comparison of the data for IR, UVC and Cd/Pb
To further explore the differences in transcriptomicsof acute and chronic groups, we compared the set of thesegenes to those regulated by UVC exposure and the expo-sure to heavy metals [17,18]. When we compared acutely
arch 624 (2007) 101–113
regulated genes, we found many similarities. There were78 up-regulated and 15 down-regulated genes upon UVCexposure and 20 up-regulated and 47 down-regulatedgenes upon exposure to heavy metals, similarly regulatedby acute exposure to IR (2 h sample; SupplementaryTable 1). This calculated to a nearly 20% (78 out of 449)of all genes that were induced upon 2 h exposure to radi-ation to be also induced by UVC. Concurrently, 10% (47out of 423) of all genes that were down-regulated at 2 hafter IR exposure were down-regulated by exposure toheavy metals. Surprisingly, there was not a single genethat was regulated in both chronic exposure to IR as wellas exposure to heavy metals or UVC. This comparisonfurther confirms the “specificity” of chronic exposure.
The above multi-stress analysis confirmed that expo-sure to chronic radiation represents a different type ofstress when compared to acute exposure to IR and evenexposure to UVC and heavy metals. The changes intranscriptome of the chronically exposed plants mayrepresent an adaptation to a milder and constant typeof stress. The higher increase in HRF in chronicallyexposed stress also supports this assumption. In animals,it is proposed that exposure to radiation leads to the shiftfrom non-homologous to homologous repair of strandbreaks [54,55]. This mechanism is especially apparentin dividing cells because they spend less time in the G1phase, when HR repair is down-regulated [54,55]. Thisshift in the utilization of the strand break repair pathwaysmay represent a protection mechanism directed to long-term survival. It seems logical that a similar mechanismmay be conserved in plants.
3.6. Data comparison to previous publications
Another important comparison we performed wasthe comparison to a set of data published by Nagata etal. [56]. They performed the microchip analysis of A.thaliana plants exposed to 2000 Gy of Co60 harvested at2 h and 24 h post-irradiation. Comparison of the set ofgenes induced at 2 h post-irradiation revealed that halfof the genes (14 out of 28) reported by Nagata et al. werealso changed in our experiment [56] (Table 3S). In con-trast, only 3 genes in our experiment out of the total 19genes reported by Nagata et al. were also induced at 24 hpost-irradiation [56] (Supplementary Table 2). The num-ber of genes similarly down-regulated was much smaller,whereby only 2 of 27 and 5 of 19 at 2 h and 24 h groups,respectively.
It is not surprising that we did not observe more sim-ilarities in the transcriptomes in both experiments. Themain reason could be that Nagata et al. exposed plantsto the dose that was 2000-fold higher than the dose used
on Rese
icstticd
3e
ayblpt((ps(mRmw
eAietFe
TR
G
cMADPDDTAP
Te
I. Kovalchuk et al. / Mutati
n our experiment (2000 versus 1 Gy) [56], theoreticallyausing exponentially higher amounts of damage andtress. Additionally, differences in growth conditions ofhe plants could also have a substantial influence on theranscriptome. To objectively compare this data, exper-mental plants would have to be grown under identicalonditions before the different doses of radiation wereealt.
.7. Real-time PCR confirmation of the genexpression
In order to confirm the validity of the microchippproach, we performed real-time PCR (RT-PCR) anal-sis of 10 genes commonly or differentially up-regulatedy the acute and chronic exposure to IR. The fol-owing genes were analyzed: cold-regulated cor15arecursor (At2g42540), MYB-related transcription fac-or (At2g46830), alternative NADH-dehydrogenase256057 at), DNA-binding protein similar to CCA1At1g01060), putative protein (At3g54500), DNAJrotein (At5g23240), DREB2A (At5g05410), tran-criptional activator CBF1 (At1g12610), At14a-1At3g28290), putative protein ethylene-responsive ele-ent binding protein homolog (At4g34410). TheT-PCR confirmed the expression pattern observed inicrochip experiment, although the degree of regulationas somewhat different (Table 1).Next, we analyzed the expression pattern of sev-
ral of these genes using the database provided byffymetrix (www.affymetrix.com) and found the major-
ty of these genes to change their expression upon
xposure to various stresses, including salt, manni-ol, drought, cold, UVB, mitomycin and bleomycin.or example, cor15a (At2g42540) did not change itsxpression at 1 h after cold exposure, but did strongly
able 1eal-time PCR data
ene
or15a precursor (At2g42540)YB-related transcription factor CCA1, LHY (At2g46830)lternative NADH-dehydrogenase (At1g07180)NA-binding protein similar to CCA1 (At1g01060)utative protein (At3g54500)NAJ protein (At5g23240)REB2A (At5g05410)ranscriptional activator CBF1 similar DREB1A (At1g12610)t14a-1 (At3g28290)utative protein ethylene-responsive element binding protein homolog (At4g3
he real-time PCR data is presented as a ratio to control. Original data was calach of two independent RNA preparations). The microchip data is in parenth
arch 624 (2007) 101–113 109
(>10-fold) at 24 h. It was strongly induced by a streamof dry air (at 1 h and 3 h after exposure), by the expo-sure to mannitol (3–24 h after exposure), and by theexposure to salt (3–24 h after exposure). At the sametime it was insignificantly changed upon UV exposure.Also, the MYB-related transcription factor CCA1, LHY(At2g46830) was over 3-fold induced by cold at 12–24 hafter exposure, down-regulated by exposure to high-light (within the first 1 h), strongly down-regulated byexposure to UVB, drought, mannitol, salt, mitomycinand bleomycin (3–12 h). DREB2A (At5g05410) wasinduced by 2–4-fold by cold, UV-B, drought, mannitol,salt, mitomycin and bleomycin at 1–24 h after exposure.The other genes such as At14a-1, ethylene-responsiveelement, CCA1 related were also similarly induced bynumber of stresses.
The above-mentioned comparison confirms how sim-ilar the transcriptomes of A. thaliana plants exposed tovariety of stresses are. This further suggests that it shouldbe possible to select the group of common “stress tran-scriptome” as well as to identify the unique IDs of eachspecific stress. This, however, was beyond the scope ofcurrent report.
3.8. Promoter analysis
We analyzed the promoter areas of genes belong-ing to “transcription factors and DNA binding”,“general stress response”, “nucleic acid metabolism”,“oxidative stress” and “signal transduction” forthe following elements: ABA-responsive elements(ABRE; PyACGTGGC), dehydration-responsive ele-
ments (DRE; TACCGACAT), DRE-related core motif(core; CCGAC), ubiquitous regulatory elements (G-box; ACCGTG), MYB (TAACGGTT and other multiplemotifs), MYC (multiple motifs), sex-determining region
110 I. Kovalchuk et al. / Mutation Research 624 (2007) 101–113
Fig. 6. Analysis of the promoter regions of radiation induced genes revealed substantial differences between acute and chronic groups. Promoterareas (2000 nucleotides) of the genes belonging to “general stress”, “transcription factors”, “signal transduction” and “oxidative stress” were analyzed
ct, SRY4 I, Ch
for the following motives: ABRE, DRE, G-box, MYB, MYC, core, Oof the motives, whereas X-axis shows different motives found in 2 I, 2
Y (SRY; AAACAAA), CdxA, chicken homeoboxgene TF (CdxA; ATTAATA), octamer protein bindingsites/X-ray-responsive elements (Oct; ATGCAAAT),and hepatocyte nuclear factor/X-ray-responsive ele-ments (HNF-3; GTTTGTTTT). A detailed analysis of2000 nts of the promoter region revealed substantial dif-ferences in distribution of the elements among the genesbelonging to “acute” or “chronic” groups (Fig. 6). At thesame time, no difference was found upon comparison of“2 h” and “24 h” groups.
In this work, differences were found in the SRY ele-ments, that commonly present in genes determining cellfate and differentiation in animals. We found that therewas substantially less SRY element-containing genesbelonging to the “general stress” group down-regulatedby chronic exposure when compared to acute exposure(60% the acute groups and only 28% in chronic group).Similarly, the “transcription factors” group of genes hadonly 20–25% of SRY elements in the chronic group andover 60% in acute group. The SRY element is commonlyfound in plant genes (Fig. 6), and despite the fact thatthe function of SRY elements in plants is not clear, it is
possible that its function is conserved in plants and thatacute stresses result in more severe changes in cell dif-ferentiation and cell fate decisions than does chronic IRstress.
, HNF-3, CdxA. Y-axis shows the percentage of genes containing oneI, 2 D, 24 D and Ch D groups.
The HNF transcription factors are involved in celldifferentiation and cell proliferation and are commonlyregulated by stress. This element is rarely found inplant genes, so it was difficult to make any comparisonbetween “acute” and “chronic” groups (Fig. 6).
The ABRE element was found to be more abundant inthe “general stress” group of genes that was up-regulatedby chronic radiation as opposed to those of acute expo-sure. This element was found in all genes in the chronicgroup and only in less than 20% of genes in the acutegroup (Fig. 6). The group of down-regulated genes alsocontained more ABRE elements in the chronic group,14% versus the 0% found in acute group. This furthersupports our theory that chronic stress regulates moregenes that are helping in plant adaptation, whereas theacute stress regulates the genes necessary for immedi-ate survival. This assumption would suggest that thechronic stress would induce more genes belonging to the“general stress” category, which would therefore containvarious regulatory elements such as ABRE, core, andDRE. We have profiled promoters of several “unknown”and “undetermined” genes and found much lower fre-
quency of the occurrence of ABRE, DRE, Oct and coreelements (data not shown).
Acute stress induced more genes belonging to “tran-scription factors” (and probably signaling pathways) that
on Rese
cCembMtac
4
ttoatrimaag
iht
A
yAma
A
cj
R
[
[
[
[
[
[
[
[
[
[
[
I. Kovalchuk et al. / Mutati
ontain various regulatory elements such as MYC, core,dxA, SRY. Indeed, as it can be seen in Fig. 6, regulatorylements like ABRE, CORE, CdxA were more com-only found in ‘general stress’ group of genes induced
y chronic exposure, whereas regulatory elements likeYC, core, CdxA and SRY were more common in
he acute group of “transcription factor” genes. Further,s can be seen from Fig. 4, acute stress more activelyhanges the expression of transcription factors.
. Conclusion
Our study suggests that plants respond to acute radia-ion in a similar way as it responds to other stresses. Thisype of response is often directed to immediate repairf the damage, activation of pro-survival mechanisms,nd perhaps inhibition of cell division/cell differentia-ion. In contrast, chronic stress leads to a totally differentesponse that reflects in adaptive responses by regulat-ng genes belonging to general stress and nucleic acid
etabolism. The latter is of great importance, as plantdaptation is associated with chromatin modificationsnd changes in methylation pattern, both having a trans-enerational nature.
The main, general conclusion that this study suggestss that acutely exposed plants have to respond quick andard to survive, whereas chronically exposed plants haveo adjust and fine-tune their physiology.
cknowledgements
We want to thank Edward Oakeley for his help in anal-sis of microchip data and Barbara Hohn, Franz Zemp,lex Boyko and Scott Greer for critical comments on theanuscript. The NSERC and Alberta Ingenuity Grants
re acknowledged for financial support.
ppendix A. Supplementary data
Supplementary data associated with this articlean be found, in the online version, at doi:10.1016/.mrfmmm.2007.04.009.
eferences
[1] S.P. Jackson, Detecting, signalling and repairing DNA double-strand breaks, Biochem. Soc. Trans. 29 (2001) 655–661.
[2] R.W. Holst, D.J. Nagel, Radiation effects on plants, in: W. Wang,J.W. Gorsuch, J.S Hughes (Eds.), Plants for Environmental Stud-
ies, CRC Press/Lewis Publishers, New York, 1997, pp. 31–87.
[3] N.P. Arkhipov, N.D. Kuchma, S. Askbrant, P.S. Pasternak, V.V.Musica, Acute and long-term effects of irradiation on pine (Pinussilvestris) stands post-Chernobyl, Sci. Total Environ. 157 (1994)383–386.
[
arch 624 (2007) 101–113 111
[4] V.Y. Dashlers, I.D. Rashals, The influence of gamma or neutronradiation on the changes of plant productivity in populations ofArabidopdsis thaliana in eight generations, Arabidopsis Inform.Serv. 14 (1977).
[5] J.P. Witherspoon, A.K. Corney, Differential and combined effectsof beta, gamma and fast neutron irradiation of soybean seedlings,Radiat. Bot. 3 (1970) 125–133.
[6] J. Paradiz, J. Skrk, B. Druskovic, Cytogenetic effects of ionizingradiation on meristem, Acta Pharm. 42 (1992) 397–401.
[7] I.G. Mednic, P.D. Usmanov, Influence of gamma-rays on the num-ber of initial cell in Arabidopsis thaliana, Arabidopsis Inform.Serv. 22 (1985) 65–70.
[8] V.V. Schevchenko, L.I. Grinikh, Spectrum of chlorophyll defi-cient mutations induced by gamma-irradiation of Arabidopsisthaliana at different stages of development and scored withembryo test, Arabidopsis Inform. Serv. 18 (1981) 127–129.
[9] I. Kovalchuk, O. Kovalchuk, A. Arkhipov, B. Hohn, Transgenicplants are sensitive bioindicators of nuclear pollution caused bythe Chernobyl accident, Nat. Biotechnol. 16 (1998) 1054–1057.
10] O. Kovalchuk, A. Arkhipov, I. Barylyak, I. Karachov, V. Titov, B.Hohn, I. Kovalchuk, Plants experiencing chronic internal expo-sure to ionizing radiation exhibit higher frequency of homologousrecombination than acutely irradiated plants, Mutat. Res. 449(2000) 47–56.
11] S. Ichikawa, C. Takahashi, Somatic mutation frequencies in thestamen hairs of stable and mutable clones of Tradescantia afteracute gamma-ray treatments with small doses, Mutat. Res. 45(1977) 195–204.
12] S. Ichikawa, Tradescantia stamen-hair system as an excellentbotanical tester of mutagenicity: its responses to ionizing radia-tion and chemical mutagens, and some synergistic effects found,Mutat. Res. 270 (1992) 3–22.
13] V.I. Abramov, O.M. Fedorenko, V.A. Shevchenko, Geneticconsequences of radioactive contamination for populations ofArabidopsis, Sci. Total Environ. 112 (1992) 19–28.
14] A.B. Syomov, S.N. Ptitsyna, S.A. Sergeeva, Analysis of DNAstrand break induction and repair in plants from the vicinity ofChernobyl, Sci. Total Environ. 112 (1992) 1–8.
15] E.L. Kordium, P.G. Sidorenko, The results of the cytogeneticmonitoring of the species of angiosperm plants growing in the areaof the radionuclide contamination after the accident at the Cher-nobyl Atomic Electric Power Station, Tsitol. Genet. 31 (1997)39–46.
16] E.I. Ziablitskaia, S.A. Geras’kin, A.A. Udalova, E.V. Spirin, Ananalysis of the genetic sequelae of the contamination of winter ryecrops by the radioactive fallout from the Chernobyl Atomic Elec-tric power station, Radiat. Biol. Radioecol. 36 (1996) 498–505.
17] I. Kovalchuk, V. Titov, B. Hohn, O. Kovalchuk, Transcriptomeprofiling reveals similarities and differences in plant responses tocadmium and lead, Mutat. Res. 570 (2005) 149–161.
18] J. Molinier, E.J. Oakeley, O. Niederhauser, I. Kovalchuk, B. Hohn,Dynamic response of plant genome to ultraviolet radiation andother genotoxic stresses, Mutat. Res. 571 (2005) 235–247.
19] T. Eulgem, Regulation of the Arabidopsis defense transcriptome,Trends Plant Sci. 10 (2005) 71–78.
20] S. Herbette, L. Taconnat, V. Hugouvieux, L. Piette, M.L. Magni-ette, S. Cuine, P. Auroy, P. Richaud, C. Forestier, J. Bourguignon,
J.P. Renou, A. Vavasseur, N. Leonhardt, Genome-wide transcrip-tome profiling of the early cadmium response of Arabidopsis rootsand shoots, Biochimie (2006).
21] I. Gadjev, S. Vanderauwera, T.S. Gechev, C. Laloi, I.N. Minkov,V. Shulaev, K. Apel, D. Inze, R. Mittler, F. Van Breusegem,
Transcriptomic footprints disclose specificity of reactive oxy-gen species signaling in Arabidopsis, Plant Physiol. 141 (2006)436–445.
22] R. Thilmony, W. Underwood, S.Y. He, Genome-wide transcrip-tional analysis of the Arabidopsis thaliana interaction with theplant pathogen Pseudomonas syringae pv. tomato DC3000 andthe human pathogen Escherichia coli O157:H7, Plant J. 46 (2006)34–53.
23] P. Swoboda, S. Gal, B. Hohn, H. Puchta, Intrachromosomalhomologous recombination in whole plants, EMBO J. 13 (1994)484–489.
24] A. Moiseev, V. Ivanov, Directory for Dosimetry and RadiationHygiene, Atomizdat, Moscow, 1991, p. 220.
25] N.I. Sanzharova, V.A. Kotik, A.N. Arkhipov, G.A. Sokolik, Iu.A.Ivanov, S.V. Fesenko, S.E. Levchuk, The quantitative parametersof the vertical migration of radionuclides in the soils in differ-ent types of meadows, Radiat. Biol. Radioecol. 36 (1996) 488–497.
26] R. Jefferson, Assaying chimeric genes in plants: the GUSgene fusion system, Plant Mol. Biol. Reporter 5 (1987) 387–405.
27] Y.E. Dubrova, V.N. Nesterov, N.G. Krouchinsky, V.A. Ostapenko,R. Neumann, D.L. Neil, A.J. Jeffreys, Human minisatellitemutation rate after the Chernobyl accident, Nature 380 (1996)683–686.
28] C. Mothersill, C. Seymour, Radiation-induced bystander andother non-targeted effects: novel intervention points in can-cer therapy? Curr. Cancer Drug Targets 6 (2006) 447–454.
29] E.G. Wright, P.J. Coates, Untargeted effects of ionizing radia-tion: implications for radiation pathology, Mutat. Res. 597 (2006)119–132.
30] U. Wittstock, B.A. Halkier, Glucosinolate research in the Ara-bidopsis era, Trends Plant Sci. 7 (2002) 263–270.
31] H. Duan, M.A. Schuler, Differential expression and evolution ofthe Arabidopsis CYP86A subfamily, Plant Physiol. 137 (2005)1067–1081.
32] N. Kitahata, S. Saito, Y. Miyazawa, T. Umezawa, Y. Shimada,Y.K. Min, M. Mizutani, N. Hirai, K. Shinozaki, S. Yoshida, T.Asami, Chemical regulation of abscisic acid catabolism in plantsby cytochrome P450 inhibitors, Bioorg. Med. Chem. 13 (2005)4491–4498.
33] K. Sun, Y. Cui, B.A. Hauser, Environmental stress alters genesexpression and induces ovule abortion: reactive oxygen speciesappear as ovules commit to abort, Planta 222 (2005) 632–642.
34] T. Ishikawa, Y. Morimoto, R. Madhusudhan, Y. Sawa, H. Shibata,Y. Yabuta, A. Nishizawa, S. Shigeoka, Acclimation to diverseenvironmental stresses caused by a suppression of cytosolic ascor-bate peroxidase in tobacco BY-2 cells, Plant Cell Physiol. 46(2005) 1264–1271.
35] G.H. Badawi, N. Kawano, Y. Yamauchi, E. Shimada, R. Sasaki,A. Kubo, K. Tanaka, Over-expression of ascorbate peroxidasein tobacco chloroplasts enhances the tolerance to salt stress andwater deficit, Physiol. Plant. 121 (2004) 231–238.
36] I. Murgia, D. Tarantino, C. Vannini, M. Bracale, S. Carravieri, C.Soave, Arabidopsis thaliana plants overexpressing thylakoidal
ascorbate peroxidase show increased resistance to Paraquat-induced photooxidative stress and to nitric oxide-induced celldeath, Plant J. 38 (2004) 940–953.
37] I. Thoma, C. Loeffler, A.K. Sinha, M. Gupta, M. Krischke, B.Steffan, T. Roitsch, M.J. Mueller, Cyclopentenone isoprostanes
[
arch 624 (2007) 101–113
induced by reactive oxygen species trigger defense gene activa-tion and phytoalexin accumulation in plants, Plant J. 34 (2003)363–375.
38] M.W. Bianchi, C. Roux, N. Vartanian, Drought regulation ofGST8, encoding the Arabidopsis homologue of ParC/Nt107glutathione transferase/peroxidase, Physiol. Plant 116 (2002)96–105.
39] A.P. Smith, S.D. Nourizadeh, W.A. Peer, J. Xu, A. Bandyopad-hyay, A.S. Murphy, P.B. Goldsbrough, Arabidopsis AtGSTF2is regulated by ethylene and auxin, and encodes a glutathione-S-transferase that interacts with flavonoids, Plant J. 36 (2003)433–442.
40] X.J. He, R.L. Mu, W.H. Cao, Z.G. Zhang, J.S. Zhang, S.Y. Chen,AtNAC2, a transcription factor downstream of ethylene and auxinsignaling pathways, is involved in salt stress response and lateralroot development, Plant J. 44 (2005) 903–916.
41] N.S. Mishra, R. Tuteja, N. Tuteja, Signaling through MAP kinasenetworks in plants, Arch. Biochem. Biophys. 452 (2006) 55–68.
42] Y.H. Cheong, H.S. Chang, R. Gupta, X. Wang, T. Zhu, S.Luan, Transcriptional profiling reveals novel interactions betweenwounding, pathogen, abiotic stress, and hormonal responses inArabidopsis, Plant Physiol. 129 (2002) 661–677.
43] K. Sax, The effect of ionizing radiation on plant growth, Am. J.Bot. 42 (1955) 360–364.
44] J.E. Gunckel, The effects of ionizing radiation on plants: mor-phological effects quarterly, Rev. Biol. 32 (1957) 46–56.
45] K. Daly, K.H. Thompson, Quantitative dose-response of growthand development in Arabidopsis thaliana exposed to chronicgamma-radiation, Int. J. Radiat. Biol. Relat. Stud. Phys. Chem.Med. 28 (1975) 61–66.
46] G. Fox, Drought and the evolution of flowering time in desertannuals, Am. J. Bot. 77 (1990) 1508–1518.
47] C. Martinez, E. Pons, G. Prats, J. Leon, Salicylic acid regu-lates flowering time and links defence responses and reproductivedevelopment, Plant J. 37 (2004) 209–217.
48] Y. Sakuma, K. Maruyama, Y. Osakabe, F. Qin, M. Seki,K. Shinozaki, K. Yamaguchi-Shinozaki, Functional analysisof an Arabidopsis transcription factor, DREB2A, involved indrought-responsive gene expression, Plant Cell 18 (2006) 1292–1309.
49] C. Temme, S. Zaessinger, S. Meyer, M. Simonelig, E. Wahle, Acomplex containing the CCR4 and CAF1 proteins is involvedin mRNA deadenylation in Drosophila, EMBO J. 23 (2004)2862–2871.
50] K.W. Mulder, G.S. Winkler, H.T. Timmers, DNA damage andreplication stress induced transcription of RNR genes is depen-dent on the Ccr4-Not complex, Nucleic Acids Res. 33 (2005)6384–6392.
51] T.J. Westmoreland, J.R. Marks, J.A. Olson Jr., E.M. Thompson,M.A. Resnick, C.B. Bennett, Cell cycle progression in G1 and Sphases is CCR4 dependent following ionizing radiation or replica-tion stress in Saccharomyces cerevisiae, Eukaryot. Cell 3 (2004)430–446.
52] Y. Yu, Y. Teng, H. Liu, S.H. Reed, R. Waters, UV irradiation stim-ulates histone acetylation and chromatin remodeling at a repressedyeast locus, Proc. Natl. Acad. Sci. U.S.A. 102 (2005) 8650–
8655.
53] M. Benhamed, C. Bertrand, C. Servet, D.X. Zhou, ArabidopsisGCN5, HD1, and TAF1/HAF2 interact to regulate histone acety-lation required for light-responsive gene expression, Plant Cell(2006) [Epub ahead of print].
on Rese
[
[
I. Kovalchuk et al. / Mutati
54] Y. Saintigny, F. Delacote, D. Boucher, D. Averbeck, B.S. Lopez,
XRCC4 in G1 suppresses homologous recombination in S/G2, inG1 checkpoint-defective cells, Oncogene (2006) [Epub ahead ofprint].
55] E. Convery, E.K. Shin, Q. Ding, W. Wang, P. Douglas, L.S. Davis,J.A. Nickoloff, S.P. Lees-Miller, K. Meek, Inhibition of homol-
[
arch 624 (2007) 101–113 113
ogous recombination by variants of the catalytic subunit of the
DNA-dependent protein kinase (DNA-PKcs), Proc. Natl. Acad.Sci. U.S.A. 102 (2005) 1345–1350.
56] T. Nagata, H. Yamada, Z. Du, S. Todoriki, S. Kikuchi, Microarrayanalysis of genes that respond to gamma-radiation in Arabidopsis,J. Agric. Food Chem. 53 (2005) 1022–1030.
